1

CH

AP

TE

R O

NE

INT

RO

DU

CT

ION

The

aim

of

this

dis

sert

atio

n is

to

el

uci

dat

e th

e det

ails

beh

ind th

e ty

rosi

ne

phosp

hory

lati

on

of

a pro

tein

in

volv

ed

in

Cdc4

2-m

edia

ted

signal

ing

pat

hw

ays,

SH

3P

X1.

SH

3P

X1,

also

know

n a

s so

rtin

g n

exin

9 (

SN

X9),

has

bee

n i

den

tifi

ed a

s a

subst

rate

of

the

tyro

sine

kin

ase,

ac

tivat

ed C

dc4

2-a

ssoci

ated

kin

ase

-2 (A

CK

2),

an

d

incr

easi

ngly

im

pli

cate

d i

n t

he

sort

ing o

f var

ious

cell

surf

ace

rece

pto

rs i

ncl

udin

g t

he

insu

lin,

EG

F,

and t

ransf

erri

n r

ecep

tors

. D

ue

to t

he

infl

uen

tial

role

of

SH

3P

X1 i

n a

num

ber

of

signal

ing p

athw

ays,

we

sought

to p

rovid

e a

com

ple

te i

llust

rati

on o

f th

e

bio

logic

al s

yst

ems

involv

ed a

nd t

he

signif

ican

ce o

f th

e des

crib

ed p

rote

ins

on c

om

ple

x

cell

ula

r pro

cess

es s

uch

as

rece

pto

r en

docy

tosi

s an

d d

egra

dat

ion.

Cd

c42 a

nd

its

reg

ula

tors

Cdc4

2,

a m

ember

of

the

Rho

fam

ily

of

smal

l G

TP

-bin

din

g

pro

tein

s (G

pro

tein

s),

has

bee

n

impli

cate

d

in

num

erous

cell

ula

r ev

ents

, in

cludin

g

cell

cy

cle

pro

gre

ssio

n [

1],

apopto

sis

[2,

3],

act

in c

yct

osk

eleto

n r

earr

angem

ent

[4,

5],

act

ivat

ion

of

the

stre

ss-r

esponsi

ve

mit

ogen

-act

ivat

ed p

rote

in (

MA

P)

kin

ases

(c-J

un k

inase

, p38)

[6],

intr

acel

lula

r tr

affi

ckin

g [

7],

and e

ndocy

tosi

s [8

].

Lik

e al

l G

pro

tein

s, C

dc4

2 a

cts

as a

mo

lecu

lar

swit

ch,

cycl

ing b

etw

een a

n a

ctiv

e G

TP

-bound s

tate

and a

n i

nac

tive,

or

bas

al G

DP

-bound s

tate

. T

he

acti

vat

ion o

f C

dc4

2 i

s in

itia

ted b

y n

um

erous

cell

surf

ace

rece

pto

rs,

incl

udin

g

pro

teogly

cans

[9],

re

cepto

r ty

rosi

ne

kin

ases

[1

0],

G

pro

tein

-

couple

d r

ecep

tors

(G

PC

Rs)

[11],

cyto

kin

e re

cepto

rs [

12],

and i

nte

gri

ns

[13].

O

nly

in

this

ac

tive

stat

e is

C

dc4

2 ab

le bin

d to

an

d act

ivat

e dow

nst

ream

ef

fect

or

pro

tein

s

(Fig

ure

1.1

).

2

Fig

ure

1.1

C

dc4

2-m

edia

ted

sig

nali

ng p

ath

ways.

Cdc4

2 G

TP

ase

acti

vit

y i

s ac

tivat

ed b

y a

num

ber

of

upst

ream

cel

l su

rfac

e re

cepto

rs

incl

udin

g

pro

teogly

cans,

re

cepto

r ty

rosi

ne

kin

ases

, G

pro

tein

-couple

d

rece

pto

rs,

cyto

kin

e re

cepto

rs an

d in

tegri

ns.

U

pon ac

tivat

ion,

Cdc4

2 is

th

en ab

le to

m

edia

te

signal

ing p

athw

ays

involv

ed i

n m

ult

iple

bio

logic

al f

unct

ions

incl

udin

g c

yto

skel

etal

org

aniz

atio

n,

mit

ogen

esis

, an

d r

ecep

tor

endocy

tosi

s th

rough i

ts d

ow

nst

ream

eff

ecto

r

pro

tein

s.

3

4

Due

to t

he

com

ple

x r

ole

of

smal

l G

pro

tein

s in

sev

eral

sig

nal

tra

nsd

uct

ion

pat

hw

ays,

the

num

ber

of

pro

tein

s im

pli

cate

d i

n t

hei

r re

gula

tion i

s not

surp

risi

ng [

14].

Thes

e re

gula

tors

are

inv

olv

ed i

n p

rom

oti

ng G

pro

tein

act

ivat

ion,

dea

ctiv

atio

n,

and

traf

fick

ing bet

wee

n th

e cy

toso

l an

d th

e pla

sma

mem

bra

ne.

T

hey

in

clude

guan

ine

nucl

eoti

de

exch

ange

fact

ors

(G

EF

s),

GT

Pas

e ac

tivat

ing p

rote

ins

(GA

Ps)

, an

d g

uan

ine

nucl

eoti

de

dis

soci

atio

n i

nhib

itors

(G

DIs

) (F

igure

1.2

).

By

cata

lyzi

ng

the

exch

ang

e of

GT

P

for

GD

P,

GE

Fs

are

resp

onsi

ble

fo

r

acti

vat

ing G

pro

tein

sig

nal

ing.

GE

Fs

that

act

ivat

e th

e R

ho

-subfa

mil

y o

f G

pro

tein

s

oft

en c

onta

in t

andem

Dbl

ho

molo

gy (

DH

) an

d p

leck

stri

n h

om

olo

gy (

PH

) dom

ains.

The

DH

dom

ain i

s la

rgel

y r

esponsi

ble

for

the

cata

lyti

c ac

tivit

y o

f th

e G

EF

[15,

16],

wher

eas

the

PH

dom

ain i

s th

ought

to b

e pri

mar

ily i

nv

olv

ed i

n l

oca

lizi

ng t

he

exch

ange

fact

or

to t

he

pla

sma

mem

bra

ne

thro

ugh t

he

bin

din

g o

f phosp

hat

idyli

nosi

tides

[17,

18].

Des

pit

e th

is w

idel

y a

ccep

ted r

ole

for

the

PH

dom

ain,

incr

easi

ng e

vid

ence

sugges

ts i

t

has

an a

ddit

ional

role

in m

edia

ting t

he a

ctiv

ity o

f th

e D

H d

om

ain o

f a s

ub

set

of

GE

Fs

[19].

E

xch

ange

fact

ors

hav

e bee

n l

argel

y f

ound t

o e

xis

t in

inac

tive

or

par

tial

ly a

ctiv

e

stat

es w

ithin

the c

ell.

T

her

e ap

pea

r to

be a

nu

mber

of

mec

han

ism

s re

sponsi

ble

for

mai

nta

inin

g t

hei

r in

acti

ve

confo

rmat

ions,

incl

udin

g i

ntr

amole

cula

r or

auto

inhib

itory

inte

ract

ions

bet

wee

n t

he

DH

and P

H d

om

ains

[19,

20],

inte

ract

ions

bet

wee

n t

he

DH

or

PH

d

om

ains

and

an

inhib

itory

dom

ain

[21,

22],

oli

gom

eriz

atio

n

thro

ugh

inte

rmole

cula

r in

tera

ctio

ns

bet

wee

n

DH

dom

ains

[23,

24],

an

d

inte

rmole

cula

r

inte

ract

ions

wit

h c

ellu

lar

pro

tein

s [2

5,

26].

T

he

inac

tive

confo

rmat

ion o

f G

EF

s in

viv

o

appea

rs t

o b

e a

pro

tect

ive

mec

han

ism

, pre

ven

ting t

he

aber

rant

or

hyper

-act

ivat

ion o

f

thei

r G

pro

tein

tar

get

s.

The

bin

din

g o

f G

EF

s to

the

GD

P-b

ound G

pro

tein

cau

ses

a

confo

rmat

ional

chan

ge

wit

hin

the

guan

ine

nucl

eoti

de-

bin

din

g p

ock

et,

intr

oduci

ng a

conse

rved

hydro

phobic

res

idue

into

the

Mg

2+ c

oord

inat

ion s

pher

e an

d e

lim

inat

ing t

he

5

Fig

ure

1.2

A

sc

hem

ati

c d

iagra

m of

the

Cd

c42 G

TP

ase

cy

cle

wit

h ass

oci

ate

d

regu

lato

ry p

rote

ins.

6

7

coord

inat

ion of

a cr

itic

al ly

sine

resi

due

wit

h th

e !

-phosp

hat

e of

GD

P [2

7].

T

his

com

bin

atio

n o

f ev

ents

lea

ds

to t

he

rele

ase

of

GD

P a

nd s

tabil

izat

ion o

f th

e nucl

eoti

de-

free

sta

te [

28],

wher

e hig

her

GT

P c

once

ntr

atio

ns

in c

ells

dri

ve

the

G p

rote

in t

o t

he

GT

P-b

ound s

tate

.

G

TP

ase-

acti

vat

ing p

rote

ins

(GA

Ps)

ser

ve

to a

tten

uat

e th

e G

pro

tein

-med

iate

d

signal

, re

turn

ing t

he

G p

rote

in b

ack t

o i

ts i

nac

tive

stat

e by

cat

alyzi

ng

the

hydro

lysi

s of

GT

P.

T

he

cata

lyti

c ac

tivit

y

of

the

GA

P

requir

es

a co

nse

rved

ar

gin

ine

resi

due,

iden

tifi

ed i

n b

ioch

emic

al a

nd s

truct

ura

l st

udie

s as

a k

ey c

om

ponen

t in

sta

bil

izin

g t

he

tran

siti

on s

tate

[29-3

2].

M

ore

spec

ific

ally

, th

e “a

rgin

ine

finger

,” i

nse

rted

by t

he

GA

P

into

the

GT

P-b

indin

g s

ite,

ser

ves

to s

tabil

ize

the

neg

ativ

e ch

arge

of

the "-

phosp

hat

e of

GT

P i

n t

he

tran

siti

on-s

tate

, as

wel

l as

po

siti

on g

luta

min

e 61 t

o h

ydro

gen

bond t

o t

he

nucl

eophil

ic w

ater

mole

cule

res

ponsi

ble

for

atta

ckin

g t

he "-

phosp

hat

e [3

1,

33].

Even

conse

rvat

ive

muta

tions

of

the

argin

ine

finger

lea

d t

o a

sig

nif

ican

t lo

ss o

f ca

taly

tic

acti

vit

y [

29].

H

ow

ever

, th

e ar

gin

ine

finger

can

not

acco

unt

for

all

of

the

GA

P a

ctiv

ity,

as G

AP

muta

nts

dev

oid

of

the

conse

rved

arg

inin

e re

sidue

are

stil

l ab

le t

o e

lici

t an

~10

-

fold

sti

mula

tion o

f G

TP

hydro

lysi

s, s

ugges

ting a

more

com

ple

x r

egula

tory

role

for

the

GA

P [

29,

34].

R

ecen

t st

udie

s su

ggest

that

an a

ddit

ional

role

for

the

GA

P i

nvolv

es

stab

iliz

ing t

he

swit

ch r

egio

ns

of

the

G p

rote

in,

a m

echan

ism

that

pro

per

ly p

osi

tions

resi

dues

in t

he

G p

rote

ins

that

are

cri

tica

l fo

r G

TP

hydro

lysi

s [2

9, 34].

G

uan

ine

nucl

eoti

de

dis

soci

atio

n i

nhib

itors

(G

DIs

) co

mpri

se t

he

thir

d c

lass

of

pro

tein

s in

volv

ed i

n t

he

regula

tion o

f R

ho

-fam

ily G

pro

tein

s.

The

Rho-f

amil

y G

DIs

(RhoG

DIs

) hav

e bee

n sh

ow

n to

b

e in

volv

ed in

at

le

ast

thre

e dis

tinct

bio

chem

ical

acti

vit

ies.

T

he

firs

t of

thes

e in

volv

es t

he

inhib

itio

n o

f G

DP

dis

soci

atio

n,

the

acti

vit

y

for

whic

h t

hey

wer

e ori

gin

ally

nam

ed [

35,

36].

R

hoG

DIs

are

als

o a

ble

to i

nhib

it G

AP

-

cata

lyze

d G

TP

hydro

lysi

s [3

7].

In

addit

ion,

GD

Is h

ave

also

bee

n s

how

n t

o s

tim

ula

te

the

rem

oval

of

post

-tra

nsl

atio

nal

ly

modif

ied

G

pro

tein

s fr

om

m

embra

nes

.

This

8

acti

vit

y h

as l

ed t

o t

he s

uggest

ion t

hat

GD

Is s

hutt

le t

hei

r G

pro

tein

tar

get

s bet

wee

n

dif

fere

nt

mem

bra

ne

loca

tio

ns.

T

he

bin

din

g o

f R

hoG

DI

to t

he

pre

nyla

ted C

-ter

min

us

of

Rho f

amil

y G

pro

tein

s hel

ps

to m

ask t

hei

r hy

dro

phobic

tai

l fr

om

the

hydro

phil

ic

cyto

pla

smic

envir

onm

ent

[38,

39].

Bio

logic

al

Ro

les

for

Cd

c42

Per

hap

s th

e m

ost

w

idel

y ac

cepte

d ro

le of

Cdc4

2 an

d oth

er R

ho-f

amil

y G

pro

tein

s is

in

the

org

aniz

atio

n o

f th

e act

in c

yto

skel

eton.

T

he

gen

e en

codin

g C

dc4

2

was

fir

st i

den

tifi

ed i

n t

his

conte

xt

in S

acc

haro

myce

s ce

rvis

iae,

wher

e pola

riza

tion o

f

the

acti

n c

yto

skel

eton i

s cr

itic

al f

or

bud f

orm

atio

n [

40].

In

thes

e as

says,

tem

per

ature

-

sensi

tive

(Ts)

muta

nts

def

ecti

ve

in t

he C

DC

42 g

ene

conti

nued

to g

row

at

a re

stri

cted

tem

per

ature

, but

fail

ed

to

bud,

resu

ltin

g

in

larg

e unbudded

ce

lls

[40].

Imm

unofl

uore

scen

ce s

tudie

s fu

rther

confi

rmed

this

role

for

Cdc4

2,

as

it w

as l

oca

lize

d

to t

he

tips

and s

ides

of

enla

rgin

g b

uds

of

S. ce

rvis

iae

[41].

In a

ddit

ion t

o a

ctin

cyto

skel

etal

rea

rran

gem

ent,

a n

um

ber

of

addit

ional

role

s

for

Cdc4

2,

incl

udin

g t

he

contr

ol

of

cell

gro

wth

, hav

e now

com

e to

be

appre

ciat

ed.

Init

ial

insi

ghts

into

a r

ole

for

Cdc4

2 i

n c

ell

gro

wth

cam

e fr

om

the

iden

tifi

cati

on o

f th

e

Dbl

onco

gen

e as

a G

EF

for

Cdc4

2 [

42].

D

bl

was

ori

gin

ally

iso

late

d f

rom

a h

um

an

dif

fuse

B-c

ell

lym

phom

a, a

nd w

as s

how

n t

o t

ransf

orm

NIH

3T

3 c

ells

and t

o c

ause

tum

ors

in n

ude

mic

e [4

3].

T

he

hyper

-act

ivat

ion o

f C

dc4

2 a

nd

rel

ated

Rho-f

amil

y

pro

tein

s has

bee

n s

how

n t

o b

e re

sponsi

ble

for

the

tran

sform

ing p

roper

ties

of

Dbl

[44].

Ear

ly s

tudie

s of

the

effe

cts

of

Rho-f

amil

y p

rote

ins

on c

ell

gro

wth

focu

sed o

n

GT

Pas

e-def

ecti

ve

muta

nts

, bas

ed o

n t

he

tran

sform

ing p

roper

ties

of

the

Ras

-Q61L

an

d

Ras

-G12V

muta

nts

. H

ow

ever

, m

ixed

res

ult

s hav

e bee

n r

eport

ed i

n t

he l

iter

ature

for

the

effe

cts

resu

ltin

g f

rom

GT

Pas

e-def

ecti

ve

muta

nts

of

Cdc4

2,

incl

udin

g t

he

inhib

itio

n

of

cell

gro

wth

rep

ort

ed b

y o

ur

labora

tory

and o

ther

s.

This

led

to t

he

hypoth

esis

that

9

the

cycl

ing o

f C

dc4

2 b

etw

een i

ts G

DP

- an

d G

TP

-bound s

tate

s w

as n

eces

sary

for

its

abil

ity t

o t

ransf

orm

cel

ls.

Indee

d,

Cdc4

2-F

28L

, a

muta

nt

whic

h e

xhib

its

an i

ncr

ease

d

capac

ity t

o e

xch

ange

nucl

eoti

de i

n t

he

abse

nce

of

GE

Fs

wit

hout

com

pro

mis

ing i

ts

abil

ity

to

under

go

GT

P

hydro

lysi

s,

tran

sform

s ce

lls.

More

sp

ecif

ical

ly,

stab

le

expre

ssio

n o

f C

dc4

2-F

28L

enab

les

cel

ls t

o g

row

in l

ow

ser

um

, as

wel

l as

ach

ieve

a

hig

h d

ensi

ty,

dem

onst

rati

ng a

loss

of

conta

ct i

nhib

itio

n [

44,

45].

A

ddit

ional

stu

die

s in

soft

agar

hav

e li

nked

Cdc4

2 t

o a

nch

ora

ge-

indep

enden

t gro

wth

[44].

Muta

tion o

f phen

yla

lanin

e 28 i

n R

as

and r

elat

ed p

rote

ins

to l

euci

ne

has

bee

n

show

n t

o i

ncr

ease

th

e ra

te o

f G

DP

dis

soci

atio

n b

y 1

50 f

old

, ac

tivat

ing t

he

GT

Pas

es i

n

vivo

[4

6].

This

m

uta

tion is

th

ought

to

stab

iliz

e th

e guan

ine

ring

moie

ty

of

the

nucl

eoti

de

thro

ugh p

i-pi

orb

ital

inte

ract

ions

contr

oll

ing G

DP

-GT

P e

xch

ange

[47-4

9].

Cdc4

2-F

28L

under

goes

sponta

neo

us

nucl

eoti

de

exch

ange,

wit

h r

ates

com

par

able

to

those

for

Dbl-

cata

lyze

d e

xch

ange o

n w

ild-t

ype

Cd

c42 [

45],

whil

e th

e G

TP

ase

acti

vit

y

rem

ains

esse

nti

ally

unch

anged

. T

he

inse

rt r

egio

n (

resi

dues

120

-139 i

n C

dc4

2),

un

ique

to

Rho-s

ubfa

mil

y

pro

tein

s,

has

bee

n

iden

tifi

ed

as

an

esse

nti

al

elem

ent

for

the

tran

sform

ing ac

tivit

y of

Cdc4

2-F

28L

. T

he

del

etio

n of

this

in

sert

re

gio

n does

not

affe

ct t

he

abil

ity o

f C

dc4

2-F

28L

to t

rigger

act

in c

yto

skel

etal

rea

rran

gem

ents

nor

to

acti

vat

e m

any d

ow

nst

ream

eff

ecto

rs.

How

ever

, re

moval

of

the

inse

rt p

reven

ts b

indin

g

to t

he

exch

ange

fact

or,

Cool-

1/!

-Pix

, an

d i

ts b

indin

g p

artn

er,

Cbl,

an E

3 u

biq

uit

in

ligas

e.

This

appea

rs t

o a

ccount

for

why C

dc4

2 m

uta

nts

lac

kin

g t

he

inse

rt r

egio

n a

re

tran

sform

atio

n-d

efec

tive

[50].

N

ot

only

do C

dc4

2 a

nd

Ras

shar

e st

ruct

ura

l an

d b

iolo

gic

al s

imil

arit

ies,

but

Cdc4

2 h

as a

lso b

een

found t

o b

e es

senti

al f

or

Ras-

induce

d t

ransf

orm

atio

n.

Expre

ssio

n

of

the

dom

inan

t-neg

ativ

e m

uta

nt,

C

dc4

2-T

17N

, has

bee

n

show

n

to

inhib

it

focu

s

form

atio

n b

y R

as i

n N

IH3T

3 c

ells

[51],

sig

nif

ican

tly d

ecre

ase

Ras-

induce

d a

nch

ora

ge-

10

indep

enden

t gro

wth

, an

d r

ever

t th

e tr

ansf

orm

ed p

hen

oty

pe

obse

rved

in

H-R

as-V

12

cell

lin

es [

51].

T

he

Cdc4

2-D

118N

m

uta

nt

has

also

bee

n

show

n

to

poss

ess

the

abil

ity

to

tran

sform

cel

ls.

This

muta

tio

n o

ccurs

at

a co

nse

rved

moti

f in

volv

ed i

n s

tabil

izin

g t

he

guan

ine

ring

of

the

nucl

eoti

de

thro

ugh

hydro

gen

bondin

g

inte

ract

ions

bet

wee

n

a

conse

rved

asp

arti

c ac

id r

esid

ue,

D118,

and t

he

ring n

itro

gen

(N

7)

[52].

T

he

D118N

muta

tion r

esult

s in

an i

ncr

ease

d r

ate o

f G

DP

-GT

P e

xch

ange

over

that

of

wil

d-t

ype

Cdc4

2,

alth

ough i

t is

slo

w r

elat

ive

to t

he

intr

insi

c nucl

eoti

de

exch

ange

rate

for

the

Cdc4

2-F

28L

muta

nt.

S

urp

risi

ngly

, th

e C

dc4

2-D

118N

muta

nt

has

pro

ven

to b

e m

ore

pote

nt

in

tran

sform

ing

NIH

3T

3

cell

s th

an

the

F28L

m

uta

nt.

The

ampli

fied

tran

sform

ing pro

per

ties

of

D118N

over

F

28L

hav

e bee

n at

trib

ute

d to

th

e D

118

N

muta

nt

bei

ng r

esis

tant

to c

asp

ase

clea

vag

e.

Giv

en th

e fu

nct

ion o

f C

dc4

2 in

m

itogen

ic pat

hw

ays,

a

role

fo

r C

dc4

2 in

infl

uen

cing

signal

ing

by

gro

wth

fa

ctor

rece

pto

rs

was

not

surp

risi

ng.

C

dc4

2

has

num

erous

dow

nst

ream

eff

ecto

rs,

incl

udin

g t

he

Co

ol-

1/!

-Pix

pro

tein

. C

ool-

1/!

-Pix

is

an i

nte

rest

ing p

rote

in i

n t

hat

it

oper

ates

both

as

an u

pst

ream

act

ivat

or

and d

ow

nst

ream

targ

et o

f R

ho-f

amil

y G

pro

tein

s.

Cool-

1 h

as b

een s

how

n t

o a

ct a

s an

exch

ang

e fa

ctor

for

Cdc4

2 in

it

s phosp

hory

late

d st

ate

[Fen

g et

al.

, in

pre

ss]

and ad

dit

ional

ly as

a

scaf

fold

li

nkin

g C

dc4

2 to

th

e E

3 ubiq

uit

in li

gas

e, C

bl.

T

hro

ugh th

is in

tera

ctio

n,

Cdc4

2 h

as

bee

n t

ied t

o C

bl,

involv

ed i

n ca

taly

zing t

he

ubiq

uit

inat

ion o

f th

e E

GF

rece

pto

r at

th

e cel

l su

rfac

e.

T

his

ubiq

uit

inat

ion ev

ent

is nec

ess

ary fo

r th

e E

GF

rece

pto

r to

be

sort

ed t

o v

ario

us

cel

lula

r co

mpar

tmen

ts f

or

deg

radat

ion.

How

ever

,

when

C

dc4

2

form

s a

tern

ary

com

ple

x

wit

h

p85C

ool-

1/!

-Pix

an

d

Cbl,

C

bl

is

seques

tere

d a

way

fro

m t

he

EG

F r

ecep

tor,

and i

s no l

onger

able

to b

ind t

o a

nd b

ecom

e

phosp

hory

late

d b

y t

he

rece

pto

r, a

nec

essa

ry s

tep i

n a

ctiv

atin

g i

ts E

3 u

biq

uit

in l

igas

e

acti

vit

y [

53].

T

he

inhib

itio

n o

f E

GF

rec

epto

r ubiq

uit

inat

ion d

isru

pts

norm

al r

ecep

tor

11

pro

cess

ing,

resu

ltin

g i

n i

ts a

ccum

ula

tion a

t th

e ce

ll s

urf

ace

and e

xce

ssiv

e m

itogen

ic

signal

ing [

54,

55][

Fen

g e

t al

., i

n p

ress

].

This

is

one

exam

ple

of

Cdc4

2 a

nd a

ssoci

ated

pro

tein

s in

volv

emen

t in

gro

wth

fac

tor

rece

pto

r re

gula

tion.

Addit

ional

exam

ple

s w

ill

be

dis

cuss

ed b

elow

.

Erb

B F

am

ily o

f G

row

th F

act

or

Rec

epto

rs

Mem

ber

s of

the

EG

F r

ecep

tor

fam

ily,

also

cal

led E

rbB

or

HE

R r

ecep

tors

, ar

e

involv

ed i

n t

he

init

iati

on o

f var

ious

signal

ing p

athw

ays

resu

ltin

g i

n d

iver

se b

iolo

gic

al

resp

onse

s in

cludin

g

pro

life

rati

on,

dif

fere

nti

atio

n,

mig

rati

on,

and

apopto

sis.

E

rbB

rece

pto

rs s

har

e a

conse

rved

str

uct

ure

consi

stin

g o

f tw

o c

yst

eine-

rich

reg

ions

in t

he

extr

acel

lula

r dom

ain a

nd a

tyro

sine

kin

ase

dom

ain w

ith t

yro

sine

auto

phosp

hory

lati

on

site

s w

ithin

the

cyto

pla

smic

port

ion o

f th

e pro

tein

s.

Thes

e tw

o d

om

ains

are

connec

ted

by a

sin

gle

alp

ha

hel

ix s

pan

nin

g t

he

pla

sma

mem

bra

ne.

E

rbB

rec

epto

rs a

re a

ctiv

ated

by l

igan

d b

indin

g t

o t

he

extr

acel

lula

r dom

ain,

lead

ing t

o r

ecep

tor

dim

eriz

atio

n a

nd

tran

sphosp

hory

lati

on o

f sp

ecif

ic c

yto

pla

smic

tyro

sine

resi

dues

(F

igure

1.3

).

Thes

e

tran

sphosp

hory

lati

on e

ven

ts p

rovid

e re

cognit

ion s

ites

for

the

recr

uit

men

t of

var

ious

pro

tein

s in

volv

ed i

n d

ow

nst

ream

sig

nal

ing c

ascades

. T

he

com

bin

atio

n o

f pat

hw

ays

acti

vat

ed

ult

imat

ely

resu

lts

in

chan

ges

in

gen

e ex

pre

ssio

n,

thus

trig

ger

ing

the

appro

pri

ate

bio

logic

al r

esponse

s to

the

extr

acel

lula

r cu

es.

The

abil

ity of

the

Erb

B re

cepto

rs to

hom

o-

and het

erodim

eriz

e off

ers

the

pote

nti

al f

or

signif

ican

t si

gnal

div

ersi

fica

tion,

as r

efle

cted

by t

he

var

iety

of

dif

fere

nt

ligan

ds

that

can

act

ivat

e th

ese

rece

pto

rs a

nd t

he

man

y d

iffe

rent

signal

ing o

utp

uts

that

they

gen

erat

e.

In t

he

case

of

the

EG

F r

ecep

tor,

a s

ingle

EG

F m

ole

cule

bin

ds

to e

ach

acti

vat

ed r

ecep

tor

[56].

Up

on

gro

wth

fac

tor

bin

din

g,

the

EG

F r

ecep

tor

is t

hough

t to

under

go a

dra

mat

ic c

onfo

rmat

ional

chan

ge

alte

ring t

he

posi

tion o

f th

e dim

eriz

atio

n

12

Fig

ure

1.3

A

rep

rese

nta

tion

of

epid

erm

al

gro

wth

fa

ctor

(EG

F)

rece

pto

r

act

ivati

on

an

d d

ow

nst

ream

sig

nali

ng.

Ad

ap

ted

fro

m B

ioch

emis

try (

Str

yer

), f

ifth

edit

ion

[1

50].

B

indin

g of

EG

F by E

GF

re

cepto

r re

sult

s in

th

e dim

eriz

atio

n an

d

subse

quen

t tr

ans-

phosp

hory

lati

on

of

cyto

pla

smic

ty

rosi

ne

resi

dues

.

This

phosp

hory

lati

on e

ven

t re

cruit

s th

e a

dap

tor

pro

tein

, G

rb2,

to t

he

pla

sma

mem

bra

ne

wher

e it

act

ivat

es

the

Ras

exch

ang

e fa

ctor,

son o

f se

ven

less

(S

os)

. S

os

then

cat

alyze

s

the

exch

ange

of

GT

P f

or

GD

P,

thus

acti

vat

ing R

as,

whic

h u

ltim

atel

y si

gnal

ing t

o

mit

ogen

act

ivat

ed p

rote

in (

MA

P)

kin

ase,

res

ult

ing i

n c

ell

pro

life

rati

on.

13

14

arm

, a

smal

l lo

op

mad

e up

of

amin

o

acid

s 2

42-2

59

involv

ed

in

rece

pto

r dim

er

form

atio

n, sh

ifti

ng f

rom

its

bu

ried

posi

tion i

n t

he i

nac

tive

confo

rmat

ion t

o a

n e

xte

nded

posi

tion i

n t

he

act

ivat

ed s

tate

[57].

D

elet

ions

or

muta

tions

in t

his

dim

eriz

atio

n a

rm

pre

ven

t li

gan

d-i

nduce

d E

GF

rec

epto

r ac

tivat

ion [

58,

59].

The

four

mem

ber

s of

the

Erb

B f

amil

y,

evolu

tionar

ily t

ied t

o t

he

C.

eleg

ans

rece

pto

r ty

rosi

ne

kin

ase

ort

holo

gue,

L

et-2

3

[60],

hav

e under

gone

consi

der

able

div

ersi

fica

tion.

L

igan

d

bin

din

g

contr

ols

th

e ac

tivit

ies

of

the

EG

F

rece

pto

r (a

lso

som

etim

es r

efer

red t

o a

s E

rbB

-1)

and E

rbB

-4,

unli

ke

Erb

B-2

(HE

R2/N

eu)

for

whic

h

no know

n li

gan

d ex

ists

[6

1]

(Fig

ure

1.4

). E

rbB

-3 bin

ds

only

to

m

ember

s of

the

neu

reguli

n f

amil

y (

also

cal

led h

ereg

uli

n)

and l

ack

s kin

ase a

ctiv

ity b

ecau

se o

f se

ver

al

crit

ical

muta

tions

in i

ts a

ctiv

e si

te.

Con

sequen

tly

, nei

ther

Erb

B-2

nor

Erb

B-3

is

able

to

signal

in

dep

enden

tly,

yet

both

re

cepto

rs

signif

ican

tly

enhan

ce

the

acti

vit

y

and

div

ersi

fica

tion of

the

oth

er tw

o m

ember

s [6

2,

63].

In

fa

ct,

Erb

B-2

ex

ists

as

th

e

pre

ferr

ed b

indin

g p

artn

er f

or

the

EG

F r

ecep

tor,

as

wel

l as

for

Erb

B-3

and E

rbB

-4,

once

thes

e re

cepto

rs h

ave

bee

n a

ctiv

ated

by t

hei

r re

spec

tive

ligan

ds.

T

he

bas

is f

or

this

obse

rved

pro

mis

cuit

y o

f E

rbB

-2 a

ppea

rs t

o l

ie i

n t

he

posi

tion o

f it

s dim

eriz

atio

n l

oop.

Wher

e oth

er E

rbB

rec

epto

rs m

ust

bin

d a

lig

and

in o

rder

to e

xte

nd t

he

dim

eriz

atio

n

loop,

the

corr

espondin

g d

imer

izat

ion l

oop o

f E

rbB

-2 i

s pre

-exte

nded

, th

ereb

y p

ois

ed

for

het

erodim

er f

orm

atio

n [

58].

T

he

tran

sform

ing c

apab

ilit

ies

of

Erb

B-2

lik

ely r

esult

s

from

th

e te

nden

cy of

Erb

B-2

het

erodim

ers

to under

go re

cycl

ing bac

k to

th

e ce

ll

surf

ace

rath

er t

han

deg

radat

ion i

n t

he

lyso

zom

e [6

4,

65].

In

gen

eral

, hom

odim

eric

rece

pto

r co

mbin

atio

ns

are

less

m

itogen

ic an

d tr

ansf

orm

ing th

an th

e co

rres

pondin

g

het

erodim

eric

com

bin

atio

ns

[62,

63].

F

or

exam

ple

, E

GF

R-E

rbB

-2 h

eter

odim

ers

are

asso

ciat

ed

wit

h

a m

ore

ro

bust

si

gnal

th

an

EG

F

rece

pto

r hom

odim

ers

[66].

Surp

risi

ngly

, th

e m

ost

tra

nsf

orm

ing o

f th

e het

erodim

eric

com

bin

atio

ns

occ

urs

when

15

Fig

ure

1.4

A

rep

rese

nta

tion

of

the

Erb

B f

am

ily o

f re

cep

tor

tyro

sin

e k

inase

s.

Ad

ap

ted

fro

m M

ose

sson

an

d Y

ard

en [

151].

16

17

the

ligan

d-f

ree

Erb

B-2

rec

epto

r co

mple

xes

wit

h t

he

kin

ase-

def

icie

nt

Erb

B-3

rec

epto

r,

resu

ltin

g f

rom

an i

nit

ial

bin

din

g e

ven

t bet

wee

n h

ereg

uli

n a

nd E

rbB

-3 [

62, 67, 68].

All

pai

rwis

e co

mbin

atio

ns

of

the

Erb

B

rece

pto

r fa

mil

y

mem

ber

s,

four

hom

odim

eric

an

d si

x het

erodim

eric

st

ates

, ca

n b

e ac

tivat

ed by th

e el

even

sp

ecif

ic

Erb

B l

igan

ds

char

acte

rize

d t

o d

ate.

A

ll h

igh

-aff

init

y l

igan

ds

are

com

pri

sed

of

an

EG

F-l

ike

dom

ain a

nd t

hre

e dis

ulf

ide-

bonded

intr

amole

cula

r lo

ops

[69].

T

hes

e pep

tide

ligan

ds

are

expre

ssed

in t

he

extr

acel

lula

r dom

ain o

f tr

ansm

embra

ne

pro

tein

s an

d a

re

gen

erat

ed b

y r

egula

ted p

rote

oly

sis

to y

ield

gro

wth

fac

tors

that

conta

in 4

9-8

5 a

min

o

acid

s.

T

he

gen

erat

ed

gro

wth

fa

ctors

dem

onst

rate

dif

fere

nt

spec

ific

itie

s fo

r th

e

rece

pto

r fa

mil

y m

ember

s.

Fo

r ex

ample

, E

GF

and T

GF

-! h

ave

a hig

h a

ffin

ity f

or

the

EG

F

rece

pto

r,

whil

e neu

reguli

ns

bin

d

to

Erb

B-3

an

d

Erb

B-4

re

cepto

rs.

Over

expre

ssio

n of

Erb

B-2

, an

d th

e su

bse

quen

t het

erodim

eriz

atio

n of

the

rece

pto

r,

seem

s to

bro

aden

lig

and s

pec

ific

ity,

wher

e li

gan

ds

that

are

bet

ter

at r

ecru

itin

g t

his

co-

rece

pto

r ca

n c

om

pet

itiv

ely r

educe

the

bin

din

g o

f le

ss e

ffec

tive-

ligan

ds.

The

bin

din

g o

f var

ious

ligan

ds

and t

he

abil

ity o

f E

rbB

rec

epto

rs t

o h

om

o-

and

het

erodim

eriz

e en

han

ce t

he

var

iati

on o

f ty

rosi

ne

phosp

hory

lati

on e

ven

ts t

hat

can

occ

ur

as a

n o

utc

om

e of

tran

sphosp

hory

lati

on.

In a

ddit

ion t

o t

he

phosp

ho-t

yro

sin

e re

sidues

gen

erat

ed by au

tophosp

hory

lati

on,

non-r

ecep

tor

tyro

sine

kin

ases

, su

ch as

S

rc,

can

phosp

hory

late

the

cyto

pla

smic

dom

ain o

f E

rbB

rec

epto

rs t

o f

urt

her

div

ersi

fy s

ignal

tran

sduct

ion p

athw

ays.

T

he

spec

ific

phosp

ho-t

yro

sine

resi

dues

det

erm

ine

the

sub

set

of

cyto

pla

smic

tar

get

s re

cruit

ed t

o t

he

cell

surf

ace

for

acti

vat

ion. T

hes

e ta

rget

pro

tein

s

gen

eral

ly f

all

into

the

cate

gory

of

Src

hom

olo

gy 2

(S

H2)-

or

phosp

ho-t

yro

sine

bin

din

g

(PT

B)

dom

ain-c

onta

inin

g p

rote

ins.

S

H2 d

om

ains

are

com

pose

d o

f ap

pro

xim

atel

y 1

00

amin

o a

cids

that

rec

ogniz

e phosp

hory

late

d t

yro

sine

resi

dues

bas

ed o

n t

he

thre

e to

six

amin

o a

cids

C-t

erm

inal

to t

he

phosp

hory

late

d s

ite.

C

onver

sely

, th

e bin

din

g o

f P

TB

dom

ains

(appro

xim

atel

y 150 am

ino ac

ids

in le

ngth

) is

det

erm

ined

by th

e re

sidues

18

pre

cedin

g th

e phosp

hory

lati

on si

te [

70].

T

hes

e S

H2-

and P

TB

-dom

ain co

nta

inin

g

pro

tein

s in

clude

adap

tor

pro

tein

s su

ch a

s, G

rb2,

Grb

7,

Crk

, an

d G

ab1,

pro

tein

and

lipid

kin

ases

, S

rc a

nd

phosp

hat

idyli

nosi

tol

3-k

inase

(P

I3K

) re

spec

tivel

y,

and p

rote

in

phosp

hat

ases

such

as

SH

P1 a

nd S

HP

2.

Man

y o

f th

e c

yto

pla

smic

tar

get

s of

Erb

B r

ecep

tors

sig

nal

to

the

MA

P k

inase

pat

hw

ays

via

R

as an

d S

hc,

re

sult

ing in

ce

ll pro

life

rati

on.

In

ad

dit

ion,

the

PI3

K-

acti

vat

ed A

KT

pat

hw

ay a

nd t

he

p70S

6K

/p85S

6K

pat

hw

ays

that

are

involv

ed i

n c

ell

surv

ival

ar

e dow

nst

ream

of

most

E

rbB

-rec

epto

r si

gnal

ing.

W

hil

e a

gre

at dea

l of

over

lap e

xis

ts a

mong t

he

cyto

pla

smic

tar

get

s of

Erb

B r

ecep

tors

, a

num

ber

of

spec

ific

targ

ets

do e

xis

t.

Thes

e in

clu

de

Cbl

and p

ho

sphat

idyli

nosi

tol

3-k

inas

e (P

I3K

), w

hic

h

dem

onst

rate

spec

ific

ity f

or

the

EG

F r

ecep

tor

and E

rbB

-3,

resp

ecti

vel

y [

71,

72].

T

he

bin

din

g s

pec

ific

ity o

f si

gnal

ing p

rote

ins

dow

nst

ream

of

the

Erb

B r

ecep

tors

lar

gel

y

acco

unts

for

the

abil

ity o

f th

ese

recep

tors

to s

tim

ula

te s

ignal

ing p

athw

ays

resu

ltin

g i

n

so m

any v

arie

d b

iolo

gic

al a

ctiv

itie

s.

Bec

ause

Erb

B r

ecep

tors

init

iate

mit

ogen

ic s

ignal

ing p

athw

ays,

over

expre

ssio

n

or

muta

tions

that

al

ter

thei

r in

trin

sic

kin

ase

acti

vit

y ar

e oft

en su

ffic

ient

to ca

use

mal

ignan

t tr

ansf

orm

atio

n.

T

ransf

orm

ing ca

pab

ilit

ies

hav

e bee

n at

trib

ute

d to

E

rbB

rece

pto

rs a

s ea

rly a

s th

e 1

980s,

when

th

e av

ian e

ryth

robla

stosi

s tu

mor

vir

us

was

found

to e

nco

de

a tr

unca

ted f

orm

of

the

hum

an E

GF

rec

epto

r [7

3].

E

rbB

-2 w

as l

ater

iso

late

d

in r

oden

t S

chw

annom

as a

s an

onco

gen

e (o

rigin

ally

cal

led N

eu)

wit

h a

n a

ctiv

atin

g

poin

t m

uta

tion i

n i

ts t

ransm

embra

ne

segm

ent

[74].

M

ore

rec

entl

y,

the

EG

F r

ecep

tor

and E

rbB

-2 h

ave

bee

n i

mpli

cate

d i

n s

ever

al h

um

an n

eopla

sms.

F

or

exam

ple

, th

e E

GF

rece

pto

r is

over

expre

ssed

in b

ladder

, bre

ast,

hea

d a

nd n

eck,

kid

ney

, non

-sm

all

cell

lung,

and p

rost

ate

cance

rs [

75-7

7],

whil

e E

rbB

-2/H

ER

2/N

eu o

ver

expre

ssio

n i

s fo

und

in s

tom

ach a

nd m

amm

ary

car

cinom

as [

78].

O

ver

expre

ssio

n o

f th

e kin

ase-

def

ecti

ve,

Erb

B-3

, has

als

o b

een l

inked

to h

um

an c

ance

rs i

ncl

udin

g b

reas

t, c

olo

n,

and g

astr

ic

19

carc

inom

as,

wher

eas

Erb

B-4

upre

gula

tion h

as b

een f

ound i

n b

oth

bre

ast

cance

r an

d

gra

nulo

se c

ell

tum

ors

of

the

ovar

y [

75].

Can

cer

cell

s ar

e ab

le to

bypass

th

e nece

ssar

y ch

eckpoin

ts in

volv

ed in

th

e

regula

tion

of

Erb

B

rece

pto

r si

gnal

ing

as

desc

ribed

bel

ow

.

Gli

obla

stom

as

and

sarc

om

as a

cquir

e th

e ab

ilit

y t

o p

roduce

gro

wth

fac

tors

to w

hic

h t

hey

are

res

pon

sive,

ther

eby c

reat

ing a

posi

tive

feed

-bac

k l

oop r

efer

red t

o a

s au

tocr

ine

stim

ula

tion [

79].

Oth

er c

ance

r ce

lls

induce

the

over

expre

ssio

n o

f E

rbB

rec

epto

rs a

t th

e ce

ll s

urf

ace,

resu

ltin

g in

hyper

sensi

tivit

y to

oth

erw

ise am

bie

nt

level

s of

gro

wth

fa

ctor.

G

ross

over

expre

ssio

n o

f E

rbB

rec

epto

rs c

an e

lici

t li

gan

d-i

ndep

enden

t si

gnal

ing [

80],

as

can

muta

tions

in t

he

cell

surf

ace

rece

pto

r.

Oft

en t

hes

e m

uta

tions

involv

e tr

unca

tions

of

the

extr

acel

lula

r dom

ain,

resu

ltin

g

in

const

ituti

ve

acti

vat

ion

of

the

rece

pto

r.

Det

erm

inin

g th

e m

echan

ism

by w

hic

h tr

ansf

orm

ed ce

lls

hav

e ex

plo

ited

th

e E

rbB

signal

ing

pat

hw

ays

can

be

extr

emel

y

val

uab

le

in

dev

elopin

g

pro

per

tr

eatm

ent

stra

tegie

s fo

r th

e dis

ease

.

M

ost

rec

ent

ther

apeu

tic

effo

rts

hav

e bee

n d

irec

ted t

ow

ards

the

EG

F r

ecep

tor

and E

rbB

-2,

giv

en t

he

link b

etw

een t

he

over

expre

ssio

n o

f th

ese

pro

tein

s an

d t

um

or

cell

form

atio

n.

The

gre

ates

t ch

alle

nge

in i

den

tify

ing t

her

apeu

tic

agen

ts a

gai

nst

pro

tein

kin

ases

li

es

in th

e fa

ct th

at th

e kin

ase

dom

ain is

hig

hly

co

nse

rved

th

roughout

all

seri

ne/

thre

onin

e an

d

tyro

sine

kin

ases

.

The

maj

ori

ty

of

smal

l m

ole

cule

kin

ase

inhib

itors

ar

e A

TP

-com

pet

itiv

e co

mpounds,

th

ereb

y ru

nnin

g th

e ri

sk o

f in

hib

itin

g

mult

iple

pro

tein

kin

ases

, an

d m

ult

iple

si

gnal

ing

pat

hw

ays.

D

esp

ite

this

ch

alle

nge,

AT

P-c

om

pet

itiv

e in

hib

itors

hav

e bee

n d

evel

op

ed a

gai

nst

the

EG

F r

ecep

tor

and E

rbB

-

2,

and a

re s

urp

risi

ngly

sel

ecti

ve

du

e to

the

rela

tiv

ely d

eep n

ucl

eoti

de-

bin

din

g p

ock

et

of

the

Erb

B p

rote

ins.

T

he

most

sel

ect

ive

inhib

itors

of

the

Erb

B r

ecep

tors

shar

e th

e

bas

ic q

uin

azoli

ne

stru

cture

[8

1,

82].

T

hes

e in

clude

com

pounds

like

Ires

sa (

ZD

-1839),

PK

I-66 a

nd T

arce

va

(OS

I-774).

Ir

essa

was

init

iall

y a

ppro

ved

in t

he

trea

tmen

t of

non-

20

smal

l-lu

ng c

ell

cance

r (N

SC

LC

), w

her

e th

e re

spo

nse

to I

ress

a w

as l

inked

to p

atie

nts

conta

inin

g sp

ecif

ic m

uta

tions

at cr

itic

al si

tes

wit

hin

th

e E

GF

re

cepto

r ac

tive

site

,

resu

ltin

g i

n a

n i

nduce

d f

it o

f th

e dru

g [

83].

C

urr

ent

monocl

onal

anti

body t

reat

men

t of

cance

r in

cludes

tre

atm

ent

of

colo

rect

ora

l ca

nce

r w

ith

an a

nti

-EG

F r

ecep

tor

anti

body,

Erb

itux

TM

, an

d

bre

ast

cance

r tr

eatm

ent

wit

h

the

Erb

B-2

m

onocl

onal

an

tibody,

Her

cepti

nT

M.

Erb

itux

TM

bin

ds

to t

he

EG

F r

ecep

tor

wit

h h

igh a

ffin

ity,

pre

ven

ting t

he

bin

din

g o

f E

GF

or

TG

F-!

and t

her

eby b

lock

ing r

ecep

tor

acti

vat

ion.

The

mec

han

ism

of

inhib

itio

n o

f E

rbB

-2 b

y H

erce

pti

nT

M i

s sl

ightl

y m

ore

com

pli

cate

d d

ue

to t

he

lack

of

an E

rbB

-2 l

igan

d. C

onse

quen

tly,

a gre

at d

eal

of

tim

e has

bee

n i

nves

ted i

n d

eter

min

ing

the

mec

han

ism

of

Her

cepti

nT

M i

nhib

itio

n.

It

is n

ow

thought

that

Her

cepti

nT

M n

ot

only

blo

cks

rece

pto

r bin

din

g

in

het

erodim

ers,

but

als

o

incr

ease

s th

e

rate

of

rece

pto

r

inte

rnal

izat

ion, re

sult

ing i

n t

he

reduct

ion o

f tu

mor

gro

wth

[84].

Des

pit

e th

ese

sig

nif

ican

t gai

ns,

sev

eral

fac

tors

con

tinue

to l

imit

the

effi

cacy

of

Erb

B-t

arget

ed t

her

apeu

tics

. F

or

exam

ple

, kin

ase-

inac

tive

Erb

B p

rote

ins

can r

etai

n

thei

r ab

ilit

y t

o s

ignal

when

com

ple

xed

to o

ther

Erb

B r

ecep

tors

. I

n a

ddit

ion,

gro

wth

fact

ors

can

act

ivat

e E

rbB

sig

nal

ing b

y a

ctiv

atin

g c

yto

pla

smic

tyro

sine

kin

ase

par

tner

s,

such

as

Src

. F

inal

ly,

man

y re

gula

tory

si

gnal

ing pat

hw

ays

involv

ed in

th

e dow

n-

regula

tion o

f E

rbB

rec

epto

r si

gnal

ing,

such

as

Cbl-

cata

lyze

d u

biq

uit

inat

ion,

requir

e

the

kin

ase

acti

vit

y o

f th

e re

cepto

r to

be

inta

ct.

Dow

n-r

egula

tion o

f E

rbB

rec

epto

r

signal

ing h

as,

and w

ill

conti

nue

to b

e a

sought

afte

r m

eans

of

inte

rven

ing a

gai

nst

unco

ntr

oll

ed c

ell

gro

wth

.

Cla

thri

n-m

edia

ted

En

docy

tosi

s

Cla

thri

n-m

edia

ted

endocy

tosi

s is

a

wel

l-st

udie

d

and

hig

hly

ch

arac

teri

zed

pro

cess

in w

hic

h c

ell

surf

ace

rece

pto

rs b

ecom

e in

tern

aliz

ed i

nto

the

cyto

pla

sm.

Not

only

is

cl

athri

n-m

edia

ted

endocy

tosi

s re

spon

sib

le

for

the

upta

ke

of

cell

su

rfac

e

21

rece

pto

rs,

but

it i

s al

so u

tili

zed b

y v

irtu

ally

all

eukar

yoti

c ce

lls

to i

nte

rnal

ize

nutr

ients

,

anti

gen

s, gro

wth

fa

ctors

, an

d pat

hogen

s fr

om

th

e pla

sma

mem

ber

to

in

trac

ellu

lar

com

par

tmen

ts [

85].

C

lath

rin-m

edia

ted e

ndocy

tosi

s ca

n e

ither

occ

ur

const

ituti

vel

y o

r

in r

esponse

to e

xte

rnal

sti

mu

li.

Cla

thri

n c

oat

ed v

esi

cles

(C

CV

s) a

re e

ncap

sula

ted i

nto

a ca

ge

form

ed b

y t

he

inte

ract

ion o

f cl

athri

n t

risk

elio

ns.

T

he

clat

hri

n t

risk

elio

n e

xis

ts

as a

thre

e-le

gged

str

uct

ure

com

pose

d o

f th

ree

hea

vy c

hai

ns

and t

hre

e li

ght

chai

ns.

T

he

carb

oxyl-

term

inal

en

ds

of

the

clat

hri

n hea

vy ch

ains

are

join

ed at

th

e hub re

gio

n,

mak

ing u

p t

he

tris

kel

ion s

truct

ure

[86].

T

hes

e hea

vy c

hai

ns

are

bound t

o o

ne

of

two

light

chai

ns,

LC

a an

d L

Cb,

wher

e th

e li

ght

chai

ns

hav

e bee

n s

ugges

ted

to r

egula

te t

he

asse

mbly

of

the

clat

hri

n tr

iskel

ions

[87].

P

roje

ctin

g aw

ay fr

om

th

e hub,

the

N-

term

inal

glo

bula

r dom

ain

ex

ists

as

a

bet

a-pro

pel

ler

and co

nta

ins

a bin

din

g m

oti

f

resp

onsi

ble

for

clat

hri

n’s

inte

ract

ions

wit

h v

ario

us

endocy

tic

pro

tein

s, i

ncl

udin

g t

he

adap

tor

pro

tein

, A

P-2

.

The

maj

ori

ty

of

clat

hri

n-b

indin

g

pro

tein

s as

soci

ate

wit

h

clat

hri

n t

hro

ugh c

onse

rved

moti

fs,

gen

eral

ly k

now

n a

s cl

athri

n b

ox m

oti

fs,

incl

udin

g

the

LL

NL

D (

Leu

-Leu

-Asn

-Leu

-Asp

) se

quen

ce o

f th

e "

2 s

ubunit

of

AP

-2.

E

ndocy

tosi

s of

cell

surf

ace

recep

tors

is

a ti

ghtl

y r

egula

ted,

sequen

tial

pro

cess

.

It i

nvolv

es s

ever

al c

yto

soli

c pro

tein

s, r

efer

red t

o a

s ac

cess

ory

pro

tein

s, a

s w

ell

as

the

mec

han

ical

m

eans

to

def

orm

th

e m

embra

ne

into

a

ves

icula

r bud

that

ult

imat

ely

pin

ches

off

to f

orm

a f

ree C

CV

(F

igure

1.5

).

This

pro

cess

beg

ins

wit

h t

he r

ecru

itm

ent

of

the

adap

tor

pro

tein

, A

P-2

, to

th

e pla

sma

mem

bra

ne.

A

P-2

is

a het

erote

tram

er

com

pri

sed o

f tw

o l

arge 1

00

-kD

a su

bunit

s, !

and "

, an

d t

wo s

mal

ler

chai

ns µ

2 a

nd

#2,

50 a

nd 2

0 k

Da,

res

pec

tivel

y.

AP

-2 s

erves

to

co

nce

ntr

ate

the

carg

o i

n t

he

emer

gin

g

bud,

as w

ell

as

to l

ink c

lath

rin t

o t

he p

lasm

a m

em

bra

ne

and c

oord

inat

e th

e st

ruct

ura

l

asse

mbly

of

the c

lath

rin c

oat

[88-9

1].

T

he "

sub

unit

of

AP

-2 i

nte

ract

s w

ith c

lath

rin

[92]

whil

e th

e µ

2

subunit

re

cogniz

es

tyro

sine-

bas

ed

endocy

tic

sort

ing

moti

fs

of

mult

iple

pro

tein

s, t

her

eby s

elec

ting t

he

appro

pri

ate

inte

gra

l pro

tein

s fo

r en

docy

tosi

s

22

Fig

ure

1.5

A

sch

emati

c d

iagra

m o

f cl

ath

rin

-med

iate

d e

nd

ocy

tosi

s of

cell

su

rface

rece

pto

rs.

A

dap

ted

fr

om

W

ate

rman

an

d

Yard

en

[152].

Cla

thri

n-m

edia

ted

endocy

tosi

s is

init

iate

d w

ith t

he

recr

uit

men

t of

the

adap

tor

pro

tein

, A

P-2

, to

the

cell

surf

ace.

A

P-2

ser

ves

to c

once

ntr

ate

the

car

go i

nto

the

emer

gin

g b

ud,

as w

ell

as r

ecru

it

clat

hri

n

to

the

pla

sma

mem

bra

ne.

Ass

embly

of

the

clat

hri

n

latt

ice

lead

s to

th

e

invag

inat

ion o

f th

e m

embra

ne

form

ing a

cla

thri

n-c

oat

ed p

it.

The

GT

Pas

e, d

ynam

in,

is

then

ass

emble

d a

t th

e nec

k o

f th

e cl

athri

n-c

oat

ed p

it,

wher

e G

TP

hydro

lysi

s re

sult

s in

fiss

ion t

o f

orm

a f

ree

clat

hri

n-c

oat

ed v

esic

le.

23

24

[93,

94].

O

nce

AP

-2 h

as b

een

rec

ruit

ed t

o t

he

cel

l m

embra

ne

and t

he

ass

embly

of

clat

hri

n

has

bee

n

init

iate

d,

the

mem

bra

ne

acquir

es

incr

easi

ng

curv

ature

unti

l an

invag

inat

ed pit

fo

rms.

A

s th

e cl

athri

n pit

m

ature

s, th

e fi

nal

st

ep in

volv

es fi

ssio

n

thro

ugh t

he

acti

on o

f th

e G

TP

ase,

dynam

in [

95].

D

ynam

in,

a 100-k

Da

pro

tein

, is

a

rath

er uniq

ue

mem

ber

of

the

GT

Pas

e su

per

fam

ily giv

en it

s lo

w af

finit

y fo

r G

TP

(~30 µ

M)

and hig

h in

trin

sic

and st

imula

ted ra

tes

of

GT

P hydro

lysi

s. It

is

now

bel

ieved

that

dynam

in i

s re

cruit

ed t

o t

he

cell

surf

ace

in t

he

GD

P-b

ound o

r nucl

eoti

de-

free

fo

rm an

d is

dis

trib

ute

d th

roughout

the

clath

rin la

ttic

e. G

DP

-GT

P ex

chan

ge

trig

ger

s dynam

in a

ssem

bly

at

the

nec

k o

f th

e cl

athri

n-c

oat

ed p

it,

form

ing a

hel

ical

coll

ar.

The

exac

t m

ole

cula

r m

echan

ism

of

fiss

ion i

s st

ill

uncl

ear,

though i

t se

ems

to

requir

e th

e hydro

lysi

s of

GT

P,

whic

h m

ay d

irec

tly o

r in

dir

ectl

y t

ighte

n t

he

asse

mble

d

dynam

in c

oll

ar r

esult

ing i

n a

fre

e cl

athri

n-c

oat

ed v

esic

le.

Once

the

carg

o h

as

bee

n

inte

rnal

ized

, en

ergy

-dep

enden

t unco

atin

g

rest

ore

s a

ves

icle

w

hic

h

can

even

tual

ly

mat

ure

into

an i

ntr

acel

lula

r co

mpar

tmen

t.

C

lath

rin-c

oat

ed v

esic

les

wer

e in

itia

lly i

den

tifi

ed b

y t

hei

r dis

tinct

ive

mole

cula

r

appea

rance

in e

lect

ron m

icro

gra

phs,

att

ribute

d t

o t

he

clat

hri

n l

atti

ces

that

mak

e u

p t

he

clat

hri

n c

oat

. T

his

ord

ered

str

uct

ure

tak

es

on t

he a

ppea

rance

of

a bask

et m

ade u

p o

f

pen

tagons

and

hex

agon

s [9

6,

97].

The

pen

tagons

appea

r to

fu

lfil

l th

e re

quir

ed

geo

met

ry o

f a

close

d p

oly

gonal

bas

ket

, tw

elve b

eing t

he

requir

ed n

um

ber

, but

the

num

ber

of

hex

agons

var

y [

97]

and a

ppea

r to

contr

ibute

to t

he

size

of

the

ves

icle

. F

or

exam

ple

, ves

icle

s in

the b

rain

are

~76

nm

and

co

nta

in 2

0 h

exag

on

s, w

her

eas

those

in

fibro

bla

sts

are

~120 n

m a

nd c

onta

in 6

0 h

exag

ons

[98].

C

oat

ed

pit

s co

ver

ap

pro

xim

atel

y

0.5

-2%

of

the

cell

su

rfac

e [9

9].

The

form

atio

n of

coat

ed pit

s ap

pea

rs to

st

art

at sp

eci

fic

asse

mbly

si

tes

on

th

e pla

sma

mem

bra

ne

call

ed ‘

pit

zones

’ [1

00].

T

he

fact

ors

that

def

ine

the

asse

mbly

sit

es

hav

e not

bee

n

full

y

iden

tifi

ed,

how

ever

, phosp

hat

idyli

nosi

tides

em

bed

ded

in

to

the

pla

sma

25

mem

bra

ne

hav

e been

found

to r

ecr

uit

AP

-2 t

o t

he

cell

surf

ace.

In

par

ticu

lar,

AP

-2 h

as

a hig

h b

indin

g a

ffin

ity f

or

phosp

hat

idyli

nosi

tol

(4,5

)-bis

phosp

hat

e (

PI(

4,5

)P2)

and

phosp

hat

idyli

nosi

tol

(4,5

,6)-

tris

pho

sphat

e (P

I(3,4

,5)P

3)

[101].

The

bin

din

g

of

PI(

4,5

)P2 r

equir

es p

osi

tivel

y c

har

ged

lysi

ne

resi

dues

on t

he !

-adap

tin s

ubunit

(am

ino

acid

s 21

-80),

wher

e m

uta

tion o

f th

ese

lysi

nes

red

uce

s th

e af

finit

y o

f A

P-2

for

the

pla

sma

mem

bra

ne

[102].

In

addit

ion,

mas

kin

g

PI(

4,5

)P2

wit

h

neo

myci

n,

a hig

h

affi

nit

y l

igan

d f

or

PI(

4,5

)P2,

or

the

PH

dom

ain o

f phosp

holi

pas

e C"

(!"PL

C")

, in

hib

its

the

recr

uit

men

t of

AP

-2 to

th

e en

doso

mes

an

d th

e pla

sma

mem

bra

ne

[103,

104].

Dynam

in-1

an

d

dynam

in-2

hav

e al

so

bee

n

report

ed

to

inte

ract

w

ith

phosp

hat

idyli

nosi

tides

PI(

4,5

)P2

and P

I(4)P

, al

bei

t w

ith l

ow

aff

init

y t

hro

ugh t

hei

r P

H

dom

ains

[105

, 106].

In

th

is ca

se,

pho

sphat

idyli

nosi

tides

ar

e not

likel

y to

re

cruit

dynam

in to

th

e ce

ll su

rfac

e, but

once

dynam

in is

co

nce

ntr

ated

in

th

e co

ated

pit

s,

subse

quen

t in

tera

ctio

ns

wit

h p

hosp

hat

idyli

nosi

tides

appea

r to

pla

y a

sig

nif

ican

t ro

le i

n

fiss

ion f

rom

the

pla

sma

mem

bra

ne.

T

his

model

has

been

support

ed b

y i

n v

itro s

tudie

s

wher

e in

tera

ctio

ns

bet

wee

n d

ynam

in a

nd P

I(4,5

)P2 r

esult

in t

he

dra

mat

ic e

nhan

cem

ent

of

dynam

in G

TP

ase

acti

vit

y [

107].

O

nce

th

e cl

athri

n-c

oat

ed

pit

has

pin

ched

off

fr

om

th

e pla

sma

mem

bra

ne

form

ing t

he

free

cla

thri

n-c

oat

ed v

esic

le,

the i

nte

rnal

ized

ves

icle

even

tual

ly u

nder

goes

unco

atin

g t

o f

orm

the

earl

y e

ndoso

me.

T

his

intr

acel

lula

r co

mpar

tmen

t dev

elops

into

a

rela

tivel

y l

arge

per

inucl

ear

ves

icle

mad

e up o

f m

ult

iple

inte

rnal

ves

icle

s [1

08],

and i

s

refe

rred

to a

s th

e m

ult

ives

icula

r body

(M

VB

s) o

r th

e la

te e

ndo

som

e.

Lat

e en

do

som

es

are

char

acte

rize

d b

y a

n a

ccum

ula

tion o

f hydro

lyti

c en

zym

es,

and a

low

inte

rnal

pH

,

suff

icie

ntl

y

acid

ic

to

dis

soci

ate

som

e

ligan

ds.

Stu

die

s per

form

ed

wit

h

recy

clin

g

rece

pto

rs

and

kin

ase

-def

ecti

ve

muta

nts

of

the

EG

F

rece

pto

r im

ply

th

at

the

late

endoso

me

is t

he

maj

or

site

of

sort

ing f

or

lyso

som

al d

egra

dat

ion.

In t

hes

e st

udie

s,

tran

sfer

rin a

nd k

inas

e-def

ecti

ve

EG

F r

ecep

tors

are

confi

ned

to t

he

ves

icula

r port

ion o

f

26

the

late

endoso

me,

whil

e ac

tive

EG

F r

ecep

tors

acc

um

ula

te i

n t

he

inte

rior

[109,

110].

It i

s th

ought

that

this

tra

nsl

oca

tion o

r co

mpar

tmen

tili

zati

on i

s cr

itic

al f

or

the

rem

oval

of

the

acti

ve

rece

pto

rs

from

th

e re

cycl

ing

pat

hw

ay

to

the

lyso

som

al

deg

radat

ion

pat

hw

ay,

thus

mai

nta

inin

g t

he

appro

pri

ate

rece

pto

r le

vel

s at

th

e ce

ll s

urf

ace.

Rec

epto

r en

docy

tosi

s an

d

deg

radat

ion

are

key

co

mponen

ts

in

the

dow

n-

regula

tion

of

gro

wth

fa

ctor-

med

iate

d

signal

ing

pat

hw

ays.

Giv

en

the

succ

ess

of

ther

apeu

tics

li

ke

Her

cepti

nT

M

in

slow

ing

tu

mor

gro

wth

by

incr

easi

ng

Erb

B-2

deg

radat

ion,

alte

ring th

e ra

tes

for

rece

pto

r en

docy

tosi

s has

bee

n sh

ow

n to

b

e an

effi

cien

t m

eans

of

inhib

itin

g t

um

ori

gen

esis

. O

ur

labora

tory

has

iden

tifi

ed t

wo o

ther

pote

nti

al p

arti

cipan

ts i

n E

GF

rec

epto

r deg

radat

ion,

nam

ely t

he

non-r

ecep

tor

tyro

sine

kin

ase,

A

CK

2,

and

its

subst

rate

, th

e so

rtin

g

nex

in

pro

tein

, S

H3P

X1.

G

iven

th

e

com

ple

xit

y

of

the

endocy

tic

pro

cess

an

d

its

pote

nti

al

import

ance

in

th

erap

euti

cs,

det

erm

inin

g t

he

role

of

AC

K2 a

nd S

H3P

X1 i

n r

ecep

tor

endocy

tosi

s an

d d

egra

dat

ion i

s

of

par

ticu

lar

inte

rest

.

Act

ivate

d C

dc4

2-a

ssoci

ate

d K

inase

-2

Act

ivat

ed

Cdc4

2-a

ssoci

ated

kin

ase-

2

(AC

K2)

is

an

83-k

Da

non-r

ecep

tor

tyro

sine

kin

ase

ori

gin

ally

clo

ned

fro

m a

bovin

e bra

in c

DN

A l

ibra

ry a

s a

targ

et o

f C

dc4

2 [

111].

AC

K2 is

th

e sm

alle

r is

ofo

rm of

AC

K1,

a 120-k

Da

pro

tein

cl

oned

fr

om

a

hu

man

hip

poca

mpal

cD

NA

lib

rary

[112].

A

CK

pro

tein

s ar

e ac

tivat

ed b

y C

dc4

2 i

n i

ts G

TP

-

bound s

tate

, an

d r

ecen

t fi

ndin

gs

confi

rm i

mport

ant

role

s fo

r both

iso

form

s in

sig

nal

tran

sduct

ion p

athw

ays.

T

he

pri

mar

y s

equen

ce of

AC

K2 i

ndic

ates

the

pre

sen

ce o

f

mult

iple

sig

nal

ing d

om

ains,

incl

udin

g a

tyro

sine k

inas

e d

om

ain,

an S

H3 d

om

ain,

a

CR

IB

(Cdc4

2/R

ac

inte

ract

ive-

bin

din

g)

moti

f,

two

pro

line-

rich

dom

ains,

an

d

a

clat

hri

n-b

indin

g d

om

ain [

111,

113]

(Fig

ure

1.6

).

A B

LA

ST

sea

rch o

f th

e N

atio

nal

Cen

ter

of

Bio

tech

nolo

gy I

nfo

rmat

ion d

atab

ase

wit

h t

he

AC

K2 s

equen

ce i

den

tifi

es t

he

27

regio

n

bet

wee

n

resi

dues

13

2

and

378

as

the

tyro

sine

kin

ase

dom

ain,

shar

ing

sim

ilar

itie

s w

ith a

num

ber

of

tyro

sine

kin

ases

, in

cludin

g T

nk1,

the

EG

F r

ecep

tor,

Hck

,

FA

K,

and P

YK

-2 [

114].

A

CK

2 t

yro

sine

phosp

hory

lati

on i

s st

imula

ted b

y a

num

ber

of

extr

acel

lula

r li

gan

ds

incl

udin

g e

pid

erm

al g

row

th f

acto

r (E

GF

), b

radykin

in,

and c

ell

adhes

ion m

ole

cule

s, d

emonst

rati

ng i

ts r

ole

in m

ult

iple

sig

nal

ing p

athw

ays

incl

udin

g

those

in

itia

ted

by

rece

pto

r ty

rosi

ne

kin

ases

an

d

those

in

itia

ted

by

hep

tahel

ical

/G

pro

tein

-couple

d r

ecep

tors

[111].

AC

K2 w

as

init

iall

y i

den

tifi

ed a

s a

hig

hly

spec

ific

tar

get

of

Cdc4

2,

bin

din

g

only

to t

he

acti

vat

ed f

orm

s of

the

G p

rote

in [

111].

A

CK

2 i

s a

spec

ific

Cdc4

2 e

ffec

tor

as i

t does

not

bin

d t

o t

he

act

ivat

ed f

orm

s of

Rac

, R

hoA

, or

Ras

. T

he

bin

din

g b

etw

een

AC

K2 a

nd C

dc4

2 o

ccurs

thro

ugh t

he

CR

IB m

oti

f of

AC

K2 (

resi

dues

454-4

77)

and a

regio

n b

etw

een s

wit

ch I

and s

wit

ch I

I of

the

G p

rote

in (

resi

dues

36-6

8),

des

ignat

ed t

he

AC

K-b

indin

g (

AB

) dom

ain [

115,

116].

B

indin

g t

o C

dc4

2 i

s not

suff

icie

nt

to a

ctiv

ate

AC

K2 i

n vit

ro,

how

ever

, co

-expre

ssio

n o

f A

CK

2 w

ith w

ild

-type

Cdc4

2 o

r C

dc4

2-

Q61L

en

han

ces

AC

K2

auto

phosp

hory

lati

on

in

CO

S-7

ce

lls.

Thes

e re

sult

s,

in

com

bin

atio

n w

ith t

he

inhib

itio

n o

f A

CK

2 a

uto

pho

sphory

lati

on w

ith o

ver

expre

ssio

n o

f

the

dom

inan

t-neg

ativ

e m

uta

nt,

Cdc4

2-D

57Y

[111],

confi

rm t

hat

the

tyro

sine

kin

ase

acti

vit

y o

f A

CK

2 i

s st

imula

ted i

n c

ells

by a

ctiv

ated

form

s of

Cdc4

2.

Cel

l ad

hes

ion h

as a

lso b

een

show

n t

o a

ctiv

ate

AC

K2 [

117].

T

his

act

ivat

ion

likel

y o

ccurs

via

inte

gri

ns

whic

h t

ransm

it s

ignal

s fr

om

extr

acel

lula

r m

atri

x p

rote

ins

and i

nit

iate

a n

um

ber

of

com

ple

x s

ignal

ing p

athw

ays

resp

onsi

ble

for

cell

moti

lity

,

gro

wth

, an

d

dif

fere

nti

atio

n

[118-1

22].

Furt

her

ev

iden

ce

of

inte

gri

n

involv

emen

t

incl

udes

co-i

mm

unopre

cipit

atio

n o

f A

CK

2 w

ith i

nte

gri

n #

1,

and a

dec

reas

e in

AC

K2

phosp

hory

lati

on u

pon t

reat

men

t w

ith a

n a

nti

-inte

gri

n #

1 a

nti

body a

nd R

GD

pep

tides

,

reag

ents

that

blo

ck t

he

inte

ract

ion b

etw

een f

ibro

nec

tin a

nd i

nte

gri

ns.

A

CK

2

28

Fig

ure

1.6

A

dep

icti

on

of

the

dom

ain

str

uct

ure

s o

f ty

rosi

ne

kin

ase

, A

CK

2 a

nd

its

sub

stra

te,

SH

3P

X1.

29

30

acti

vat

ion,

unli

ke

that

of

the

foca

l ad

hes

ion

kin

ase

(FA

K),

does

not

requir

e ce

ll

spre

adin

g,

as no dif

fere

nce

w

as obse

rved

in

A

CK

2 phosp

hory

lati

on bet

wee

n ce

lls

atta

ched

to

poly

lysi

ne-

an

d

fibro

nec

tin-c

oat

ed

pla

tes

[117].

Inte

rest

ingly

, A

CK

2

acti

vat

ion h

as b

een s

ugges

ted t

o d

ecr

ease

the

rate

of

cell

spre

adin

g o

f H

eLa

cell

s onto

fibro

nec

tin [

123].

T

hes

e ef

fect

s ap

pea

r to

be

mod

ula

ted b

y C

rkII

, p130C

as,

Rac

1 a

nd

Rap

1,

wher

e over

expre

ssio

n o

f C

rkII

, P

130C

as a

nd R

ac1 r

ever

se t

he

effe

cts

of

AC

K2

on c

ell

spre

adin

g,

and o

ver

expre

ssio

n o

f a

dom

inan

t-neg

ativ

e fo

rm o

f R

ap1 r

esto

res

the

abil

ity o

f A

CK

2 t

o s

low

cel

l sp

read

ing.

Giv

en t

he

inver

se r

elat

ionsh

ip b

etw

een

cell

spre

adin

g a

nd

cel

l m

oti

lity

, th

e in

crea

sed

moti

lity

exhib

ited

by A

CK

2-e

xpre

ssin

g

cell

s w

as c

onfi

rmed

[123].

Chan

ges

in c

ell

shap

e, c

ell

gro

wth

, an

d t

he f

orm

atio

n o

f fo

cal

com

ple

xes

hav

e

bee

n

attr

ibute

d

to

the

expre

ssio

n

of

AC

K2

in

induci

ble

ce

ll

lines

[1

24].

T

he

morp

holo

gy c

han

ges

rel

ated

to t

he e

xpre

ssio

n o

f A

CK

2 i

n N

IH3T

3 c

ells

incl

ude

a

signif

ican

t deg

ree

of

bra

nch

ing,

wit

h s

om

e of

the

bra

nch

es e

xte

ndin

g t

o a

len

gth

of

200 µ

m,

acco

mpan

ied b

y l

ong s

pik

es e

xte

ndin

g f

rom

the

cell

body.

Alo

ng w

ith

this

morp

holo

gic

al c

han

ge,

induci

ble

expre

ssio

n o

f A

CK

2 i

nhib

ited

cel

l gro

wth

in s

ever

al

cell

li

nes

. T

hes

e ch

anges

in

m

orp

holo

gy

and

cell

pro

life

rati

on

appea

r to

be

indep

enden

t of

AC

K2

kin

ase

act

ivit

y

as

the

induci

ble

ex

pre

ssio

n

of

kin

ase

-dea

d

AC

K2-K

158R

sh

ow

s si

mil

ar

bra

nch

ing

and

inhib

itio

n

of

gro

wth

, th

ough

less

pro

nounce

d w

hen

com

par

ed t

o w

ild-t

ype

AC

K2.

The

kin

ase

acti

vit

y o

f A

CK

2 d

oes,

how

ever

, ap

pea

r to

infl

uen

ce t

he

acti

n c

yto

skel

eton,

resu

ltin

g i

n t

he

dis

ass

embly

of

stre

ss f

iber

s an

d f

oca

l ad

hes

ion

com

ple

xes,

agai

n a

ssoci

atin

g A

CK

2 w

ith c

ell

moti

lity

and c

han

ges

in c

ell

morp

holo

gy.

Of

thes

e obse

rvat

ions,

per

hap

s th

e in

hib

itio

n o

f ce

ll

gro

wth

in A

CK

2 e

xpre

ssin

g c

ells

is

the

mo

st i

ntr

iguin

g,

bec

ause

as

desc

ribed

bel

ow

, it

is c

onsi

sten

t w

ith a

role

for

AC

K2 i

n E

GF

rec

epto

r dow

n-r

egula

tion.

31

Furt

her

evid

ence

for

this

role

surf

aced

wit

h t

he

iden

tifi

cati

on o

f cl

athri

n a

s a

bin

din

g p

artn

er f

or

AC

K2.

Cla

thri

n w

as i

den

tifi

ed i

n G

ST

pull

-dow

n e

xper

imen

ts

usi

ng

GS

T

fusi

on

pro

tein

s th

at

conta

ined

th

e tw

o

carb

oxyl-

term

inal

pro

line-

rich

dom

ains

of

AC

K2 [

113].

C

lath

rin w

as f

ound t

o i

nte

ract

wit

h t

he

over

lappin

g r

egio

ns

bet

wee

n t

he

two c

onst

ruct

s an

d a

seq

uen

ce

alig

nm

ent

confi

rmed

that

AC

K2 c

onta

ined

a co

nse

rved

cla

thri

n-b

indin

g m

oti

f, L

IDF

, sh

ared

by a

ll e

ndocy

tic

adap

tor

pro

tein

s.

Pre

dic

tably

, over

expre

ssio

n of

AC

K2 blo

cks

tran

sfer

rin en

docy

tosi

s by co

mpet

ing

wit

h t

he

endocy

tic

adap

tor

AP

-2,

for

clat

hri

n b

indin

g.

Inte

rest

ingly

, C

dc4

2 n

egat

ivel

y

regula

tes

the

bin

din

g o

f A

CK

2 t

o c

lath

rin a

nd c

on

sequen

tly r

ever

ses

the i

nhib

itio

n o

f

tran

sfer

rin r

ecep

tor

endocy

tosi

s by A

CK

2.

The

iden

tifi

cati

on o

f th

e so

rtin

g n

exin

, S

H3P

X1/s

ort

ing n

exin

9 (

SN

X9),

as

a

bin

din

g p

artn

er f

or

AC

K2 e

xpan

ded

the

role

of

the

non-r

ecep

tor

tyro

sine

kin

ase

to

incl

ude

rece

pto

r so

rtin

g a

nd

deg

radat

ion [

8].

T

he

AC

K2-S

H3P

X1 i

nte

ract

ion o

ccurs

bet

wee

n t

he

pro

line-

rich

dom

ain (

PR

D1)

of

AC

K2 a

nd t

he

SH

3 d

om

ain o

f S

H3P

X1.

SH

3P

X1 i

s not

only

an A

CK

2-b

indin

g p

artn

er,

but

also

a s

ubst

rate

, under

goin

g E

GF

-

dep

enden

t phosp

hory

lati

on

m

edia

ted via

th

e C

dc4

2-p

rom

ote

d ac

tivat

ion of

AC

K2,

wher

e th

e phosp

hory

lati

on

of

SH

3P

X1 is

ab

rogat

ed by th

e over

expre

ssio

n of

the

kin

ase-

def

icie

nt

muta

nt,

AC

K2-K

158R

.

In a

ddit

ion,

AC

K2,

SH

3P

X1,

and c

lath

rin

hav

e bee

n

show

n

to

form

a

com

ple

x

in

cell

s w

her

e th

e A

CK

2-d

epen

den

t

phosp

hory

lati

on o

f S

H3P

X1 r

esult

s in

EG

F r

ecep

tor

deg

radat

ion.

Thes

e fi

ndin

gs

are

of

par

ticu

lar

inte

rest

as

man

y c

ance

rs a

re l

inked

to t

he

over

expre

ssio

n o

f th

e E

GF

rece

pto

r or

EG

F re

cepto

r fa

mil

y m

ember

s an

d re

cepto

r dow

n-r

egula

tion is

a

key

mec

han

ism

in c

ontr

oll

ing r

ecep

tor

level

s at

the

cell

surf

ace.

32

SH

3P

X1

S

H3P

X1,

also

know

n a

s so

rtin

g n

exin

9 (

SN

X9),

was

iden

tifi

ed a

s a

bin

din

g

par

tner

for

AC

K2 i

n m

amm

alia

n c

ells

thro

ugh a

ser

ies

of

GS

T p

ull

-dow

n a

ssay

s [8

].

The

77

-kD

a S

H3P

X1 i

s a

mem

ber

of

the

sort

ing n

exin

fam

ily,

a gro

win

g f

amil

y o

f

cyto

soli

c an

d

mem

bra

ne-

asso

ciat

ed

pro

tein

s in

volv

ed

in

var

ious

asp

ects

of

endocy

tosi

s an

d s

ort

ing o

f ce

ll s

urf

ace

rece

pto

rs.

The

hal

lmar

k o

f th

is f

amil

y i

s th

e

pre

sence

of

the

PX

dom

ain,

a se

quen

ce o

f ap

pro

xim

atel

y 1

00-1

30 a

min

o a

cids

that

has

rece

ntl

y

bee

n

show

n

to

bin

d

var

ious

phosp

hat

idyli

nosi

tol

phosp

hat

es

(Ptd

InsP

s),

ther

eby s

pec

ific

ally

tar

get

ing t

hes

e pro

tein

s to

cel

lula

r m

embra

nes

enri

ched

in t

hes

e

phosp

holi

pid

s [1

25,

126].

Sev

eral

m

ember

s of

the

sort

ing

nex

in

fam

ily

conta

in

addit

ional

sig

nal

ing d

om

ains,

contr

ibuti

ng t

o t

he

loca

liza

tion o

f th

ese

pro

tein

s an

d

thei

r ab

ilit

y t

o f

orm

lar

ge

com

ple

xes

wit

hin

the

cell

. C

urr

entl

y,

twen

ty-f

ive

hu

man

sort

ing n

exin

s hav

e bee

n i

den

tifi

ed a

nd s

tudie

s ar

e u

nder

way

to d

eter

min

e th

e ro

les

of

thes

e pro

tein

s in

the r

egula

tion o

f m

embra

ne

traf

fick

ing.

Mem

ber

s of

the

sort

ing

nex

in f

amil

y c

onta

in l

ittl

e pri

mar

y s

equen

ce h

om

olo

gy,

wit

h t

he

exce

pti

on o

f th

e P

X

dom

ain.

This

dom

ain w

as o

rigin

ally

iden

tifi

ed a

s a

conse

rved

moti

f in

the p

40

ph

ox a

nd

p47

ph

ox

subunit

s of

the

neu

trophil

N

AD

PH

oxid

ase

(phox)

super

oxid

e-gen

erat

ing

com

ple

x [

127].

P

X d

om

ains

hav

e li

mit

ed s

equen

ce h

om

olo

gy a

side

from

a p

roli

ne-

rich

m

oti

f,

PxxP

, en

abli

ng

the

pro

tein

to

in

tera

ct

wit

h

SH

3-d

om

ain

conta

inin

g

pro

tein

s.

Upst

ream

of

the

pro

line-

rich

dom

ain i

s a

phosp

holi

pid

-inte

ract

ion m

oti

f,

whic

h c

an i

nte

ract

wit

h a

wid

e ra

nge

of

phophosp

holi

pid

s.

In a

ddit

ion t

o t

he

sort

ing

nex

ins,

PX

dom

ains

are

also

found i

n c

ell

signal

ing p

rote

ins

such

as

pho

spholi

pas

e D

(PL

D),

phosp

hat

idyli

nosi

tol

3-k

inas

e (P

I3K

), a

s w

ell

as y

east

pro

tein

s in

volv

ed i

n b

ud

emer

gen

ce a

nd

cel

l pola

rity

, B

em1 a

nd B

em3 [

128

, 129].

T

he

stru

cture

of

the

PX

dom

ain o

f p40

ph

ox w

as r

ecen

tly s

olv

ed a

nd h

as b

een s

how

n t

o a

dopt

a novel

fold

that

33

consi

sts

of

thre

e !

-sheet

s pack

ed a

gai

nst

a h

elic

al s

ubdom

ain,

mad

e up o

f fo

ur

alpha

hel

ices

("

2, "

3, "

4 a

nd "

4’)

, a

31

0 h

elix

and a

type

II p

oly

pro

line

hel

ix (

PP

II)

[130].

The

sort

ing nex

in fa

mil

y has

bee

n div

ided

in

to th

ree

sub

-cla

sses

base

d on

conse

rved

dom

ain s

truct

ure

. S

NX

1-2

, S

NX

4-8

, S

NX

15,

and S

NX

16 c

om

pri

se t

he

firs

t cl

ass.

They

al

l hav

e lo

ng

carb

oxyl-

term

inal

ex

tensi

ons

conta

inin

g

1-3

char

acte

rist

ic c

oil

ed-c

oil

mo

tifs

all

ow

ing

for

hom

o-

or

het

ero-o

ligom

eriz

atio

n w

ith

oth

er s

ort

ing n

exin

s, o

r pro

tein

-pro

tein

inte

ract

ions

wit

h n

on

-SN

X p

rote

ins

[131].

T

he

seco

nd su

b-c

lass

, m

ade u

p o

f S

NX

3,

SN

X10,

SN

X11,

SN

X12,

and S

NX

22

-24,

is

char

acte

rize

d

by

the

abse

nce

of

addit

ional

si

gn

alin

g

dom

ains

asid

e fr

om

th

e P

X

dom

ain (

ie.

SH

2,

SH

3,

pro

line-

rich

dom

ains)

. T

he

final

sub-c

lass

of

sort

ing n

exin

s is

mad

e up

of

the

rem

ainin

g

fam

ily

mem

ber

s co

nta

inin

g

var

ious

pro

tein

-pro

tein

inte

ract

ion m

oti

fs su

ch as

S

H3 dom

ains

[132,

133],

m

embra

ne-

targ

etin

g dom

ains

incl

udin

g h

ydro

phobic

seq

uen

ces

[134, 135],

and G

pro

tein

reg

ula

tory

seq

uen

ces

[136,

137].

SH

3P

X1 f

alls

into

this

las

t su

b-c

lass

of

sort

ing n

exin

s.

It c

onta

ins

an a

min

o-

term

inal

SH

3 d

om

ain,

whic

h h

as b

een i

mpli

cate

d i

n i

nte

ract

ions

wit

h A

CK

2 a

nd t

he

Wis

kott

-Ald

rich

sy

ndro

me

pro

tein

(W

AS

P),

th

e la

tter

b

ein

g

involv

ed

in

acti

n

poly

mer

izat

ion a

nd c

yto

skel

etal

org

aniz

atio

n [

138],

and m

ore

rec

entl

y,

the

endocy

tic

pro

tein

, dynam

in-2

[139].

S

H3P

X1 h

as a

lso

bee

n s

how

n t

o b

ind

to c

lath

rin a

nd

AP

-2,

thus

linkin

g t

he

sort

ing n

exin

to c

lath

rin

-med

iate

d e

ndocy

tosi

s an

d c

ell

traf

fick

ing

[139].

T

he

bin

din

g o

f S

H3P

X1 t

o c

lath

rin a

nd A

CK

2 w

as f

irst

dem

onst

rate

d i

n C

OS

-

7 c

ells

[8].

In

this

syst

em,

the

SH

3P

X1-c

lath

rin i

nte

ract

ion a

ppea

rs t

o b

e m

edia

ted

thro

ugh A

CK

2 a

s th

e cl

athri

n-b

indin

g d

efec

tive

muta

nt,

AC

K2

-LI2

A,

and t

he

SH

3

dom

ain-d

efec

tive

mu

tant,

A

CK

2-2

W2A

, dim

inis

hed

S

H3P

X1

bin

din

g

[8].

More

rece

ntl

y,

SH

3P

X1 d

elet

ion m

uta

nts

hav

e bee

n s

how

n t

o b

ind t

o c

lath

rin a

nd t

he

AP

-2

34

"-a

ppen

dag

e in

vit

ro [

139].

T

his

bin

din

g o

ccurs

thro

ugh t

he

regio

n b

etw

een t

he

SH

3

and P

X d

om

ains

of

SH

3P

X1 [

139].

H

ow

ever

, th

is d

irec

t bin

din

g i

nte

ract

ion i

s gre

atly

dim

inis

hed

when

full

-len

gth

SH

3P

X1 i

s use

d a

s th

e bai

t.

Thes

e ob

serv

atio

ns

sugg

est

that

the

bin

din

g s

ites

for

clat

hri

n a

nd A

P-2

may

be

obst

ruct

ed i

n f

ull

-len

gth

SH

3P

X1,

only

to b

e ex

pose

d i

n t

he d

elet

ion m

uta

nts

, per

haps

acco

unti

ng

for

the

dif

fere

nce

s in

bin

din

g b

etw

een i

n v

itro

and i

n v

ivo s

yst

ems.

Dow

nst

ream

of

the

SH

3

dom

ain

in S

H3P

X1

lies

a

Phox

hom

olo

gy (P

X)

dom

ain.

PX

dom

ains

are

hig

hly

conse

rved

in t

he

sort

ing n

exin

fam

ily a

nd a

re t

hought

to m

edia

te t

he

bin

din

g o

f phosp

hoin

osi

tide

seco

nd m

esse

nger

s, c

onse

quen

tly t

arget

ing

thes

e pro

tein

s to

th

e pla

sma

mem

bra

ne.

A

seri

es

of

lipid

-bin

din

g

exper

imen

ts

det

erm

ined

S

H3P

X1

to

be

pro

mis

cuous

in

the

bin

din

g

of

var

ious

phosp

hat

idyli

nosi

tides

[1

39].

SH

3P

X1

has

been

sh

ow

n

to

bin

d

PE

, P

S,

PI3

P,

PI(

3,4

)P2,

PI(

4,5

)P2,

and P

I(3,4

,5)P

3.

More

over

, th

e P

X d

om

ain o

f S

H3P

X1 s

eem

s

to r

equir

e at

lea

st o

ne

expose

d p

hosp

horo

us

gro

up a

s th

ere

was

no o

bse

rved

bin

din

g o

f

PI

[139].

F

inal

ly,

the

last

tw

enty

-eig

ht

amin

o a

cids

are

pre

dic

ted to

hav

e a

hig

h

pro

bab

ilit

y fo

r a

coil

ed-c

oil

fo

rmat

ion,

whic

h is

im

pli

cate

d in

fo

rmin

g fu

nct

ional

mult

imer

ic

com

ple

xes

to

th

is

C-t

erm

inal

re

gio

n.

Rec

ent

studie

s su

gges

t th

at

the

dim

eriz

atio

n o

f S

H3P

X1 m

ay b

e c

riti

cal

for

ph

osp

hory

lati

on o

f th

e so

rtin

g n

exin

,

show

n b

y a

loss

of

AC

K2

-bin

din

g a

ctiv

ity a

nd p

hosp

hory

lati

on o

f tr

unca

tion m

uta

nts

of

as l

ittl

e as

13 a

min

o a

cids

[140].

T

he

Dro

sophil

a

ort

holo

gue

of

SH

3P

X1,

DS

H3P

X1,

is

a 63-k

Da

pro

tein

involv

ed i

n a

xon g

uid

ance

[141].

T

his

pro

tein

was

ori

gin

ally

iden

tifi

ed a

s th

e sm

alle

st

mem

ber

of

a pro

tein

com

ple

x p

uri

fied

fro

m S

2 i

nse

ct c

ells

by t

he S

H2 d

om

ain o

f th

e

adap

tor

pro

tein

, D

ock

. D

ock

con

sist

s of

thre

e ta

ndem

SH

3 d

om

ains

foll

ow

ed b

y a

n

SH

2 d

om

ain a

nd w

as o

rigin

ally

iden

tifi

ed a

s an

ess

enti

al p

rote

in i

n t

he g

uid

ance

of

photo

rece

pto

r ax

ons

in

thir

d

inst

ar

larv

a [1

42].

DS

H3P

X1

has

bee

n

show

n

to

35

asso

ciat

e w

ith

the

Dro

sophil

a

ort

holo

gue

of

the

Dow

ns

Syndro

me

cell

ad

hes

ion

mole

cule

(D

scam

) re

cepto

r, a

s w

ell

as t

he

Dro

sophil

a o

rtholo

gue

of

WA

SP

and t

he

clat

hri

n-c

oat

adap

tor

pro

tein

, A

P-5

0 [

141].

A

ddit

ional

stu

die

s id

enti

fied

DS

H3P

X1 a

s

the

subst

rate

for

Dro

sophil

a A

CK

(D

Ack

) an

d d

efin

ed a

maj

or

site

of

phosp

hory

lati

on

as Y

56 [

143].

T

his

phosp

ho

ryla

tion e

ven

t ap

pea

rs t

o m

edia

te t

he

bin

din

g b

etw

een

com

pet

ing p

artn

ers

WA

SP

and D

ock

in D

roso

phil

a a

xonal

guid

ance

syst

ems.

W

hil

e th

e pre

cise

role

of

SH

3P

X1 p

ho

sphory

lati

on r

emai

ns

to b

e e

stab

lish

ed,

var

ious

lines

of

evid

ence

poin

t to

a s

ort

ing f

unct

ion i

nvolv

ing t

he

EG

F r

ecep

tor

and

rela

ted f

amil

y m

ember

s.

Sort

ing n

exin

1 (

SN

X1)

was

the

firs

t m

amm

alia

n s

ort

ing

nex

in t

o b

e ch

arac

teri

zed [

144].

It

was

iso

late

d i

n a

yea

st-t

wo-h

ybri

d s

cree

n u

sing t

he

kin

ase

dom

ain a

nd t

he

lyso

som

al t

arget

ing s

equen

ce (

tyro

sine

or

di-

leuci

ne

moti

fs)

of

the

EG

F r

ecep

tor

as b

ait.

T

he

port

ion o

f S

NX

1 t

hat

was

iso

late

d i

n t

his

scr

een,

the

carb

oxyl-

term

inal

co

iled

-coil

se

quen

ces,

in

tera

cted

sp

ecif

ical

ly w

ith th

e ly

soso

mal

targ

etin

g m

oti

f, T

yr-

Leu

-Val

-Ile

. I

mport

antl

y,

over

expre

ssio

n o

f fu

ll-l

ength

SN

X1 i

n

CV

1 (

Afr

ican

gre

en m

onkey

kid

ney

) ce

lls

mar

ked

ly d

ow

n-r

egula

ted E

GF

rec

epto

rs.

Oth

er

sort

ing

nex

ins,

in

cludin

g

SN

X2

and

SN

X4,

hav

e bee

n

show

n

to

co-

imm

unopre

cipit

ate

wit

h

rece

pto

rs

that

bin

d

EG

F,

insu

lin,

pla

tele

t-der

ived

gro

wth

fact

or

(PD

GF

) an

d t

he

long f

orm

of

the

lepti

n r

ece

pto

r [1

45],

whil

e S

NX

6 h

as b

een

show

n t

o a

ssoci

ate

wit

h t

he

tran

sform

ing g

row

th f

acto

r !! (

TG

F-!

) re

cepto

r [1

46].

Rec

ent

work

su

gges

ts th

at S

H3P

X1 pla

ys

a ro

le in

m

edia

ting th

e ex

pre

ssio

n an

d

acti

vit

y o

f th

e in

suli

n r

ecep

tor

[147].

S

ubce

llula

r fr

acti

onat

ion s

tudie

s, c

onfi

rmed

by

imm

unofl

uore

scen

ce

exper

imen

ts,

show

th

at

insu

lin

trea

tmen

t st

imula

tes

the

movem

ent

of

SH

3P

X1 fr

om

th

e cy

toso

l to

th

e m

embra

ne.

In

ad

dit

ion,

SH

3P

X1

asso

ciat

es

wit

h t

he

insu

lin r

ecep

tor

in v

itro

and o

ver

expre

ssio

n l

eads

to a

red

uct

ion i

n

cell

surf

ace

insu

lin r

ecep

tors

. S

imil

arly

, re

cent

dat

a fr

om

our

labora

tory

sugges

t th

at

the

AC

K2-c

atal

yze

d p

hosp

hory

lati

on o

f S

H3P

X1 s

tim

ula

tes

EG

F r

ecep

tor

deg

radat

ion

36

in

CO

S-7

ce

lls

[8].

The

phosp

hory

lati

on

of

SH

3P

X1

appea

rs

to

be

crit

ical

in

dec

reas

ing E

GF

re

cepto

r le

vel

s as

th

e kin

ase

-defi

cien

t m

uta

nt,

A

CK

2-K

158R

, has

litt

le

effe

ct.

M

ore

re

cent

work

has

im

pli

cate

d

SH

3P

X1

in

the

endocy

tosi

s of

tran

sfer

rin

rece

pto

r.

A

lth

ou

gh

full

-len

gth

S

H3P

X1

has

no

det

ecta

ble

ef

fect

, th

e

carb

oxyl-

term

inal

tr

unca

tion

muta

nts

, #

C141

and #

C412,

aboli

sh

the

upta

ke

of

tran

sfer

rin

in

HeL

a ce

lls

[139].

Q

uan

tify

ing

tran

sfer

rin

upta

ke

thro

ugh

imm

unofl

uore

scen

ce a

nal

ysi

s of

K562

cel

ls c

onfi

rmed

this

res

ult

[139].

T

he

obse

rved

effe

cts

of

SH

3P

X1 o

n r

ecep

tor

endocy

tosi

s hav

e bee

n a

ttri

bute

d t

o t

he

inte

ract

ion

bet

wee

n th

e S

H3 an

d pro

line-

rich

dom

ains

of

SH

3P

X1 an

d dynam

in,

resp

ecti

vel

y

[148].

R

econst

ituti

on a

ssay

s det

aili

ng t

he

liposo

me

bin

din

g o

f dynam

in a

nd S

H3P

X1

show

that

thes

e tw

o p

rote

ins

hav

e a

pre

fere

nce

for

phosp

hat

idyli

nosi

tols

. I

n t

hes

e

exper

imen

ts,

dynam

in is

un

able

to

bin

d phosp

hat

idyli

nosi

tols

in

ce

lls

dep

lete

d of

endogen

ous

SH

3P

X1,

how

ever

bin

din

g i

s re

store

d u

pon t

he a

ddit

ion o

f re

com

bin

ant

SH

3P

X1.

Support

ing e

vid

ence

show

s dep

leti

on o

f S

H3P

X1 i

n H

eLa

cell

s by R

NA

inte

rfer

ence

(R

NA

i) r

esult

s in

mis

loca

liza

tion o

f dynam

in-2

(D

yn2)

from

the

pla

sma

mem

bra

ne.

A

ddit

ional

ly,

SH

3P

X1 has

bee

n sh

ow

n to

st

imula

te dynam

in’s

bas

al

GT

Pas

e ac

tivit

y b

y s

even

fold

[149].

G

iven

thes

e new

rep

ort

s, w

e ar

e in

tere

sted

in

dev

elopin

g

a sy

stem

in

w

hic

h

we

can

furt

her

as

sess

th

e ro

le

of

SH

3P

X1

phosp

hory

lati

on

in

med

iati

ng

the

com

ple

x

pro

ces

ses

of

rece

pto

r en

docy

tosi

s an

d

deg

radat

ion.

Over

vie

w o

f d

isse

rtati

on

stu

die

s

Init

iall

y,

the

obje

ctiv

es o

f th

is d

isse

rtat

ion i

ncl

uded

iden

tify

ing t

he

tyro

sine

phosp

hory

lati

on s

ites

on S

H3P

X1,

dev

elopin

g i

n v

itro

kin

ase

assa

ys

thro

ugh w

hic

h

smal

l m

ole

cule

in

hib

itors

of

AC

K-c

atal

yze

d

SH

3P

X1

phosp

hory

lati

on

could

b

e

37

iden

tifi

ed,

and d

evel

opin

g a

syst

em i

n w

hic

h t

hese

dom

inan

t-neg

ativ

e re

agen

ts c

ould

be

use

d i

n s

tudyin

g t

he

effe

cts

of

SH

3P

X1 p

hosp

hory

lati

on o

n r

ecep

tor

pro

cess

ing.

To

do

so,

I des

crib

e th

e sy

nth

esis

an

d

use

of

SH

3P

X1

carb

oxyl-

term

inal

del

etio

n m

uta

nts

an

d ty

rosi

ne-

to-p

hen

yla

lanin

e p

oin

t m

uta

nts

in

ef

fort

s to

id

enti

fy

AC

K2-c

atal

yze

d

pho

sphory

lati

on

site

s in

ch

apte

r tw

o.

In

ad

dit

ion,

I det

ail

the

gen

erat

ion an

d puri

fica

tion of

reco

mbin

ant

pro

tein

s, H

is-A

CK

2 an

d H

is-S

H3P

X1,

from

in

sect

ce

ll an

d bac

teri

al ce

ll ex

pre

ssio

n sy

stem

s, re

spec

tivel

y.

I

incl

ude

the

pre

par

atio

n

of

the

mas

s sp

ectr

om

etry

sa

mple

th

rough

whic

h

tyro

sine

287

was

iden

tifi

ed a

s th

e m

ajor

AC

K2

-cat

alyze

d s

ite

on S

H3P

X1,

and t

he

condit

ions

for

the

in

vitr

o k

inas

e ass

ay u

sed i

n t

he

hig

h-t

hro

ughput

scre

ens

at M

erck

& C

o.

As

a re

sult

of

thes

e sc

reen

s, P

arke-

Dav

is c

om

pound,

PD

158780,

was

show

n t

o i

nhib

it A

CK

2 k

inas

e

acti

vit

y w

ith a

n I

C5

0 o

f ~

80 p

M.

In c

hap

ter

thre

e, I

go o

n t

o d

escr

ibe

the

iden

tifi

cati

on

of

two

pre

vio

usl

y

unre

port

ed

tyro

sine

kin

ases

fo

r S

H3P

X1,

FA

K

and

Src

.

I

char

acte

rize

thei

r bin

din

g i

nte

ract

ions

and m

odes

of

SH

3P

X1 p

hosp

hory

lati

on,

and

show

that

they

var

y c

on

sider

ably

fro

m r

esult

s se

en w

ith A

CK

2.

More

over

, I

des

crib

e

tech

niq

ues

use

d t

o i

den

tify

tyro

sine

resi

du

es 1

77,

239,

269,

294,

and 5

61 f

rom

Src

-

cata

lyze

d S

H3P

X1 p

hosp

hory

lati

on i

n c

ells

.

38

Ref

eren

ces

1.

Ols

on,

M.F

., A

. A

shw

ort

h,

and A

. H

all,

An e

ssenti

al

role

for

Rho,

Rac,

and

Cdc4

2

GT

Pase

s in

ce

ll

cycl

e p

rogre

ssio

n

thro

ugh

G1.

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54

CH

AP

TE

R T

WO

RE

GU

LA

TIO

N O

F A

CK

2-C

AT

AL

YZ

ED

SH

3P

X1 P

HO

SP

HO

RY

LA

TIO

N

Ab

stra

ct

Act

ivat

ed C

dc4

2-a

sso

ciat

ed k

inas

e-2 (

AC

K2)

is a

non-r

ecep

tor

tyro

sine

kin

ase

that

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dow

nst

ream

tar

get

of

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Rho-f

amil

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pro

tein

, C

dc4

2.

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83

-

kD

a is

ofo

rm o

f A

CK

was

ori

gin

ally

clo

ned

fro

m a

bovin

e bra

in l

ibra

ry a

nd b

ased

on

pri

mar

y s

equen

ce a

nal

ysi

s, i

s co

mpri

sed

of

sever

al

signal

ing d

om

ains,

in

cludin

g S

H3,

pro

line-

rich

, an

d

clat

hri

n-b

indin

g

dom

ains.

N

ot

surp

risi

ngly

, A

CK

2

has

bee

n

impli

cate

d i

n m

edia

ting t

he

acti

vat

ion o

f num

erous

cell

sig

nal

ing p

athw

ays

that

res

ult

in

dif

fere

nt

bio

logic

al

acti

vit

ies.

Of

thes

e,

an

emer

gin

g

role

fo

r A

CK

2

in

the

endocy

tosi

s an

d t

raff

ickin

g o

f gro

wth

fac

tor

rece

pto

rs f

or

deg

radat

ion,

hold

s par

ticu

lar

inte

rest

. T

his

role

for

AC

K2 l

ikel

y i

nvolv

es i

ts a

bil

ity t

o p

hosp

hory

late

the

sort

ing

nex

in,

SH

3P

X1.

Thus

far,

SH

3P

X1 i

s th

e only

know

n c

ellu

lar

phosp

ho-s

ubst

rate

for

AC

K2,

and p

hosp

hory

lati

on o

f S

H3P

X1 h

as b

een l

inked

to t

he

deg

radat

ion o

f E

GF

rece

pto

rs i

n C

OS

-7 c

ells

. I

n o

rder

to f

urt

her

elu

cidat

e th

e m

ole

cula

r m

echan

ism

s

under

lyin

g g

row

th f

acto

r re

cepto

r en

docy

tosi

s an

d d

egra

dat

ion,

and m

ore

pre

cise

ly

def

ine

the

role

of

AC

K2 a

nd S

H3P

X1 i

n t

his

com

ple

x c

ellu

lar

pro

cess

, w

e hav

e se

t

out

to g

ener

ate

reag

ents

, nam

ely,

phosp

hory

lati

on-d

efec

tive

muta

nts

of

SH

3P

X1 a

nd

smal

l m

ole

cule

inhib

itors

agai

nst

the

tyro

sine

kin

ase

acti

vit

y o

f A

CK

2.

To t

his

end,

we

hav

e gen

erat

ed a

pan

el o

f ca

rboxyl-

term

inal

del

etio

n c

onst

ruct

s of

SH

3P

X1 t

o h

elp

def

ine

the

regio

n o

f S

H3P

X1 p

hosp

hory

late

d b

y A

CK

2.

Our

findin

gs

dem

onst

rate

that

AC

K2 l

ose

s it

s ab

ilit

y t

o p

hosp

hory

late

SH

3P

X1

in c

onst

ruct

1-5

11

wher

e th

e C

-

term

inal

84 a

min

o a

cids

hav

e bee

n r

emoved

. M

ass

spect

rom

etry

anal

ysi

s of

AC

K2-

cata

lyze

d

pho

sphory

lati

on

of

SH

3P

X1

revea

ls

tyro

sine

287

as

the

maj

or

site

of

phosp

hory

lati

on.

T

hes

e dat

a,

toget

her

w

ith

oth

er

report

s,

argue

for

a re

gula

tory

55

mec

han

ism

that

involv

es t

he

dim

eriz

atio

n o

f S

H3P

X1 t

hro

ugh t

he

C-t

erm

inal

BA

R

dom

ain.

In a

ddit

ion,

we

hav

e i

den

tifi

ed a

com

pound,

PD

158780,

whic

h a

cts

as a

pote

nt

inhib

itor

of

AC

K2 k

inas

e ac

tivit

y i

n v

itro

(IC

50 ~

80 p

M).

T

he

gen

erat

ion o

f

phosp

hory

lati

on-d

efec

tive

SH

3P

X1 m

uta

nts

and

the

iden

tifi

cati

on o

f A

CK

2 i

nhib

itors

should

hel

p p

rovid

e a

bet

ter

under

stan

din

g o

f th

e m

echan

ism

s by w

hic

h g

row

th f

acto

r

rece

pto

r le

vel

s in

the

pla

sma

mem

bra

ne

are

tightl

y r

egula

ted.

Intr

od

uct

ion

Reg

ula

tion o

f gro

wth

fac

tor

rece

pto

r ex

pre

ssio

n a

nd k

inas

e a

ctiv

ity l

evel

s is

esse

nti

al fo

r th

e pro

pag

atio

n of

sever

al

signal

tr

ansd

uct

ion pat

hw

ays,

par

ticu

larl

y

those

that

med

iate

mit

ogen

ic r

esp

onse

s in

cel

ls.

Norm

al c

ell

gro

wth

is

achie

ved

by

mai

nta

inin

g t

he

pro

per

bal

ance

bet

wee

n g

row

th f

acto

r re

cepto

r ac

tivat

ion,

signal

ing,

endocy

tosi

s, d

egra

dat

ion,

and r

ecycl

ing.

As

a r

esult

, th

e over

expre

ssio

n o

f gro

wth

fact

or

rece

pto

rs o

r m

uta

tions

that

lea

d t

o i

ncr

ease

s in

thei

r ty

rosi

ne

kin

ase

act

ivit

y

dis

rupt

this

del

icat

e bal

ance

, an

d i

s su

ffic

ient

to c

ause

mal

ignan

t tr

ansf

orm

atio

n.

Of

the

var

ious

know

n

gro

wth

fa

ctor

rece

pto

rs,

the

epid

erm

al

gro

wth

fa

ctor

(EG

F)

rece

pto

r an

d

oth

er

Erb

B

fam

ily

mem

ber

s ar

e th

e bes

t st

udie

d

and

most

hig

hly

char

acte

rize

d.

O

ver

expre

ssio

n of

the

Erb

B fa

mil

y of

rece

pto

rs ap

pea

rs to

pla

y a

causa

tive

role

in

v

ario

us

form

s of

cance

r. A

w

ell-

docu

men

ted ex

ample

in

volv

es

Erb

B-2

/HE

R2/N

eu,

as t

he

over

expre

ssio

n o

f th

is E

rbB

fam

ily m

ember

has

bee

n f

ound

in 2

5-3

0%

of

all

hum

an b

reas

t ca

nce

r ca

ses

and

appea

rs t

o b

e pre

dic

tive

of

a poor

pro

gnosi

s fo

r th

is d

isea

se [

1].

C

urr

entl

y,

the

use

of

monocl

onal

anti

bodie

s to

blo

ck

the

Erb

B-2

/HE

R2/N

eu r

ecep

tor,

pai

red w

ith t

radit

ional

chem

oth

erap

y,

has

bee

n s

how

n

to b

e ef

fica

cious

in t

he

trea

tmen

t of

bre

ast

cancer

pat

ients

. C

on

sequen

tly,

ther

e is

much

in

tere

st in

el

uci

dat

ing

th

e m

echan

ism

under

lyin

g th

is im

munoth

erap

y.

It

is

curr

entl

y b

elie

ved

that

Her

cepti

nT

M n

ot

only

blo

cks

the

form

atio

n o

f het

erodim

ers

56

bet

wee

n E

rbB

-2 a

nd E

GF

rec

epto

rs,

a st

ep t

hat

is

esse

nti

al f

or

rece

pto

r ac

tivit

y,

but

also

incr

ease

s th

e ra

te o

f E

rbB

-2 i

nte

rnal

izat

ion a

nd s

ubse

quen

t deg

radat

ion o

f th

e

rece

pto

r, t

hus

resu

ltin

g i

n t

he

reduct

ion o

f tu

mor

gro

wth

[2].

H

erce

pti

nT

M s

tudie

s hav

e

pro

vid

ed s

ubst

anti

al i

nsi

ght

into

the

abil

ity o

f re

cepto

r en

docy

tosi

s an

d d

egra

dat

ion t

o

counte

r th

e ab

erra

nt

gro

wth

of

tran

sform

ed c

ells

.

As

a re

sult

, m

uch

focu

s has

bee

n o

n n

ot

only

ch

arac

teri

zing t

he

mec

han

ism

s

beh

ind r

ecep

tor

endocy

tosi

s an

d d

egra

dat

ion b

ut

also

in i

den

tify

ing a

nd c

har

acte

rizi

ng

key

pro

tein

s in

volv

ed i

n t

hes

e ev

ents

.

Rec

entl

y,

two n

ew p

arti

cipan

ts h

ave

bee

n

impli

cate

d in

E

GF

re

cepto

r deg

radat

ion,

nam

ely th

e non-r

ecep

tor

tyro

sine

kin

ase,

AC

K2,

and i

ts p

hosp

ho-s

ubst

rate

, th

e so

rtin

g n

exin

pro

tein

, S

H3P

X1.

Pre

vio

usl

y,

AC

K2 h

as

bee

n a

ssoci

ated

wit

h s

uch

cel

lula

r pro

cess

es

as i

nte

gri

n s

ignal

ing [

3],

cel

l

gro

wth

re

gula

tion

[4

], an

d ax

on guid

ance

sy

stem

s in

D

roso

phil

a [5

]. A

CK

2 an

d

SH

3P

X1 h

ave

since

bee

n l

inked

to e

ndocy

tosi

s th

rough t

hei

r ab

ilit

y t

o a

ssoci

ate

wit

h

var

ious

pro

tein

s in

volv

ed i

n t

his

pro

cess

, an

d a

lter

cel

l su

rfac

e re

cepto

r num

ber

s w

hen

over

expre

ssed

. S

pec

ific

ally

, A

CK

2 a

nd S

H3P

X1 h

ave

bee

n s

how

n t

o f

orm

com

ple

xes

wit

h e

ndocy

tic

pro

tein

s, i

ncl

udin

g c

lath

rin,

dynam

in,

and A

P-2

[6-9

].

Not

only

do

thes

e pro

tein

s as

soci

ate

wit

h k

ey e

ndocy

tic

par

tner

s, b

ut

over

expre

ssio

n o

f A

CK

2,

eith

er w

ith w

ild-t

ype

SH

3P

X1 o

r var

ious

SH

3P

X1 d

elet

ion m

uta

nts

, has

bee

n s

how

n

to a

ffec

t th

e pro

cess

ing a

nd t

raff

ickin

g o

f both

EG

F a

nd t

ransf

erri

n r

ecep

tors

[7,

10].

Spec

ific

ally

, dat

a fr

om

our

labora

tory

su

gges

t th

at

the

AC

K2-m

edia

ted

phosp

hory

lati

on o

f S

H3P

X1 s

tim

ula

tes

EG

F r

ecep

tor

deg

radat

ion i

n C

OS

-7 c

ells

[10].

Her

e,

the

phosp

hory

lati

on

of

SH

3P

X1

appea

rs

to

be

crit

ical

in

dec

reas

ing

EG

F

rece

pto

r le

vel

s as

the

kin

ase-

def

icie

nt

muta

nt,

AC

K2-K

158R

, has

lit

tle

effe

ct.

More

rece

nt

work

has

im

pli

cate

d S

H3P

X1 i

n t

he

endocyto

sis

of

tran

sfer

rin r

ecep

tors

, w

her

e

the

carb

oxyl-

term

inal

tru

nca

tion m

uta

nts

, #

C141 a

nd #

C412,

aboli

sh t

he

upta

ke o

f

tran

sfer

rin i

n H

eLa

cell

s [7

].

57

Whil

e th

e pre

cise

ro

le

of

AC

K2

-dep

enden

t p

hosp

hory

lati

on

of

SH

3P

X1

rem

ains

to

be

esta

bli

shed

, var

ious

lines

of

evid

ence

poin

t to

a

sort

ing

funct

ion

involv

ing t

he

EG

F r

ecep

tor

and r

elat

ed f

amil

y m

ember

s.

SH

3P

X1 i

s a

mem

ber

of

the

gro

win

g f

amil

y o

f so

rtin

g n

exin

s, c

yto

soli

c pro

tein

s in

volv

ed i

n t

he

traf

fick

ing a

nd

deg

radat

ion o

f ce

ll s

urf

ace

rece

pto

rs.

Wit

hin

this

fam

ily,

sort

ing n

exin

1 (

SN

X1)

serv

es a

s a

pro

toty

pe

show

n t

o b

ind t

o t

he

cyto

soli

c dom

ain o

f th

e E

GF

rec

epto

r in

yea

st-t

wo-h

ybri

d s

cree

ns

and t

o d

ow

n-r

egula

te E

GF

rec

epto

rs i

n C

V1 (

Afr

ican

gre

en

monkey

kid

ney

) ce

lls

[11].

O

ther

sort

ing n

exin

mem

ber

s hav

e b

een a

ssoci

ated

wit

h

the

pro

cess

ing o

f G

pro

tein

-couple

d r

ecep

tors

, pro

teas

e-ac

tivat

ed r

ecep

tor-

1 (

PA

R1)

[12,

13],

low

-den

sity

lip

opro

tein

rec

epto

r (L

DL

R)

[14],

pla

tele

t-der

ived

gro

wth

fac

tor

(PD

GF

) re

cepto

r [1

5],

and l

epti

n r

ecep

tor

[15].

R

ecen

t w

ork

suggest

s th

at S

H3P

X1

pla

ys

a ro

le

in

med

iati

ng

the

expre

ssio

n

and

ac

tivit

y

of

the

insu

lin

rece

pto

r,

dem

onst

rate

d i

n s

ub

cell

ula

r fr

acti

onat

ion a

nd i

mm

unofl

uore

scen

ce s

tudie

s [1

6].

T

his

,

toget

her

wit

h i

ts e

ffec

ts o

n t

he

traf

fick

ing o

f E

GF

and t

ransf

erri

n r

ecep

tors

, ar

gues

for

a gen

eral

role

for

SH

3P

X1

in p

roce

ssin

g t

ransm

embra

ne

rece

pto

rs.

In o

rder

to d

efin

itiv

ely l

ink t

he

phosp

hory

lati

on o

f S

H3P

X1 t

o g

row

th f

acto

r

rece

pto

r dow

n-r

egula

tion

and

deg

radat

ion,

it

wil

l ult

imat

ely

be

nec

essa

ry

to

man

ipula

te it

s phosp

hory

lati

on st

atus

thro

ugh th

e use

of

phosp

hory

lati

on-d

efec

tive

muta

nts

, an

d i

dea

lly,

smal

l m

ole

cule

inhib

itors

. A

s a

firs

t st

ep t

ow

ard a

chie

vin

g t

hes

e

goal

s, w

e hav

e u

sed a co

mbin

atio

n of

bio

chem

ical

ap

pro

aches

, in

cludin

g del

etio

n

anal

ysi

s, s

ite-

dir

ecte

d m

uta

gen

esi

s, a

nd m

ass

spec

trom

etry

, to

more

full

y c

har

acte

rize

the

inte

ract

ions

bet

wee

n

AC

K2

and

SH

3P

X1,

and

the

site

s of

AC

K2

-cat

alyze

d

phosp

hory

lati

on.

We

hav

e a

lso i

nit

iate

d a

sea

rch f

or

smal

l m

ole

cule

s th

at a

re c

apab

le

of

inhib

itin

g t

hes

e phosp

hory

lati

on e

ven

ts.

58

Mate

rials

an

d M

eth

od

s

Mate

rials

—A

nti

-HA

an

d

anti

-Myc

anti

bodie

s w

ere

from

C

ovan

ce.

A

nti

-

phosp

hoty

rosi

ne

(4G

10)

anti

body w

as f

rom

Upst

ate

Bio

tech

nolo

gy,

Inc.

T

4 D

NA

ligas

e, L

ipofe

ctam

ine

Tra

nsf

ecti

on R

eagen

t, P

rote

in G

agar

ose

bea

ds,

and t

he

Bac

-to-

Bac

Bacu

lovir

us

Expre

ssio

n S

yst

em w

ere

from

Invit

rogen

. T

he

Quik

Chan

ge

Sit

e-

Dir

ecte

d M

uta

gen

esis

kit

was

fro

m S

trat

agen

e an

d t

he

Qia

quic

k G

el E

xtr

acti

on k

it

was

fro

m Q

iagen

. [

32P

]-A

TP

was

fro

m P

erkin

Elm

er.

Con

stru

ctio

n

of

SH

3P

X1

Del

etio

n

Mu

tan

ts—

The

pcD

NA

3-S

H3P

X1

wil

d-t

ype

pla

smid

was

use

d a

s a t

empla

te t

o c

onst

ruct

the c

arboxyl-

term

inal

del

etio

n m

uta

nts

.

Bri

efly

, D

NA

pri

mer

s to

gen

erat

e ea

ch d

elet

ion

const

ruct

(S

H3P

X1

-#C

84, #

C197,

#C

340, #

C395, #

C447)

wer

e en

gin

eere

d w

ith B

amH

1 a

nd E

coR

1 r

estr

icti

on s

ites

,

and t

hen

thes

e pri

mer

s w

ere

use

d i

n a

PC

R r

eact

ion (

PC

R S

pri

nt,

Hybai

d).

T

he

PC

R

pro

duct

s w

ere

ligat

ed in

to th

e pC

R2-T

OP

O vec

tor,

foll

ow

ed by r

estr

icti

on d

iges

ts

wit

h B

amH

1 a

nd E

coR

1.

These

DN

A p

roduct

s w

ere

reso

lved

on a

1%

agar

ose

gel

conta

inin

g 8

0 n

g/m

L e

thid

ium

bro

mid

e, a

nd t

hen

the

inse

rts

wer

e ex

cise

d f

rom

the

gel

and p

uri

fied

wit

h t

he

Qia

quic

k G

el E

xtr

acti

on k

it.

The

resu

ltin

g D

NA

inse

rts

wer

e

then

lig

ated

in

to t

he

HA

-tag

ged

pcD

NA

3 e

xpre

ssio

n v

ecto

r usi

ng T

4 D

NA

lig

ase.

Con

stru

ctio

n o

f S

H3P

X1 P

oin

t M

uta

nts

—S

H3P

X1 t

yro

sine-

to-p

hen

yla

lanin

e poin

t

muta

nts

wer

e al

so g

ener

ated

in

a p

cDN

A3-S

H3P

X1 w

ild

-type

bac

kgro

und.

Conse

rved

muta

nts

of

Dro

sophil

a a

nd h

um

an o

rtholo

gs:

Y9F

, Y

56F

, Y

287F

, Y

496F

, Y

546F

,

Y578F

, as

w

ell

as m

ult

iple

m

uta

nts

: Y

546F

/Y578F

, Y

546F

/Y561F

/Y563F

/Y578F

,

and

Y546F

/Y561F

/Y563

F/Y

570F

/Y578F

w

ere

gen

erat

ed

by

PC

R

usi

ng

the

Quik

Chan

ge

Sit

e-D

irec

ted M

uta

gen

esis

kit

fro

m S

trat

agen

e.

SH

3P

X1 w

ild-t

ype

and p

oin

t m

uta

nts

wer

e su

bcl

oned

into

the

V5-p

cDN

A 3

.1

vec

tor

usi

ng t

he

Dir

ecti

onal

TO

PO

Expre

ssio

n kit

fro

m I

nvit

rogen

. P

rim

ers

wer

e

des

igned

bas

ed

on

the

man

ufa

cture

r’s

inst

ruct

ions

and

SH

3P

X1

wil

d-t

ype

and

59

tyro

sine-

to-p

hen

yla

lanin

e m

uta

nt

DN

A i

nse

rts

were

gen

erat

ed b

y P

CR

usi

ng t

he H

A-

tagged

pcD

NA

3-S

H3P

X1 co

nst

ruct

s as

bac

kgro

und.

The

blu

nt-

end P

CR

pro

duct

s

wer

e re

solv

ed o

n a

1%

agar

ose

gel

conta

inin

g 8

0 n

g/m

L e

thid

ium

bro

mid

e, a

nd t

hen

the

DN

A

inse

rts

wer

e ex

cise

d

from

th

e gel

an

d

puri

fied

w

ith

the

Qia

quic

k

Gel

Extr

acti

on k

it.

The

resu

ltin

g D

NA

inse

rts

wer

e th

en l

igat

ed i

nto

the

pcD

NA

3.1

/V5-

His

-TO

PO

pla

smid

.

Cel

l C

ult

ure

, T

ran

sfec

tion

, an

d P

rep

ara

tion

of

Lysa

tes—

CO

S-7

an

d H

EK

293

cell

s w

ere c

ult

ure

d i

n D

ulb

ecco

’s m

odif

ied E

agle

’s m

ediu

m (

DM

EM

) co

nta

inin

g 1

0%

feta

l bo

vin

e se

rum

(F

BS

).

The

cel

l li

nes

wer

e m

ainta

ined

in 5

% C

O2 a

t 37°C

. T

o

expre

ss t

he

var

ious

form

s of

AC

K2,

SH

3P

X1,

and d

ynam

in-2

in c

ells

, M

yc-

, H

A-,

or

V5-t

agged

co

nst

ruct

s en

codin

g

the

pro

tein

s w

ere

tran

sfec

ted

into

ce

lls

usi

ng

Lip

ofe

ctam

ine

Tra

nsf

ecti

on

Rea

gen

t acc

ord

ing

to

the

man

ufa

cture

r’s

inst

ruct

ions

(Invit

rogen

).

Tw

enty

-four

ho

urs

foll

ow

ing t

he

tran

sfec

tion,

the

cell

s w

ere

rinse

d w

ith

phosp

hat

e-buff

ered

sal

ine

(PB

S)

and l

yse

d w

ith c

old

mam

mal

ian c

ell

lysi

s buff

er (

10

mM

Tri

s (p

H 7

.4),

5 m

M M

gC

l 2,

150 m

M N

aC

l, 1

% T

rito

n X

-100,

1 m

M s

odiu

m

ort

hovan

adat

e,

5

mM

bet

a-gly

cero

l phosp

hat

e,

10 µ

g/m

L

apro

tinin

, 10 µ

g/m

L

leupep

tin)

foll

ow

ed

by

cell

sc

rapin

g.

T

he

cell

ly

sate

s w

ere

then

cl

eare

d

by

centr

ifugat

ion a

t 16,1

00 r

pm

in a

mic

rofu

ge

for

12 m

inute

s.

Pro

tein

conce

ntr

atio

ns

wer

e det

erm

ined

by d

iluti

ng t

he

lysa

tes

1:2

50 w

ith 1

x B

radfo

rd r

eagen

t an

d m

easu

ring

the

abso

rban

ce b

y v

isib

le l

amp o

n t

he

spec

trophoto

met

er.

Imm

un

op

reci

pit

ati

on

—C

ell

lysa

tes

wer

e in

cubat

ed w

ith M

yc,

HA

or

V5 a

nti

bodie

s

for

2-2

0 h

ours

, w

ith r

ota

tion a

t 4°C

. P

rote

in G

agar

ose

bea

ds

wer

e ad

ded

and r

ota

ted

for

an a

ddit

ional

hour.

T

he

bea

ds

wer

e th

en p

reci

pit

ated

in a

mic

rofu

ge

and w

ashed

3-

4 t

imes

wit

h c

old

mam

mal

ian

cel

l ly

sis

buff

er.

The

bea

ds

wer

e re

susp

ended

in 5

x

SD

S l

oad

ing b

uff

er a

nd t

he

asso

ciat

ed p

rote

ins

were

res

olv

ed b

y S

DS

-PA

GE

. T

he

gel

was

then

tra

nsf

erre

d t

o a

PV

DF

mem

bra

ne

and s

ubje

cted

to W

este

rn b

lott

ing a

nal

ysi

s.

60

Inse

ct

Cel

l E

xp

ress

ion

—S

f21

cell

s w

ere

main

tain

ed

in

Gra

ces

Inse

ct

Med

ia

(Invit

rogen

) su

pple

men

ted w

ith 1

0%

fet

al b

ovin

e se

rum

. V

iruse

s en

codin

g H

is-t

agged

and unta

gged

ver

sions

of

AC

K2,

AC

K2-K

158R

, an

d S

H3P

X1 w

ere

pre

par

ed fo

r

inse

ct ce

ll ex

pre

ssio

n usi

ng th

e In

vit

rogen

B

ac-t

o-B

ac B

aculo

vir

us

Expre

ssio

n kit

.

Sf2

1

cell

s w

ere

infe

cted

w

ith

the

vir

use

s an

d

expre

ssio

n

of

each

co

nst

ruct

w

as

det

erm

ined

by W

este

rn b

lott

ing a

nal

ysi

s fo

llow

ing 2

4-,

48-,

and 7

2-h

our

infe

ctio

n

per

iods.

A

CK

2 k

inas

e ac

tivit

y w

as

assa

yed

by c

o-i

nfe

ctin

g S

f21 c

ells

wit

h A

CK

2 a

nd

SH

3P

X1 vir

use

s, re

solv

ing ce

ll ly

sate

s by S

DS

-PA

GE

, tr

ansf

erri

ng to

P

VD

F,

and

imm

unoblo

ttin

g f

or

tyro

sine-

phosp

hory

late

d S

H3P

X1.

Rec

om

bin

an

t P

rote

in E

xp

ress

ion

an

d P

uri

fica

tion

—E

xpre

ssio

n of

His

-SH

3P

X1

was

car

ried

out

in t

he

BL

21 E

. co

li s

trai

n.

Wil

d-t

ype

SH

3P

X1 w

as c

loned

into

the

pE

T28A

vec

tor

for

reco

mbin

ant

expre

ssio

n.

O

ver

nig

ht

cult

ure

s fr

om

co

lonie

s of

BL

21 c

ells

(N

ovag

en)

tran

sform

ed w

ith t

he

expre

ssio

n v

ecto

r w

ere

gro

wn a

t 37°C

.

Thes

e w

ere

use

d t

o i

nocu

late

one-

lite

r cu

lture

s of

super

bro

th,

an e

nri

ched

bac

teri

al

gro

wth

med

ia.

Bac

teri

al c

ult

ure

s w

ere

gro

wn t

o a

n O

D6

00 o

f 0.8

at

37°C

and i

nduce

d

wit

h

200 µ

M

IPT

G

over

nig

ht

at

room

te

mpera

ture

.

Cel

ls

wer

e har

ves

ted

by

centr

ifugat

ion at

4000 rp

m fo

r 10 m

inute

s an

d fr

oze

n at

"

80°C

. A

ll su

bse

quen

t

puri

fica

tion s

teps

wer

e c

arri

ed o

ut

at 4

°C.

Bac

teri

al p

elle

ts w

ere

resu

spen

ded

in

Ni2

+

bin

din

g

buff

er

(20

mM

T

ris

(pH

7.9

),

500

mM

N

aCl,

20

mM

im

idaz

ole

, 10%

gly

cero

l) su

pple

men

ted w

ith pro

teas

e in

hib

itors

(1 m

M P

MS

F,

10 µ

g/m

L ea

ch of

apro

tinin

and l

eupep

tin,

10

µM

ben

zam

idin

e) a

nd l

yse

d b

y t

hre

e pas

sag

es t

hro

ugh a

Fre

nch

Pre

ssu

re C

ell.

T

he

extr

acts

wer

e th

en s

onic

ated

for

5 m

inute

s an

d t

he

lysa

tes

wer

e cl

arif

ied by ult

race

ntr

ifugat

ion at

40,0

00 rp

m fo

r 45 m

inute

s. T

he

clar

ifie

d

lysa

tes

wer

e in

cubat

ed f

or

30

min

ute

s w

ith N

i2+ b

ead

s (A

mer

sham

) at

4°C

. T

he

bea

ds

wer

e th

en w

ash

ed w

ith

bin

din

g b

uff

er a

nd t

he p

rote

in r

ecover

ed w

ith e

luti

on b

uff

er

(20 m

M T

ris

(pH

7.9

), 5

00 m

M N

aCl,

200 m

M i

mid

azole

, 10%

gly

cero

l).

Sam

ple

s

61

from

th

e pro

tein

puri

fica

tion

w

ere

then

re

solv

ed by S

DS

-PA

GE

an

d st

ained

w

ith

Coom

assi

e blu

e.

In

V

itro K

inase

Rea

ctio

ns—

Pre

lim

inar

y k

inas

e re

acti

ons

wer

e per

form

ed w

ith H

is-

AC

K2 a

nd H

is-S

H3P

X1 i

n a

kin

ase

reac

tion b

uff

er (

10 m

M H

epes

(pH

7.4

), 5

mM

MgC

l 2,

150 m

M N

aCl)

in t

he p

rese

nce

of

1 m

M N

a 3V

O4 a

nd 1

mM

AT

P f

or

0.5

-18

hours

at

30°C

. T

he kin

ase re

acti

ons

wer

e quen

ched

w

ith th

e ad

dit

ion of

5x

S

DS

load

ing buff

er.

T

he

reac

tio

n sa

mple

s w

ere

boil

ed,

reso

lved

by S

DS

-PA

GE

, an

d

tran

sfer

red t

o a

PV

DF

mem

bra

ne

wher

e su

bst

rate

phosp

hory

lati

on w

as m

easu

red b

y

imm

unoblo

ttin

g w

ith a

nti

-phosp

hoty

rosi

ne

anti

body.

AC

K2

Hig

h-t

hro

ugh

pu

t S

cree

n—

The

LA

NC

ET

M

tim

e-re

solv

ed

fluore

scen

t

reso

nan

ce e

ner

gy t

ransf

er s

yst

em f

rom

Per

kin

Elm

er w

as i

nit

iall

y u

sed f

or

scre

enin

g

for

smal

l m

ole

cule

inhib

itors

of

AC

K2 a

ctiv

ity.

LA

NC

ET

M i

s base

d o

n t

he

ener

gy

tran

sfer

bet

wee

n

donor

(euro

piu

m

chel

ate)

an

d

acce

pto

r (a

llophyco

cyan

in—

AP

C)

reag

ents

that

are

bro

ught

wit

hin

pro

xim

ity b

y a

spec

ific

bin

din

g e

ven

t.

In t

he

case

of

the

kin

ase

reac

tion,

euro

piu

m i

s co

nju

gat

ed t

o a

n a

nti

-phosp

hoty

rosi

ne

anti

body a

nd

AP

C i

s co

nju

gat

ed t

o s

trep

tavid

in b

eads.

E

ner

gy t

ransf

er o

ccurs

in t

he

pre

sence

of

a

tyro

sine

phosp

hory

late

d,

bio

tinyla

ted s

ubst

rate

. U

sing t

his

syst

em,

kin

ase

reac

tions

wer

e ca

rrie

d o

ut

wit

h 4

.15 µ

g/m

L A

CK

2,

200 n

M S

H3P

X1,

2 m

M A

TP

in 5

0 m

M

Hep

es,

10 m

M M

gC

l 2,

and 0

.1%

Tri

ton X

-100 f

or

0-2

hours

at

room

tem

per

ature

. T

he

reac

tion

sam

ple

s w

ere

then

quen

ched

w

ith

the

addit

ion

of

AP

C-c

onju

gat

ed

stre

pta

vid

in an

d E

uro

piu

m-c

hel

ated

an

ti-p

hosp

hoty

rosi

ne

anti

body.

S

ample

s w

ere

then

rea

d b

y a

flu

ore

scen

t pla

te r

eader

.

In s

ubse

quen

t sc

reen

s, a

[3

3P

]-A

TP

fil

ter

assa

y w

as u

sed t

o s

impli

fy d

etec

tion.

The

condit

ions

wer

e sl

ightl

y a

lter

ed b

y u

sing 8

.3 µ

g/m

L A

CK

2,

200 n

M S

H3P

X1,

50 µ

M A

TP

, an

d 0

.2 µ

Ci/

wel

l fo

r 0-1

8 h

ours

at

room

tem

per

ature

. F

oll

ow

ing t

he

kin

ase

reac

tions,

pro

tein

s w

ere

pre

cipit

ated

wit

h 3

0%

tri

cholo

roac

etic

aci

d (

TC

A)

and

62

left

on i

ce f

or

1 h

our.

T

he

reac

tion m

ixtu

re w

as

then

tra

nsf

erre

d t

o a

pre

-wet

ted 9

6-

wel

l fi

lter

pla

te,

foll

ow

ed b

y 5

was

hes

wit

h a

sodiu

m p

yro

phosp

hat

e/15%

TC

A w

ash

buff

er.

The

filt

er

was

dri

ed

and

scin

till

atio

n

fluid

w

as

added

, al

low

ing

for

the

mea

sure

men

t of

[33P

]O4

3-

inco

rpora

tion

into

ad

her

ent

pro

tein

s.

L

ater

, 1:3

se

rial

dil

uti

ons

wer

e ca

rrie

d o

ut

wit

h t

yrp

host

in,

AG

1478,

an E

GF

rec

epto

r in

hib

itor,

as

wel

l

as w

ith t

he

Par

ke-

Dav

is p

yri

do-p

yri

mid

ine,

PD

158780,

and t

he

SU

Gen

com

pound,

SU

6656,

in r

epli

cate

s of

four.

Res

ult

s

Much

inte

rest

has

bee

n d

irec

ted a

t dis

sect

ing t

he

mec

han

ism

beh

ind A

CK

2-d

epen

den

t

phosp

hory

lati

on o

f S

H3P

X1 a

nd i

ts e

ffec

ts o

n E

GF

rec

epto

r deg

radat

ion i

n c

ells

. A

s a

firs

t st

ep t

ow

ard g

ener

atin

g r

eagen

ts t

o s

tudy t

his

com

ple

x p

roce

ss,

we

wer

e in

tere

sted

in i

den

tify

ing t

he

tyro

sine

pho

sphory

lati

on s

ite(

s) o

n t

he

sort

ing n

exin

.

Carb

oxyl-

term

inal

Tru

nca

tion

M

uta

nts

of

SH

3P

X1

are

D

efec

tive

for

AC

K2-

dep

end

ent

Ph

osp

hory

lati

on

To d

elin

eate

the

regio

n o

n S

H3P

X1 t

hat

conta

ins

the

phosp

hory

lati

on s

ite(

s)

for

AC

K2,

a se

ries

of

C-t

erm

inal

tru

nca

tion m

uta

nts

was

const

ruct

ed.

Eac

h o

f th

ese

const

ruct

s w

as d

esig

ned

to

co

nta

in t

he

N-t

erm

inal

SH

3 d

om

ain,

as w

e hav

e sh

ow

n t

hat

this

reg

ion i

s ess

enti

al f

or

AC

K2-S

H3P

X1 b

indin

g (

Fig

ure

2.1

).

Phosp

hory

lati

on o

f

the

HA

-epit

ope

tagged

muta

nts

#C

84, #

C197, #

C340, #

C395, #

C447 w

as a

sses

sed

by

co-e

xpre

ssio

n

wit

h

the

Myc-

AC

K2

const

ruct

in

C

OS

-7

cell

s,

foll

ow

ed

by

imm

unopre

cipit

atio

n w

ith a

nti

-HA

anti

body,

SD

S-P

AG

E,

and W

este

rn b

lott

ing w

ith

anti

-phosp

hoty

rosi

ne

anti

body.

Whil

e A

CK

2-c

ataly

zed t

yro

sine

pho

sphory

lati

on w

as

det

ecte

d in

w

ild-t

ype

SH

3P

X1 (F

igure

2.2

A,

lane

3),

it

w

as not

obse

rved

in

th

e

carb

oxyl-

term

inal

tru

nca

tion m

uta

nts

#C

84, #

C197 (

Fig

ure

2.2

A,

lanes

1 a

nd 2

) an

d

63

Fig

ure

2.1

C

arb

oxyl-

term

inal

Del

etio

n M

uta

nts

of

SH

3P

X1.

Car

boxyl-

term

inal

del

etio

n m

uta

nts

of

SH

3P

X1 w

ere

des

igned

so t

hat

they

mai

nta

ined

the

amin

o-t

erm

inal

SH

3 d

om

ain t

hat

has

bee

n s

ho

wn t

o b

e cr

itic

al f

or

bin

din

g A

CK

2.

Muta

nts

#

C84, #

C197, #

C340, #

C395, #

447 w

ere

engin

eere

d

wit

h

Bam

H1

and

Eco

R1 r

estr

icti

on s

ites

, w

ith

the

resu

ltin

g P

CR

pro

duct

s li

gat

ed i

nto

the

pC

R2-T

OP

O

vec

tor.

T

he

ligat

ion p

roduct

s w

ere

then

dig

este

d w

ith B

amH

1 a

nd E

coR

1 r

est

rict

ion

enzy

mes

.

Thes

e D

NA

pro

duct

s w

ere

reso

lved

on

a 1%

ag

arose

gel

co

nta

inin

g

80 n

g/m

L e

thid

ium

bro

mid

e, a

nd t

he

inse

rts

wer

e ex

cise

d f

rom

the

gel

and p

uri

fied

wit

h t

he

Qia

quic

k G

el E

xtr

acti

on k

it.

The

resu

ltin

g D

NA

inse

rts

wer

e th

en l

igat

ed

into

the

HA

-tag

ged

pcD

NA

3 e

xpre

ssio

n v

ecto

r usi

ng T

4 D

NA

lig

ase.

64

65

Fig

ure

2.2

Th

e tr

un

cati

on

m

uta

nt !

C84

is

def

ecti

ve

for

AC

K2-d

epen

den

t

ph

osp

hory

lati

on

.

Myc-

AC

K2

and

HA

-SH

3P

X1

trunca

tion

muta

nts

w

ere

co-

tran

sfec

ted i

n C

OS

-7 c

ells

. T

wen

ty-f

our

to s

eventy

-tw

o h

ours

aft

er t

ransf

ecti

on,

the

cell

s w

ere

lyse

d

and

the

whole

ce

ll

lysa

tes

wer

e re

solv

ed

by

SD

S-P

AG

E

and

tran

sfer

red t

o P

VD

F m

embra

ne.

T

he

sam

ple

s w

ere

then

subje

cted

to

West

ern b

lott

ing

anal

ysi

s w

ith a

nti

-phosp

hoty

rosi

ne

(A)

and a

nti

-HA

anti

bodie

s (B

).

66

67

!C

339 (

dat

a not

show

n).

T

hes

e dat

a in

dic

ate

that

the

del

etio

n o

f th

e la

st 8

4 a

min

o

acid

s fr

om

th

e C

-ter

min

al en

d of

SH

3P

X1 re

sult

s in

a

com

ple

te lo

ss of

AC

K2-

cata

lyze

d t

yro

sine

phosp

hory

lati

on.

Giv

en t

his

res

ult

, w

e th

en s

et o

ut

to m

uta

te e

ach

of

the

tyro

sine

resi

dues

in t

he

C-t

erm

inal

84

amin

o

acid

re

gio

n

of

SH

3P

X1

to

phen

yla

lanin

e.

S

H3P

X1

poin

t

muta

nts

Y546F

, Y

561F

, Y

56

3F

, Y

570F

and Y

578F

wer

e gen

erat

ed u

sing s

ite-

dir

ecte

d

muta

gen

esis

, an

d m

ult

iple

rounds

of

muta

gen

esis

wer

e per

form

ed t

o y

ield

mult

iple

poin

t m

uta

nts

Y546F

/Y578

F a

nd Y

546F

/Y561F

/Y563F

/Y570F

/Y578F

. T

he

resu

ltin

g

muta

nts

w

ere

co-e

xpre

ssed

w

ith A

CK

2 in

C

OS

-7 ce

lls.

F

oll

ow

ing ce

ll ly

sis,

th

e

SH

3P

X1 m

uta

nts

wer

e pre

cipit

ated

fro

m w

hole

-cel

l ly

sate

s w

ith a

nti

-HA

anti

body,

and t

hen

im

munoblo

tted

wit

h a

nti

-phosp

hoty

rosi

ne

anti

body.

Unex

pec

tedly

, A

CK

2-

dep

enden

t phosp

hory

lati

on w

as s

till

obse

rved

wit

h e

ach o

f th

ese m

uta

nts

, in

cludin

g

the

const

ruct

that

conta

ins

subst

ituti

ons

for

each

of

the

C-t

erm

inal

tyro

sine

resi

dues

(SH

3P

X1-F

546/F

561/F

563/F

570/F

578),

co

mpar

ed

to

the

neg

ativ

e co

ntr

ol,

A

CK

2

kin

ase-

def

icie

nt

muta

nt

AC

K2-K

158R

wit

h S

H3P

X1 (

Fig

ure

2.3

, co

mpar

e la

ne

2 w

ith

lanes

3-8

).

Ther

e ap

pea

r to

be

two p

oss

ible

expla

nat

ions

for

this

obse

rvat

ion:

eit

her

AC

K2 i

s unab

le t

o b

ind t

o t

hes

e C

-ter

min

al d

elet

ion m

uta

nts

, th

us

acco

unti

ng f

or

the

loss

of

phosp

hory

lati

on o

f th

e !

C84 c

on

stru

ct,

or

AC

K2 i

s cap

able

of

phosp

hory

lati

ng

mult

iple

tyro

sine

resi

dues

, in

cludin

g t

ho

se t

hat

lie

at

posi

tion

s upst

ream

fro

m t

he

C-

term

inal

tyro

sine

resi

dues

.

To a

ddre

ss t

he

firs

t poss

ibil

ity,

we

car

ried

out

in viv

o

exper

imen

ts t

o a

ssay

the

bin

din

g b

etw

een A

CK

2 a

nd t

he

var

iou

s S

H3P

X1 d

elet

ion

muta

nts

. M

yc-

AC

K2 a

nd t

he

HA

-tag

ged

tru

nca

tion m

uta

nts

wer

e co

-expre

ssed

in

CO

S-7

cel

ls,

foll

ow

ed b

y i

mm

unopre

cipit

atio

n o

f w

hole

cel

l ly

sate

s w

ith a

nti

-Myc

anti

body a

nd i

mm

unoblo

ttin

g w

ith a

nti

-HA

anti

body

. H

ow

ever

, th

ese

exper

imen

ts

gav

e m

ixed

res

ult

s an

d d

id n

ot

per

mit

a d

efin

itiv

e in

terp

reta

tion.

We

then

subcl

oned

AC

K2 i

nto

the

V5-e

pit

ope

tagged

pcD

NA

3.1

vec

tor,

giv

en t

hat

this

pla

smid

appea

rs

68

Fig

ure

2.3

A

CK

2 p

hosp

hory

late

s C

-ter

min

al

tyro

sin

e-to

-ph

enyla

lan

ine

mu

tan

ts.

Myc-

AC

K2 w

as c

o-t

ransf

ecte

d w

ith t

he

indic

ated

HA

-SH

3P

X1 p

oin

t m

uta

nts

in C

OS

-

7 ce

lls.

T

wen

ty-f

our

to se

ven

ty-t

wo hours

af

ter

tran

sfec

tion,

the

SH

3P

X1 m

uta

nts

wer

e im

munopre

cipit

ated

wit

h a

nti

-HA

anti

body.

The

resu

ltin

g p

reci

pit

ated

sam

ple

s

wer

e th

en

reso

lved

by

SD

S-P

AG

E,

tran

ferr

ed

to

PV

DF

m

embra

ne,

an

d

Wes

tern

blo

tted

wit

h a

nti

-phosp

hoty

rosi

ne

and a

nti

-HA

anti

bodie

s.

69

70

to g

ive

rise

to a

more

robust

expre

ssio

n o

f A

CK

2.

The

resu

ltin

g V

5-A

CK

2 c

onst

ruct

was

co

-expre

ssed

in

C

OS

-7

cell

s w

ith

th

e S

H3P

X1

del

etio

n

muta

nts

, w

her

e th

e

AC

K2-S

H3P

X1 i

nte

ract

ions

wer

e as

sess

ed b

y p

reci

pit

atin

g A

CK

2 f

rom

cel

l ly

sate

s

wit

h an

ti-V

5 an

tibody,

and im

munoblo

ttin

g fo

r as

soci

ated

H

A-S

H3P

X1 co

nst

ruct

s

usi

ng a

n a

nti

-HA

anti

body.

As

show

n i

n F

igure

2.4

, under

condit

ions

wher

e fu

ll-

length

HA

-SH

3P

X1 w

as c

lear

ly a

ble

to b

e co

-im

munopre

cipit

ated

wit

h V

5-A

CK

2,

all

of

the

C-t

erm

inal

SH

3P

X1 d

elet

ion m

uta

nts

wer

e in

effe

ctiv

e in

bin

din

g t

o A

CK

2.

Thus,

thes

e fi

ndin

gs

indic

ate

that

the

inab

ilit

y o

f th

e C

-ter

min

al t

runca

tion m

uta

nts

of

SH

3P

X1 t

o b

e phosp

hory

late

d b

y A

CK

2 i

s a

dir

ect

conse

quen

ce

of

thei

r in

abil

ity t

o

bin

d t

o t

he

tyro

sine

kin

ase.

All

Con

serv

ed T

yro

sin

e R

esid

ues

of

SH

3P

X1 A

re S

usc

epti

ble

to A

CK

2-c

ata

lyze

d

Ph

osp

hory

lati

on

Giv

en t

he

resu

lts

des

crib

ed i

n t

he

pre

cedin

g s

ecti

on w

hic

h i

ndic

ated

that

th

e C

-

term

inal

dom

ain o

f S

H3P

X1 d

id n

ot

conta

in t

he

phosp

hory

lati

on s

ite(

s) f

or

AC

K2,

we

then

exam

ined

those

tyro

sine

resi

dues

of

SH

3P

X1 k

now

n t

o b

e co

nse

rved

bet

wee

n

dif

fere

nt

spec

ies.

Speci

fica

lly,

we

gen

erat

ed

tyro

sine-

to-p

hen

yla

lanin

e poin

t

muta

tions

of

conse

rved

res

idues

bet

wee

n h

um

an,

mouse

and Drosophila o

rtholo

gues

.

The

foll

ow

ing S

H3P

X1 m

uta

nts

, Y

9F

, Y

56F

, Y

287F

, Y

496F

, Y

546F

, an

d Y

578F

wer

e gen

erat

ed t

hro

ugh s

ite-

dir

ecte

d m

uta

gen

esis

. T

he

abil

ity o

f th

ese

muta

nts

to b

e

phosp

hory

late

d

in

an

AC

K2-d

epen

den

t m

ann

er

was

ev

alu

ated

upon

thei

r co

-

expre

ssio

n

wit

h

AC

K2

in

CO

S-7

ce

lls.

The

cell

ly

sate

s w

ere

subje

ct

to

imm

unopre

cipit

atio

n w

ith a

nti

-HA

anti

body,

foll

ow

ed b

y i

mm

unoblo

ttin

g w

ith a

nti

-

phosp

hoty

rosi

ne

anti

body.

We

mea

sure

d t

he

AC

K2

-cat

alyze

d p

hosp

hory

lati

on o

f th

e

poin

t m

uta

nts

agai

nst

wil

d-t

ype

SH

3P

X1,

usi

ng c

ells

co-e

xpre

ssin

g k

inas

e-def

ecti

ve

AC

K2 (

AC

K2

-K158R

) w

ith S

H3P

X1 a

s a

neg

ativ

e co

ntr

ol

for

thes

e ex

per

imen

ts.

71

Fig

ure

2.4

AC

K2

is

un

ab

le

to

bin

d

to

SH

3P

X1

del

etio

n

mu

tan

ts.

A s

chem

atic

rep

rese

nta

tion o

f th

e S

H3P

X1 d

elet

ion c

onst

ruct

s (A

).

CO

S-7

cel

ls w

ere

tran

sfec

ted

wit

h

V5-A

CK

2

and

HA

-SH

3P

X1

carb

oxyl-

term

inal

del

etio

n

muta

nts

.

Tw

enty

-four

to s

even

ty-t

wo h

ours

aft

er t

ransf

ecti

on,

AC

K2 w

as i

mm

unopre

cipit

ated

wit

h a

nti

-V5 a

nti

body a

nd t

he s

ample

s w

ere r

esolv

ed b

y S

DS

-PA

GE

. T

he

gel

was

then

tra

nsf

erre

d t

o P

VD

F m

embra

ne

and s

ubje

cted

to W

este

rn b

lott

ing w

ith a

nti

-HA

anti

body (

B).

72

73

Tyro

sine

56

has

bee

n

report

ed

to

be

the

maj

or

phosp

hory

lati

on

site

in

Dro

sophil

a [

5].

H

ow

ever

, w

e fo

und t

hat

eac

h o

f th

e si

ngle

poin

t m

uta

nts

ret

ained

the

abil

ity t

o b

e ty

rosi

ne

phosp

hory

late

d i

n a

n A

CK

2-d

epen

den

t m

anner

(F

igure

2.5

, la

nes

4-1

0).

In

fac

t, t

her

e w

as n

o d

etec

table

dec

reas

e in

phosp

hory

lati

on s

ignal

of

any o

f th

e

poin

t m

uta

nts

com

par

ed t

o w

ild-t

ype.

T

his

sugges

ted t

hat

ther

e m

ay b

e m

ult

iple

sit

es

on S

H3P

X1 f

or

AC

K2-c

atal

yze

d p

hosp

hory

lati

on, as

we

hav

e not

bee

n a

ble

to d

etec

t a

signif

ican

t dec

reas

e in

phosp

hory

lati

on f

or

any o

f th

e su

bst

ituti

ons

thus

far

exam

ined

.

Ther

e ar

e 21 t

yro

sine

amin

o a

cids

in t

he h

um

an o

rtholo

gue

of

SH

3P

X1.

This

,

couple

d

wit

h

the

fact

th

at

ther

e ar

e no re

port

ed

conse

nsu

s se

quen

ces

fo

r A

CK

2

tyro

sine

phosp

hory

lati

on,

contr

ibute

s to

th

e

chal

lenge

of

usi

ng

site

-dir

ecte

d

muta

gen

esis

to i

den

tify

the

maj

or

tyro

sine

phosp

hory

lati

on s

ites

on S

H3P

X1.

Thus,

as

an a

lter

nat

ive

appro

ach,

we

hav

e tu

rned

to m

ass

spec

trom

etry

. T

his

req

uir

es t

hat

we

dev

elop

reco

mbin

ant

sourc

es

of

AC

K2

and

S

H3P

X1

in

ord

er

to

per

form

th

e

phosp

hory

lati

on

assa

ys

and

pho

spho-p

epti

de

map

pin

g

anal

ysi

s in

a

wel

l-def

ined

,

puri

fied

syst

em.

Tyro

sin

e re

sid

ue

287 w

as

Iden

tifi

ed a

s a P

hosp

ho-s

ite

by M

ass

Sp

ectr

om

etry

We

init

iall

y s

et o

ut

to i

den

tify

the

AC

K2-c

atal

yzed

phosp

hory

lati

on s

ites

on

SH

3P

X1,

usi

ng

our

inse

ct c

ell

pre

par

atio

n o

f re

com

bin

ant

AC

K2 a

nd

the

E.

coli

-

expre

ssed

His

-SH

3P

X1.

How

ever

, w

e so

on f

ound t

hat

we

wer

e unab

le t

o g

ener

ate

suff

icie

nt

level

s of

(AC

K2-c

atal

yze

d)

phosp

hory

late

d

SH

3P

X1

from

our

in

vitr

o

assa

ys

to p

erm

it a

def

init

ive

iden

tifi

cati

on o

f th

e phosp

hory

lati

on s

ite(

s).

As

a r

esult

,

we

looked

for

condit

ions

whic

h w

ould

giv

e an

enhan

ced p

hosp

hory

lati

on o

f S

H3P

X1,

hopin

g

that

w

e co

uld

w

ork

bac

kw

ards

tow

ard

iden

tify

ing

the

AC

K2-c

atal

yze

d

phosp

hory

lati

on s

ites

. T

his

led

us

to e

xam

ine

FA

K a

nd S

rc a

s poss

ible

kin

ases

for

SH

3P

X1 g

iven

the

sim

ilar

itie

s bet

wee

n t

hei

r re

spec

tive

kin

ase

dom

ains

and t

hat

of

74

Fig

ure

2.5

T

he

AC

K2-d

epen

den

t p

hosp

hory

lati

on

of

SH

3P

X1 c

on

stru

cts

bea

rin

g

sub

stit

uti

on

s at

the

con

serv

ed t

yro

sin

e re

sid

ues

is

com

para

ble

to

that

of

wil

d-

typ

e S

H3P

X1.

C

OS

-7 ce

lls

wer

e tr

ansf

ecte

d w

ith M

yc-

AC

K2 an

d H

A-S

H3P

X1

tyro

sine-

to-p

hen

yla

lanin

e poin

t m

uta

nts

.

Tw

enty

-four

to

seven

ty-t

wo

hours

af

ter

tran

sfec

tion,

SH

3P

X1 a

nd t

he

indic

ated

poin

t m

uta

nts

wer

e im

munopre

cipit

ated

wit

h

anti

-HA

anti

body a

nd r

esolv

ed b

y S

DS

-PA

GE

. T

he

gel

was

then

tra

nsf

erre

d t

o P

VD

F

mem

bra

ne

and s

ubje

cted

to i

mm

unoblo

ttin

g w

ith a

nti

-phosp

hoty

rosi

ne

anti

body.

75

76

AC

K2.

Spec

ific

ally

, an

im

munopre

cipit

ated

sam

ple

fro

m H

EK

293

cel

ls e

xpre

ssin

g

Src

an

d S

H3P

X1 w

as su

bm

itte

d to

th

e C

orn

ell

Mas

s S

pec

trom

etry

C

ore

F

acil

ity.

Liq

uid

chro

mat

ogra

phy M

S/M

S w

as u

sed t

o i

denti

fy t

yro

sine

resi

dues

Y177,

Y239,

Y269,

Y294, an

d Y

561 a

s S

rc-c

atal

yze

d p

hosp

hory

lati

on s

ites

(F

igure

2.6

).

We

then

tes

ted t

he

abil

ity o

f A

CK

2 t

o p

ho

sphory

late

the

resu

ltin

g t

yro

sine-

to-

phen

yla

lanin

e m

uta

nts

. A

s sh

ow

n i

n F

igure

2.7

, ea

ch o

f th

e p

oin

t m

uta

nts

, in

cludin

g

muta

nt

Y17

7F

/Y239F

/Y269F

/Y294F

/Y561

(Fig

ure

2.7

. la

nes

4-9

),

wer

e

phosp

hory

late

d b

y A

CK

2,

indic

atin

g t

hat

AC

K2

and S

rc p

hosp

hory

late

dis

tinct

sit

es

on S

H3P

X1.

Conse

quen

tly

, w

e use

d s

imil

ar c

ondit

ions

to s

ubm

it a

sam

ple

fro

m H

EK

293 c

ells

tra

nsf

ecte

d w

ith A

CK

2 a

nd S

H3P

X1.

The

cell

s w

ere

lyse

d 2

4 h

ours

aft

er

tran

sfec

tion a

nd V

5-S

H3P

X1 w

as p

reci

pit

ated

fro

m t

he

pre

par

ed l

ysa

tes

(~10 m

g t

ota

l

pro

tein

) w

ith a

nti

-V5 a

nti

bod

y.

The

pre

cipit

ated

sam

ple

was

res

olv

ed b

y S

DS

-PA

GE

,

stai

ned

wit

h C

oom

assi

e, a

nd e

xci

sed (

Fig

ure

2.8

A).

A

liquots

fro

m t

his

sam

ple

wer

e

set

asid

e an

d t

yro

sine

pho

sphory

lati

on w

as c

onfi

rmed

by W

este

rn B

lott

ing (

Fig

ure

2.8

B).

T

his

sam

ple

was

then

sub

mit

ted t

o t

he

Corn

ell

Bio

tech

nolo

gy R

eso

urc

e C

ente

r

Pro

teom

ics

and M

ass

Spec

tro

met

ry C

ore

Fac

ilit

y.

An a

ppro

ach u

sing

mas

s re

acti

on

monit

ori

ng

(MR

M-I

DA

) te

chniq

ues

id

enti

fied

ty

rosi

ne

287

as

a si

te

of

AC

K2-

cata

lyze

d p

hosp

hory

lati

on.

Tyro

sine

287 i

s a

conse

rved

tyro

sine

resi

due

loca

ted i

n t

he

PX

dom

ain

of

SH

3P

X1.

W

e

curr

entl

y

bel

iev

e th

at

ther

e ar

e

addit

ional

A

CK

2-

cata

lyze

d

phosp

hory

lati

on

site

s as

earl

ier

muta

gen

esis

an

aly

ses

indic

ated

th

at

the

Y287F

muta

nt

was

sti

ll p

hosp

hory

late

d b

y A

CK

2.

As

a r

esult

, ad

dit

ional

sam

ple

s ar

e

bei

ng p

repar

ed t

o c

onfi

rm a

nd

pote

nti

ally

iden

tify

new

sit

es.

77

Fig

ure

2.6

S

equ

ence

ali

gn

men

t of

hu

man

, m

ou

se a

nd

Drosophila o

rth

olo

gu

es o

f

SH

3P

X1.

78

------------------+-------------------+-------------------+-------------------+-

10 20 30 40

M A T K A R V M Y D F A A E P G N N E L T V N E G E I I T I T N P D V G G G W L SH3PX1 homo

M A T K A R V M Y D F A A E P G N N E L T V T E G E I I T V T N P N V G G G W L SH3PX1 mouse

M T S Y V R A M Y D F T G E P G S S E L S I A T G D V L S V T R S D V G E G W W SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

50 60 70 80

E G R N I K G E R G L V P T D Y V E I L P S D G K D Q F S C G N S V A D Q A F L SH3PX1 homo

E G K N N K G E Q G L V P T D Y V E I L P N D G K D P F S C G N S V A D Q A F L SH3PX1 mouse

E G K N A R G Q I G L F P A A Y V E V M S A A E A Q K L S A S G A T S V P Q V P SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

90 100 110 120

D S L S A S T A H A S S S A A S N N H Q V G S G N D P W S A W S A S K S G N W E SH3PX1 homo

D S L T A S T A Q T N S S S A N S N N Q V G G G N D P W T A W N A P K P G N W D SH3PX1 mouse

D P F A S P P L P R Y D Q T A D D D - - - - - - - - - - - G - - S - - - - N W D SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

130 140 150 160

S S E G W G A Q P E G A G A Q R N T N T P N N W D T A F G H P Q A Y Q G P A T G SH3PX1 homo

S S D A W G S R T D G T S A Q R N S - S A N N W D T G F G H P Q A Y Q G P A T G SH3PX1 mouse

D E D D W S D D N D T Y S E I G P G - - - - - - - - - - G Q K S A G Q S A R A G SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

170 180 190 200

D D D D W D E D W D G P K S S S - Y F K D S E S A D A G G A Q R G N S R A S S S SH3PX1 homo

D D D E W D E D W D D P K S S S P Y F K D S E P A E A G G I Q R G N S R A G A S SH3PX1 mouse

G L S H S T S D Y D N K H L P I A P N D D T Q S L A S V A G G T G A A G T V K K SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

210 220 230 240

S M K I P L N K F P G F A K P G T E Q Y L L A K Q L A K P K E K I P I I V G D Y SH3PX1 homo

S M K L P L N K F P G F A K P G M E Q Y L L A K Q L A K P K E K I A I I V G D Y SH3PX1 mouse

G M F A K S S D S Y I L G L S S T K E K I P E C E M A Y I T Q - - - - - V E D S SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

250 260 270 280

G P M W V Y P T S T F D C V V A D P R K G S K M Y G L K S Y I E Y Q L T P T N T SH3PX1 homo

G P M W V Y P T S T F D C V V A D P R K G S K M Y G L K S Y I E Y Q L T P T N T SH3PX1 mouse

I Y Q W T Q N H S P Y S V V V A S P K K E S K F K G M K T F I A Y Q L T P S F N SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

290 300 310 320

N R S V N H R Y K H F D W L Y E R L L V K F G S A I P I P S L P D K Q V T G R F SH3PX1 homo

N R S V N H R Y K H F D W L Y E R L L V K F G S A I P I P S L P D K Q V T G R F SH3PX1 mouse

N I S V S R R Y K H F D W L H E R L V D K F - C L I P V P P L P D K Q I S G R Y SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

330 340 350 360

E E E F I K M R M E R L Q A W M T R M C R H P V I S E S E V F Q Q F L N F R D E SH3PX1 homo

E E E F I K M R M E R L Q A W M T R M C R H P V V S E S E V F Q Q F L N F R D E SH3PX1 mouse

E E Q F V E H R R V Q L Q E F V D W V C R H P V I S K C E V W Y H F L T C R D E SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

370 380 390 400

K E W K T G K R K A E R D E L A G V M I F S T M E P E A P D L D L V E I E Q K C SH3PX1 homo

K E W K T G K R K A E K D E L V G V M I F S T M E P E A P D L D L I E I E Q K C SH3PX1 mouse

K I W K S G K R K A E R D P Y M G V N Y C L A I S P P D K N L L H S K V D A Q V SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

410 420 430 440

E A V G K F T K A M D D G V K E L L T V G Q E H W K R C T G P L P K E Y Q K I G SH3PX1 homo

D A V G K F T K A M D D G V K E L L T V G Q E H W K R C T G P L P K E Y Q K I G SH3PX1 mouse

E L G T Q F I H S M D V A V R N L N N I S N D M A K R S M S Q S K K E F Q R I G SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

450 460 470 480

K A L Q S L A T V F S S S G Y Q G - - - - E T D L N D A I T E A G K T Y E E I A SH3PX1 homo

K A L Q S L A A V F S S S G Y Q G - - - - E T D L N D A I T E A G K T Y E E I A SH3PX1 mouse

D G L S D L A K A L A I D E R R A P T R N A V P L S E S V G R I G G I F I G I G SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

490 500 510 520

S L V A E Q P K K D L H F L M E C N H E Y K G F L G C F P D I I G T H K G A I E SH3PX1 homo

S L V A E Q P K K D L H F L M E C N H E Y K G F L G C F P D I I G A H K G A I E SH3PX1 mouse

Q A F G D Q P K H D W I P L S D R L H I Y R G V L N C F P D I F S T H K G A I Q SH3PX1 drosophila

79

Fig

ure

2.6

(C

on

tin

ued

)

------------------+-------------------+-------------------+-------------------+-

530 540 550 560

K V K E S D K L V A T S K I T L Q D K Q N M V K R V S I M S Y A L Q A E M N H F SH3PX1 homo

K V K E S D K L V A T S K I T P Q D K Q T M V K R V G T M S Y A L Q A E M N H F SH3PX1 mouse

K R K D C E K L A G E G R M N N P Q L H D V N R R T D V V S Y T V L A E L T H F SH3PX1 drosophila

------------------+-------------------+-------------------+-------------------+-

570 580 590 600

H S N R I Y D Y N S V I R L Y L E Q Q V Q F Y E T I A E K L R Q A L S R F P V M SH3PX1 homo

H S N R I Y D Y N S V I R L Y L E Q Q V Q F Y E T I A E K L R Q A L S R F P V M SH3PX1 mouse

K S E R D T H L K H T L K N F I A E Q I K F Y Q G V V A R L Q E A S R Q I E SH3PX1 drosophila

Y.....Conserved tyrosine residues between human, mouse and Drosophila

Y.....Src-catalyzed phosphorylation sites identified by mass spectrometry

Y.....ACK2-catalyzed phosphorylation site identified by mass spectrometry

80

Fig

ure

2.7

A

CK

2-c

ata

lyze

d p

hosp

hory

lati

on

sit

es o

n S

H3P

X1 a

re d

isti

nct

fro

m

those

of

Src

. M

yc-

AC

K2 w

as

co-t

ransf

ecte

d w

ith t

he

indic

ated

HA

-SH

3P

X1 p

oin

t

muta

nts

in

C

OS

-7 ce

lls.

T

wen

ty-f

our

to se

ven

ty-t

wo hours

af

ter

tran

sfec

tion,

the

SH

3P

X1 m

uta

nts

w

ere im

munopre

cipit

ated

w

ith

an

ti-H

A an

tibody.

T

he

resu

ltin

g

pre

cipit

ated

sa

mple

s w

ere

then

re

solv

ed

by

SD

S-P

AG

E,

tran

ferr

ed

to

PV

DF

mem

bra

ne,

and W

este

rn b

lott

ed w

ith a

nti

-phosp

hoty

rosi

ne

and a

nti

-HA

anti

bodie

s.

81

82

Fig

ure

2.8

P

rep

ara

tion

of

the

AC

K2-c

ata

lyze

d S

H3P

X1 p

hosp

hory

lati

on

sam

ple

for

mass

sp

ectr

om

etry

an

aly

sis.

M

yc-

AC

K2 a

nd V

5-S

H3P

X1 w

ere

co-e

xpre

ssed

in

HE

K 2

93 c

ells

. V

5-S

H3P

X1 w

as i

mm

unopre

cipit

ated

fro

m t

he

whole

cel

l ly

sate

s

wit

h a

nti

-V5 a

nti

body,

reso

lved

by S

DS

-PA

GE

, an

d s

tain

ed w

ith C

oom

ass

ie (

A).

T

he

ban

d

conta

inin

g

SH

3P

X1

was

ex

cise

d

and

su

bm

itte

d

to

the

Corn

ell

Mass

Spec

trom

etry

Core

Fac

ilit

y.

Ali

quots

fro

m t

his

sam

ple

was

als

o r

esolv

ed b

y S

DS

-

PA

GE

, tr

ansf

erre

d t

o P

VD

F,

and s

ubje

cted

to W

este

rn b

lott

ing a

nal

ysi

s w

ith a

nti

-

phosp

hoty

rosi

ne

and a

nti

-V5 a

nti

bodie

s (B

).

83

84

Rec

om

bin

an

t P

rote

in G

ener

ati

on

In o

rder

to d

evel

op t

he

assa

y t

o s

cree

n f

or

smal

l m

ole

cule

inhib

itors

of

AC

K2

kin

ase

acti

vit

y,

it w

as fi

rst

nec

essa

ry to

ex

pre

ss an

d puri

fy re

com

bin

ant

form

s of

AC

K2 a

nd

SH

3P

X1.

Due t

o t

he s

ize o

f A

CK

2 a

nd S

H3P

X1 (

83 k

Da a

nd 7

7 k

Da,

resp

ecti

vel

y),

we

init

iall

y s

et o

ut

to e

xpre

ss t

hes

e pro

tein

s in

inse

ct c

ells

. V

iruse

s

enco

din

g

His

-tag

ged

as

wel

l as

unta

gged

ver

sions

of

AC

K2

and

SH

3P

X1

wer

e

dev

eloped

for

expre

ssio

n o

f th

ese

pro

tein

s in

Sf2

1 c

ells

usi

ng t

he

Invit

rogen

Bac

-to-

Bac

kit

. W

e fo

und t

hat

SH

3P

X1,

when

expre

ssed

as

a H

is-t

agged

pro

tein

in i

nse

ct

cell

s,

reta

ined

a

basa

l le

vel

of

tyro

sine

pho

sphory

lati

on.

G

iven

re

port

s th

at

Dro

sophil

a S

H3P

X1 i

s pho

sphory

late

d b

y D

roso

phil

a A

CK

2 [

5],

we

thought

that

the

bas

al

phosp

hory

lati

on

of

SH

3P

X1

could

poss

ibly

be

due

to

an

isofo

rm

of

AC

K

expre

ssed

endogen

ou

sly i

n t

he

Sf2

1 c

ells

. C

onse

quen

tly,

we

sought

to e

lim

inat

e th

is

phosp

hory

lati

on

signal

by

dev

elopin

g

a kin

ase

-def

ecti

ve

AC

K2-K

158R

vir

us.

How

ever

, co

-infe

ctin

g S

f21 c

ells

wit

h A

CK

2-K

158R

and S

H3P

X1 d

id n

ot

rever

se t

his

effe

ct.

Sti

ll,

by c

o-e

xpre

ssin

g A

CK

2 k

inas

e to

get

her

wit

h S

H3P

X1 i

n S

f21 c

ells

, w

e

wer

e ab

le t

o o

bse

rve

an i

ncr

ease

in t

he p

hosp

hory

lati

on o

f S

H3P

X1 r

ealt

ive

to t

hat

for

SH

3P

X1 a

lone

(Fig

ure

2.9

B,

com

par

e la

nes

1 a

nd 2

), t

hus

confi

rmin

g t

he

acti

vit

y o

f

AC

K2 f

rom

inse

ct c

ells

.

To t

ry t

o m

inim

ize

any a

mbig

uit

y a

risi

ng f

rom

the

bas

al p

hosp

hory

lati

on o

f

SH

3P

X1,

we

then

expre

ssed

it

as

a G

ST

fusi

on p

rote

in i

n E

. co

li.

Full

-len

gth

SH

3P

X1

was

clo

ned

into

the

pG

EX

-KG

vec

tor

for

reco

mbin

ant

expre

ssio

n.

Expre

ssio

n o

f th

e

GS

T-S

H3P

X1 f

usi

on p

rote

in w

as c

arri

ed o

ut

in t

he

BL

21 E

. co

li s

trai

n,

wher

e one-

lite

r cu

lture

s w

ere

gro

wn t

o a

n O

D6

00 o

f 0.8

in s

uper

bro

th a

nd i

nduce

d w

ith 2

00

µM

IPT

G

over

nig

ht.

C

ells

w

ere

har

ves

ted

by

ce

ntr

ifugat

ion

and

GS

T-S

H3P

X1

was

puri

fied

on glu

tath

ione-

agar

ose

bea

ds

(Sig

ma)

. P

reli

min

ary kin

ase

reac

tions

wer

e

carr

ied o

ut

by i

ncu

bat

ing H

is-A

CK

2 (

puri

fied

fro

m i

nse

ct c

ells

) w

ith G

ST

-SH

3P

X1

85

Fig

ure

2.9

S

H3P

X1 c

an

be

ph

osp

hory

late

d b

y A

CK

2 u

pon

co

-exp

ress

ion

of

thes

e

pro

tein

s in

in

sect

ce

lls.

Vir

use

s en

codin

g

His

-AC

K2

and

His

-SH

3P

X1

wer

e

gen

erat

ed u

sing t

he

Bac

-to-B

ac B

aculo

vir

us

expre

ssio

n s

yst

em.

The

resu

ltin

g v

iruse

s

wer

e use

d t

o i

nfe

ct S

f21 c

ells

for

24

-72 h

ours

pri

or

to c

ell

lysi

s.

The

whole

cel

l

lysa

tes

wer

e re

solv

ed b

y S

DS

-PA

GE

and t

ransf

erre

d t

o P

VD

F m

embra

ne.

H

is-A

CK

2

expre

ssio

n

was

sh

ow

n

by

West

ern

blo

ttin

g

whole

-cel

l ly

sate

s w

ith

anti

-AC

K2

anti

body (

A).

T

he

kin

ase

acti

vit

y o

f re

com

bin

ant

AC

K2 w

as a

ssess

ed b

y c

o-i

nfe

ctin

g

AC

K2 a

nd S

H3P

X1 i

n S

f21 c

ells

. T

wen

ty-f

our

to s

even

ty-t

wo h

ours

aft

er i

nfe

ctio

n

the

cell

s w

ere

lyse

d

and

SH

3P

X1

was

im

munopre

cipit

ated

w

ith

anti

-SH

3P

X1

anti

body,

and s

ample

s w

ere

reso

lved

by S

DS

-PA

GE

. T

he

gel

was

then

tra

nsf

erre

d t

o

PV

DF

mem

bra

ne

and i

mm

unoblo

tted

wit

h a

nti

-phosp

hoty

rosi

ne

anti

body (

B).

86

87

on g

luta

thio

ne-

agar

ose

bea

ds

in k

inas

e re

acti

on b

uff

er (

10 m

M H

epes

(pH

7.4

), 5

mM

MgC

l 2,

150 m

M N

aCl,

1 m

M N

a 3V

O4,

1 m

M A

TP

) fo

r 30 m

inute

s at

30°C

. K

inas

e

reac

tion s

ample

s w

ere

quen

ched

wit

h 5

x S

DS

loadin

g b

uff

er,

and p

hosp

hory

lati

on o

f

GS

T-S

H3P

X1 w

as d

etec

ted b

y i

mm

unoblo

ttin

g w

ith a

nti

-phosp

hoty

rosi

ne

anti

body

(dat

a not

show

n).

A

ltho

ugh th

e phosp

hory

lati

on of

GS

T-S

H3P

X1 co

nfi

rmed

th

e

acti

vit

y o

f th

e re

com

bin

ant

pro

tein

, w

e fo

und t

hat

GS

T-S

H3P

X1 e

xis

ts i

n m

onom

eric

and v

ario

us

oli

gom

eric

com

ple

xes

as

det

erm

ined

by p

uri

fica

tion o

n a

siz

e ex

clusi

on

colu

mn (

wit

h t

he

oli

gom

eric

com

ple

xes

elu

ting a

s a

bro

ad p

eak j

ust

aft

er t

he

void

volu

me)

.

To d

eter

min

e if

the

oli

gom

eric

com

ple

xes

of

SH

3P

X1 r

efle

cted

an i

nher

ent

abil

ity o

f th

e pro

tein

to u

nder

go s

elf-

asso

ciat

ion (

e.g.

dim

eriz

atio

n o

f th

e C

-ter

min

al

BA

R m

oti

f),

we

expre

ssed

S

H3P

X1

as

a

His

-tag

ged

pro

tein

. S

H3P

X1 w

as

firs

t

cloned

into

the

pE

T28A

vec

tor

and t

ransf

orm

ed i

nto

BL

21 c

ells

. O

ne-

lite

r cu

lture

s

wer

e gro

wn t

o a

n O

D6

00 o

f 0.8

in s

up

er b

roth

at

37°C

and i

nduce

d w

ith 2

00 µ

M I

PT

G

over

nig

ht

at r

oom

tem

per

ature

. C

ells

wer

e har

ves

ted a

nd t

he

pro

tein

was

puri

fied

on a

nic

kel

colu

mn.

Furt

her

puri

fica

tion b

y s

ize

excl

usi

on r

evea

led t

he

monom

eric

nat

ure

of

the

His

-tag

ged

S

H3P

X1.

A

s sh

ow

n in

F

igu

re 2.1

0,

His

-SH

3P

X1 re

com

bin

ant

pro

tein

is

able

to b

e phosp

hory

late

d b

y H

is-A

CK

2 f

rom

inse

ct c

ells

(F

igure

2.1

0B

,

lane

1).

G

iven

the

pro

pen

sity

of

GS

T-S

H3P

X1 t

o a

ggre

gat

e, w

e dec

ided

that

the

His

-

tagged

SH

3P

X1 w

ould

be

use

d i

n f

utu

re s

tudie

s.

Park

e-D

avis

co

mp

ou

nd

P

D158780 w

as

iden

tifi

ed in

a H

igh

-th

rou

gh

pu

t A

CK

2

Kin

ase

Scr

een

As

an a

lter

nat

ive

mea

ns

for

exam

inin

g t

he

cell

ula

r an

d b

iolo

gic

al c

onse

quen

ces

of

the

AC

K2-c

atal

yze

d p

hosp

hory

lati

on o

f S

H3P

X1,

we

wer

e in

tere

sted

in i

den

tify

ing s

mal

l

mole

cule

inhib

itors

of

AC

K2 k

inas

e ac

tivit

y. F

or

this

purp

ose

, w

e use

d t

he

hig

h-

88

Fig

ure

2.1

0

E

xp

ress

ion

an

d

act

ivit

y

of

reco

mb

inan

t S

H3P

X1

in

E.

coli

.

Over

nig

ht

cult

ure

s fr

om

co

lonie

s of

BL

21

cell

s (N

ovag

en)

tran

sform

ed

wit

h

the

expre

ssio

n v

ecto

r w

ere

gro

wn a

t 37°C

. T

hes

e w

ere

use

d t

o i

nocu

late

one-

lite

r cu

lture

s

of

super

bro

th.

Bac

teri

al c

ult

ure

s w

ere

gro

wn t

o a

n O

D6

00 o

f 0.8

at

37°C

and i

nduce

d

wit

h

200 µ

M

IPT

G

over

nig

ht

at

room

te

mpera

ture

.

Cel

ls

wer

e har

ves

ted

by

centr

ifugat

ion at

4000 rp

m fo

r 10 m

inute

s an

d fr

oze

n at

!

80°C

. A

ll su

bse

quen

t

puri

fica

tion s

teps

wer

e c

arri

ed o

ut

at 4

°C.

Bac

teri

al p

elle

ts w

ere

resu

spen

ded

in

Ni2

+

bin

din

g

buff

er

(20

mM

T

ris

(pH

7.9

),

500

mM

N

aCl,

20

mM

im

idaz

ole

, 10%

gly

cero

l) su

pple

men

ted w

ith pro

teas

e in

hib

itors

(1 m

M P

MS

F,

10 µ

g/m

L ea

ch of

apro

tinin

and l

eupep

tin,

10

µM

ben

zam

idin

e) a

nd l

yse

d b

y t

hre

e pas

sag

es t

hro

ugh a

Fre

nch

Pre

ssu

re C

ell.

T

he

extr

acts

wer

e th

en s

onic

ated

for

5 m

inute

s an

d t

he

lysa

tes

wer

e cl

arif

ied by ult

race

ntr

ifugat

ion at

40,0

00 rp

m fo

r 45 m

inute

s. T

he

clar

ifie

d

lysa

tes

wer

e in

cubat

ed f

or

30

min

ute

s w

ith N

i2+ b

ead

s (A

mer

sham

) at

4°C

. T

he

bea

ds

wer

e th

en w

ash

ed w

ith

bin

din

g b

uff

er a

nd t

he p

rote

in r

ecover

ed w

ith e

luti

on b

uff

er

(20 m

M T

ris

(pH

7.9

), 5

00 m

M N

aCl,

200 m

M i

mid

azole

, 10%

gly

cero

l).

Sam

ple

s

from

th

e pro

tein

puri

fica

tion

w

ere

then

re

solv

ed by S

DS

-PA

GE

an

d st

ained

w

ith

Coom

assi

e b

lue

(A).

T

he

abil

ity o

f S

H3P

X1 t

o b

e phosp

hory

late

d b

y A

CK

2 w

as

exam

ined

by i

ncu

bat

ing

the

puri

fied

pro

tein

wit

h r

ecom

bin

ant

His

-AC

K2 f

rom

inse

ct

cell

s fo

r 30 m

inute

s at

30°C

in H

EP

ES

/MgC

l 2/N

aCl 2

buff

er.

The

kin

ase

react

ion w

as

quen

ched

w

ith 5x S

DS

-PA

GE

sa

mple

buff

er an

d sa

mple

s w

ere

reso

lved

by S

DS

-

PA

GE

and l

ater

subje

cted

to W

este

rn b

lott

ing w

ith a

nti

-phosp

hoty

rosi

ne

anti

body (

B).

89

90

thro

ughput

smal

l m

ole

cule

sc

reen

ing

faci

lity

in

th

e A

uto

mat

ed

Bio

tech

nolo

gy

dep

artm

ent

of

Mer

ck

&

Co.

In

itia

lly,

we

set

out

to

use

th

e w

ell-

char

acte

rize

d

det

ecti

on s

yst

em,

LA

NC

ET

M,

a ti

me-

reso

lved

flu

ore

scen

t re

son

ance

ener

gy t

ransf

er

syst

em,

from

Per

kin

Elm

er.

LA

NC

ET

M i

s bas

ed o

n t

he

ener

gy t

ransf

er b

etw

een d

onor

(euro

piu

m c

hel

ate)

and a

ccep

tor

(all

ophyco

cyan

in—

AP

C)

reag

ents

that

are

bro

ught

wit

hin

pro

xim

ity by a

spec

ific

bin

din

g ev

ent.

I

n th

e ca

se of

the

kin

ase

reac

tion,

euro

piu

m i

s co

nju

gat

ed t

o a

n a

nti

-phosp

hoty

rosi

ne

anti

body a

nd A

PC

is

conju

gat

ed t

o

stre

pta

vid

in

bea

ds.

Ener

gy

tran

sfer

occ

urs

in

th

e pre

sence

of

a ty

rosi

ne

phosp

hory

late

d,

bio

tinyla

ted s

ubst

rate

(F

igure

2.1

1).

Opti

mal

kin

ase

reac

tion co

ndit

ions

wer

e det

erm

ined

by per

form

ing a

tim

e

cours

e fo

r th

e A

CK

2-c

atal

yze

d p

hosp

hory

lati

on r

eact

ion.

The

gre

ates

t ch

alle

nge

was

gen

erat

ing

enough

SH

3P

X1

phosp

hory

lati

on

to

be

read

out

by

th

e fl

uore

scen

t-

det

ecti

on

syst

em.

W

e ad

dre

ssed

th

is

lim

itat

ion

by

alte

ring

reac

tion

condit

ions

incl

udin

g,

incr

easi

ng t

he

kin

ase c

once

ntr

atio

n a

nd

chan

gin

g t

he

buff

er s

alin

ity,

whil

e

at th

e sa

me ti

me,

per

form

ing posi

tive

contr

ols

w

ith re

com

bin

ant

insu

lin re

cepto

r.

Thro

ugh t

hes

e ad

just

men

ts,

we

ob

serv

ed t

hat

the o

pti

mal

AC

K2 k

inas

e ac

tivit

y w

as

obse

rved

in 5

0 m

M H

epes

/10 m

M M

gC

l 2/0

.1%

Tri

ton X

-100 b

uff

er,

wher

e so

diu

m

chlo

ride

was

fo

und

to

sever

ely

inhib

it

sub

stra

te

phosp

hory

lati

on.

U

nder

th

ese

condit

ions,

the

reac

tion r

each

ed c

om

ple

tion

aft

er 5

-10 m

inute

s at

room

tem

per

ature

(Fig

ure

2.1

2).

T

o i

nhib

it p

ote

nti

al n

onsp

ecif

ic i

nte

ract

ions

bet

wee

n A

CK

2 a

nd t

he

reac

tion p

late

, w

e in

cluded

det

ergen

ts a

nd B

SA

in s

ubse

quen

t en

zym

e ti

trat

ions.

When

the

addit

ion

of

det

ergen

t an

d

BS

A

did

not

alte

r our

resu

lts,

w

e per

form

ed

a

stau

rosp

ori

ne

dose

re

sponse

to

co

nfi

rm th

at th

e si

gnal

w

as due

to kin

ase

acti

vit

y

(Fig

ure

2.1

3).

91

Fig

ure

2.1

1

L

AN

CE

TM

ti

me-

reso

lved

fl

uore

scen

t re

son

an

ce

ener

gy

tr

an

sfer

syst

em.

E

xci

tati

on

of

a E

uro

piu

m-c

onju

gat

ed

anti

-phosp

hoty

rosi

ne

anti

body

in

pro

xim

ity

of

the

fluore

scen

t pro

be

allo

phyco

cyan

in

(AP

C),

re

sult

s in

an

en

ergy

tran

sfer

that

can

be

read

at

the

emis

sion w

avel

ength

of

AP

C b

y a

flu

ore

scen

t pla

te

read

er.

92

93

Fig

ure

2.1

2 A

CK

2 a

ctiv

ity i

n t

he

LA

NC

ET

M d

etec

tion

syst

em.

Rec

om

bin

ant

His

-AC

K2 (

4.1

5 µ

g/m

L)

and H

is-S

H3P

X1 (

200 n

M)

wer

e ad

ded

to a

kin

ase

reac

tion m

ixtu

re c

onta

inin

g 2

mM

AT

P,

50 m

M H

EP

ES

, 10 m

M M

gC

l 2,

and

0.1

% T

rito

n X

-100.

The

kin

ase

reac

tion w

as a

llow

ed t

o p

roce

ed a

t ro

om

tem

per

ature

for

the

indic

ated

tim

e in

terv

als

afte

r w

hic

h t

he

reac

tion s

ample

s w

ere

then

quen

ched

wit

h

the

addit

ion

of

AP

C-c

onju

gat

ed

stre

pta

vid

in

and

Euro

piu

m-c

hel

ated

an

ti-

phosp

hoty

rosi

ne

anti

body. S

ample

s w

ere

then

rea

d b

y a

flu

ore

scen

t pla

te r

eader

.

94

95

Fig

ure

2.1

3

T

he

kin

ase

act

ivit

y

of

AC

K2

is

inh

ibit

ed

by

stau

rosp

ori

ne.

The

kin

ase

reac

tions

wer

e ca

rrie

d o

ut

wit

h 4

.15 µ

g/m

L A

CK

2 a

nd 2

00 n

M S

H3P

X1 i

n

kin

ase

buff

er (

2 m

M A

TP

, 50 m

M H

EP

ES

, 10 m

M M

gC

l 2,

and 0

.1%

Tri

ton X

-100)

in

the

pre

sence

of

20 µ

M-8

0

nM

st

auro

spori

ne.

Rea

ctio

ns

wer

e quen

ched

af

ter

5

min

ute

s at

room

tem

per

ature

wit

h t

he

addit

ion o

f A

PC

-conju

gat

ed s

trep

tavid

in a

nd

Euro

piu

m-c

hel

ated

an

ti-p

hosp

hoty

rosi

ne

anti

body.

S

ample

s w

ere

then

re

ad

by

a

fluore

scen

t pla

te r

eader

.

96

97

Ult

imat

ely,

we

moved

to a

[3

3P

]-A

TP

fil

ter

assa

y t

o s

impli

fy d

etec

tion (

Fig

ure

2.1

4).

Foll

ow

ing

the

kin

ase

reac

tion,

the

pro

tein

s w

ere

pre

cipit

ated

w

ith

tric

hlo

roac

etic

aci

d. T

he

reac

tion m

ixtu

re w

as t

hen

tra

nsf

erre

d t

o a

fil

ter

pla

te w

her

e

[3

3P

] in

corp

ora

tion o

f ad

her

ent

pro

tein

s w

as m

easu

red.

A c

om

par

ativ

e ti

me

cours

e

was

ca

rrie

d

out

under

th

e sa

me

condit

ions

as

the

LA

NC

ET

M

syst

em.

H

ow

ever

,

inco

rpora

tion of

[33P

]-A

TP

w

as ver

y lo

w,

attr

ibute

d to

th

e re

lati

vel

y poor

kin

ase

acti

vit

y o

f A

CK

2.

We

then

exte

nded

the

reac

tion t

ime

to o

ver

nig

ht

condit

ions

to

max

imiz

e th

e si

gnal

-to-b

ack

gro

und

rati

o

to

2.5

:1,

wit

hin

th

e ra

nge

of

scre

enin

g

(Fig

ure

2.1

5).

T

he

nex

t st

ep w

as

to a

uto

mat

e t

he

assa

y,

wit

h e

ach r

eact

ion c

om

ponen

t

dis

trib

ute

d

by

an

auto

mat

ed

inst

rum

ent.

Unfo

rtunat

ely,

the

DM

SO

co

ntr

ol

pla

te

show

ed s

ever

al s

pik

es i

n s

ignal

, m

ost

lik

ely

due

to t

he

low

sig

nal

-to

-bac

kgro

und r

atio

as w

ell

as to

a

man

ual

st

ep nec

essa

ry in

th

e pro

cess

. W

e did

not

pro

ceed

to

an

auto

mat

ed

scre

en

bec

ause

w

e fe

lt

that

th

e ar

tifa

cts

would

sk

ew

any

true

hit

s.

How

ever

, th

e si

gnal

-to-b

ackg

round r

atio

was

hig

h e

nough t

o s

cree

n c

om

pound

s in

repli

cate

s.

Dose

-res

ponse

pro

file

s w

ere

per

form

ed u

sing t

yrp

host

in A

G1478 (

a know

n

EG

F r

ecep

tor

kin

ase

inhib

itor)

, P

arke-

Dav

is p

yri

do-p

yro

mid

ine,

PD

158780,

and t

he

SU

Gen

com

pound,

SU

6656.

We

found t

hat

the

Par

k-D

avis

com

pound w

as a

hig

hly

pote

nt

inhib

itor

of

AC

K2 k

inas

e ac

tivit

y i

n v

itro w

ith a

n I

C50 o

f ~

80 p

M (

Fig

ure

2.1

6).

Dis

cuss

ion

Due

to t

he

role

of

Erb

B r

ecep

tors

in i

nit

iati

ng m

itog

enic

sig

nal

ing p

athw

ays,

over

expre

ssio

n o

r m

uta

tions

that

alt

er t

he

intr

insi

c kin

ase

acti

vit

y o

f th

ese

rece

pto

rs

are

oft

en s

uff

icie

nt

to c

ause

mal

ignan

t tr

ansf

orm

atio

n.

Can

cer

cell

s ar

e ab

le t

o b

ypas

s

the

nec

essa

ry c

heck

poin

ts i

nvolv

ed i

n t

he

regula

tion o

f E

rbB

rec

epto

r si

gnal

ing i

n

sever

al w

ays,

in

cludin

g th

e over

expre

ssio

n of

gro

wth

fa

ctor

rece

pto

rs at

th

e ce

ll

surf

ace,

res

ult

ing i

n h

yper

sensi

tivit

y t

o o

ther

wis

e am

bie

nt

level

s of

gro

wth

fac

tor.

98

Fig

ure

2.1

4 F

ilte

r ass

ay f

or

ph

osp

hory

lati

on

rea

ctio

ns.

Foll

ow

ing

in

vit

ro

kin

ase

reac

tions,

th

e pro

tein

s ar

e pre

cipit

ated

w

ith

chil

led

tric

hlo

roac

etic

aci

d (

TC

A)

and t

ransf

erre

d t

o 9

6-w

ell

filt

er p

late

s.

The

sam

ple

s ar

e

was

hed

w

ith

TC

A

in

the

filt

er

pla

tes

and

inco

rpora

ted

radio

acti

ve

phosp

hat

e is

mea

sure

d i

n t

he

pre

sence

of

scin

till

atio

n f

luid

.

99

100

Fig

ure

2.1

5

AC

K2-c

ata

lyze

d k

inase

rea

ctio

ns

ass

ayed

by f

iltr

ati

on

.

Kin

ase

reac

tion

s w

ere

carr

ied o

ut

wit

h 8

.3 µ

g/m

L A

CK

2,

200 n

M S

H3P

X1,

50 µ

M

AT

P,

0.2

µC

i/w

ell

for

18-h

our

reac

tion a

t ro

om

tem

per

ature

. F

oll

ow

ing i

n v

itro k

inas

e

reac

tions,

the

pro

tein

s w

ere

pre

cipit

ated

wit

h c

hil

led

tri

chlo

roac

etic

aci

d (

TC

A)

and

tran

sfer

red t

o 9

6-w

ell

filt

er p

late

s.

The

sam

ple

s w

ere

was

hed

wit

h T

CA

in t

he

filt

er

pla

tes

and

inco

rpora

ted

radio

acti

ve

phosp

hat

e w

as

mea

sure

d

in

the

pre

sence

of

scin

till

atio

n f

luid

.

101

102

Fig

ure

2.1

6

Park

e-D

avis

pyri

dop

yri

mid

ine

inh

ibit

s A

CK

2 k

inase

act

ivit

y w

ith

an

IC5

0 ~

80 p

M.

Str

uct

ure

of

Par

ke-

Dav

is c

om

pound P

D158780 (

A).

K

inas

e re

acti

ons

wer

e ca

rrie

d o

ut

wit

h 8

.3 µ

g/m

L A

CK

2,

200 n

M S

H3P

X1,

50 µ

M A

TP

+ 0

.2 µ

Ci/

wel

l

for

18 h

ours

at

room

tem

per

ature

. F

oll

ow

ing i

n vit

ro k

inas

e re

acti

on

s, t

he

pro

tein

s

wer

e pre

cipit

ated

w

ith ch

ille

d tr

ichlo

roac

etic

ac

id an

d tr

ansf

erre

d to

96

-wel

l fi

lter

pla

tes.

T

he

sam

ple

s w

ere

was

hed

w

ith T

CA

in

th

e fi

lter

pla

tes

and in

corp

ora

ted

radio

acti

ve

ph

osp

hat

e w

as m

easu

red i

n t

he

pre

sence

of

scin

till

atio

n f

luid

(B

).

103

104

Gro

ss

over

expre

ssio

n

of

Erb

B

rece

pto

rs

has

ev

en

bee

n

show

n

to

induce

li

gan

d-

indep

enden

t si

gnal

ing [

17],

thus

enhan

cing c

ell

pro

life

rati

on i

n t

he

abse

nce

of

exte

rnal

signal

s. A

noth

er

pote

nti

al

cause

of

can

cer

is

the

loss

of

gro

wth

fa

ctor

rece

pto

r

deg

radat

ion.

In c

ells

wit

h e

levat

ed l

evel

s of

gro

wth

fac

tor

rece

pto

rs a

t th

e ce

ll s

urf

ace,

one

mec

han

ism

fo

r re

stori

ng norm

al pro

life

rati

ve

rate

s in

volv

es th

e upta

ke

of

the

rece

pto

rs f

rom

the

pla

sma

mem

bra

ne.

A

wel

l-st

ud

ied m

echan

ism

for

dow

n-r

egula

ting

gro

wth

fa

ctor

rece

pto

rs

is

clat

hri

n-m

edia

ted

endocy

tosi

s,

a hig

hly

ch

arac

teri

zed

pro

cess

in

w

hic

h

cel

l su

rfac

e re

cepto

rs

beco

me

inte

rnal

ized

in

to

the

cyto

pla

sm.

Endocy

tosi

s of

cell

su

rfac

e re

cepto

rs is

a

tightl

y re

gula

ted,

sequen

tial

pro

cess

. It

involv

es s

ever

al c

yto

soli

c pro

tein

s, r

efer

red t

o a

s ac

cess

ory

pro

tein

s, a

s w

ell

as t

he

mec

han

ical

m

eans

to

def

orm

th

e m

embra

ne

into

a

ves

icula

r bud

that

ult

imat

ely

pin

ches

off

fro

m t

he

mem

bra

ne

to f

orm

the

free

cla

thri

n-c

oat

ed v

esic

le.

The

pri

nci

pal

pro

tein

s in

cl

athri

n-m

edia

ted en

docy

tosi

s in

clude,

cl

athri

n,

a

tris

kel

ion p

rote

in m

akin

g u

p t

he

cage o

f th

e cl

athri

n-c

oat

ed v

esic

le,

AP

-2,

resp

onsi

ble

for

conce

ntr

atin

g t

he

carg

o a

nd r

ecru

itin

g c

lath

rin t

o t

he

pla

sma

mem

bra

ne,

and t

he

GT

Pas

e,

dynam

in,

involv

ed

in

pro

vid

ing

the

mec

han

ical

fo

rce

from

w

hic

h

the

clat

hri

n-c

oat

ed v

esic

le p

ropel

s it

self

fro

m t

he

mem

bra

ne.

In

tere

stin

gly

, A

CK

2 a

nd

SH

3P

X1 c

onta

in s

ignal

ing d

om

ains

that

all

ow

them

to i

nte

ract

wit

h k

ey a

ccess

ory

pro

tein

s in

the

endocy

tic

pro

cess

. A

CK

2 h

as b

een s

how

n t

o b

ind t

o c

lath

rin i

n G

ST

pull

-dow

n

exper

imen

ts

wher

e th

e tw

o

carb

oxy

l-te

rmin

al

pro

line-

rich

dom

ains

of

AC

K2 w

ere

show

n t

o m

edia

te t

his

act

ion [

6].

A

cla

thri

n-b

indin

g m

oti

f, L

IDF

, has

since

bee

n

iden

tifi

ed

bet

wee

n

the

two

pro

line-

rich

m

oti

fs.

S

H3P

X1

conta

ins

an

amin

o-t

erm

inal

SH

3 d

om

ain,

whic

h h

as b

een i

mpli

cate

d i

n i

nte

ract

ions

wit

h A

CK

2,

the

Wis

kott

-Ald

rich

sy

ndro

me

pro

tein

(W

AS

P),

an

d m

ore

re

centl

y,

the

endocy

tic

105

GT

Pas

e, d

ynam

in-2

[7].

T

his

sort

ing n

exin

has

als

o b

een s

how

n t

o b

ind t

o c

lath

rin

and A

P-2

, th

us

stre

ngth

enin

g t

he

link t

o c

lath

rin-m

edia

ted e

nd

ocy

tosi

s an

d s

ort

ing [

7].

Whil

e th

e ex

act

funct

ion o

f S

H3P

X1 i

n r

ecep

tor

endocy

tosi

s an

d d

egra

dat

ion

is s

till

not

under

stood,

one

can m

ake

a re

asonab

le a

ssum

pti

on b

ased

on w

hat

has

bee

n

det

erm

ined

for

oth

er m

ember

s of

the

sort

ing n

exin

fam

ily.

Sort

ing n

exin

1 (

SN

X1),

the

firs

t m

amm

alia

n s

ort

ing n

exin

to b

e ch

arac

teri

zed,

has

bee

n s

how

n t

o i

nte

ract

wit

h

EG

F r

ecep

tors

and r

egula

te a

ctiv

ated

rec

epto

r le

vel

s in

CV

1 (

Afr

ican

gre

en m

onkey

kid

ney

) ce

lls

[11].

O

ther

sort

ing n

exin

s, S

NX

2 a

nd S

NX

4,

co-i

mm

unopre

cipit

ate

wit

h

rece

pto

rs t

hat

bin

d E

GF

, in

suli

n,

pla

tele

t-der

ived

gro

wth

fac

tor

(PD

GF

) an

d t

he

long

form

of

the

lepti

n r

ecep

tor

[15].

M

ore

over

, over

expre

ssio

n o

f S

H3P

X1 t

oget

her

wit

h

AC

K2 in

C

OS

-7 ce

lls

was

sh

ow

n to

al

ter

the

pro

cess

ing an

d tr

affi

ckin

g of

EG

F,

insu

lin,

and t

ransf

erri

n r

ecep

tors

[10]

[7,

16].

In o

rder

to u

ltim

atel

y u

nder

stan

d t

he r

ole

s of

AC

K2 a

nd S

H3P

X1 i

n g

row

th

fact

or

rece

pto

r deg

radat

ion,

it w

ill

be

import

ant

to c

har

acte

rize

the

AC

K2-S

H3P

X1

inte

ract

ion,

iden

tify

the

site

(s)

on t

he

sort

ing n

exin

that

are

phosp

hory

late

d b

y A

CK

2,

and d

eter

min

e th

e si

gnif

ican

ce o

f th

e phosp

hory

lati

on e

ven

t to

cel

lula

r pro

cess

ing.

To

dat

e, w

e hav

e dem

onst

rate

d t

he l

oss

of

AC

K2-c

atal

yze

d p

hosp

hory

lati

on i

n t

he !

C84

muta

nt

of

SH

3P

X1 b

y d

elet

ion a

nal

ysi

s.

Our

init

ial

hypoth

esis

was

th

at t

his

car

boxyl-

term

inal

reg

ion o

f S

H3P

X1 c

onta

ined

the

AC

K2 p

hosp

hory

lati

on s

ites

. I

nst

ead,

we

det

erm

ined

that

the

loss

of

pho

sphory

lati

on w

as a

res

ult

of

the

inab

ilit

y o

f A

CK

2 t

o

bin

d t

o t

his

car

boxyl-

term

inal

tru

nca

tion m

uta

nt

of

SH

3P

X1.

Rec

ent

report

s cl

aim

ing

that

the

carb

oxyl-

term

inal

BA

R m

oti

f is

cri

tica

l fo

r dim

eriz

atio

n o

f th

e so

rtin

g n

exin

[18]

may

expla

in t

his

obse

rvat

ion,

as d

imer

izat

ion m

ay b

e cr

itic

al f

or

the

bin

din

g o

f

AC

K2 a

nd s

ubse

quen

t phosp

hory

lati

on o

f th

e so

rtin

g n

exin

.

Pre

vio

usl

y,

the

tyro

sine

phosp

hory

lati

on

site

of

the

Dro

sophil

a

SH

3P

X1

ort

holo

gue

has

bee

n re

port

ed as

Y

56

[5].

H

ow

ever

, m

uta

ting th

e co

rres

pondin

g

106

resi

dues

in

th

e hum

an

isofo

rm

of

SH

3P

X1

did

n

ot

blo

ck

the

AC

K2-c

atal

yze

d

phosp

hory

lati

on o

f S

H3P

X1.

This

and o

ther

exper

imen

ts l

ed u

s to

the

real

izat

ion t

hat

AC

K2 li

kel

y phosp

hory

late

s S

H3P

X1 on m

ult

iple

ty

rosi

ne

resi

dues

. A

ttem

pts

to

iden

tify

the

AC

K2-c

atal

yze

d s

ites

by m

ass

spec

trom

etry

hav

e le

d t

o t

he

iden

tifi

cati

on

of

tyro

sine

287 a

s a

site

of

pho

sphory

lati

on.

This

tyro

sine

resi

due

is l

oca

ted

in t

he

PX

dom

ain o

f S

H3P

X1,

resp

onsi

ble

for

targ

etin

g t

he

pro

tein

to t

he

pla

sma

mem

bra

ne

thro

ugh

inte

ract

ions

wit

h

phosp

hat

idyli

nosi

tols

.

One

could

im

agin

e th

e

phosp

hory

lati

on o

f Y

287 a

s a

regula

ting f

acto

r in

dic

tati

ng t

he

loca

liza

tion o

f th

e

sort

ing n

exin

in t

he

cell

. D

espit

e th

is p

osi

tive

iden

tifi

cati

on,

our

dat

a sh

ow

that

the

SH

3P

X1 m

uta

nt

Y287F

ret

ains

som

e l

evel

of

ph

osp

hory

lati

on w

hen

co-e

xpre

ssed

in

cell

s.

As

a re

sult

, w

e bel

ieve

ther

e ar

e ad

dit

ional

sit

es o

f m

odif

icat

ion a

nd a

re t

akin

g

mea

sure

s to

id

enti

fy th

ese

site

s. In

th

e fu

ture

, w

e bel

ieve

that

th

e gen

erat

ion of

SH

3P

X1 m

uta

nts

def

ecti

ve

for

AC

K2-c

atal

yze

d p

hosp

hory

lati

on s

hould

be

inval

uab

le

in e

stab

lish

ing t

he

role

for

SH

3P

X1 p

hosp

hory

lati

on i

n r

ecep

tor

endocy

tosi

s.

As

a

com

ple

men

tary

ap

pro

ach

to

war

d

pro

bin

g

the

import

ance

of

AC

K2

acti

vit

y a

nd i

ts p

hosp

hory

lati

on o

f S

H3P

X1,

we

set

out

to i

den

tify

sm

all

mole

cule

inhib

itors

of

AC

K2.

Thro

ugh a

ver

y g

ener

ous

rela

tionsh

ip w

ith M

erck

, w

e w

ere

able

to

uti

lize

th

e hig

h-t

hro

ughput

scre

enin

g

faci

lity

of

the

Auto

mat

ed

Bio

tech

nolo

gy

dep

artm

ent.

H

ere

we

wer

e ab

le t

o i

den

tify

the

Park

e-D

avis

com

pound,

PD

158780 a

s

an i

n v

itro

inhib

itor

of

AC

K2 k

inas

e ac

tivit

y.

PD

158780 i

s a

pyri

dopyri

mid

ine,

a

der

ivia

tive

of

the

quin

azoli

ne

fam

ily o

f co

mpounds.

T

hes

e a

rom

atic

sm

all

mole

cule

s

hav

e bee

n

show

n

to

effe

ctiv

ely

inhib

it

EG

F

rece

pto

r fa

mil

y

pro

tein

s.

In

dee

d,

PD

158780 i

nhib

its

EG

F r

ecep

tor

kin

ase

acti

vit

y w

ith a

n I

C50 ~

8 p

M [

19].

T

hus,

it

wil

l

be

dif

ficu

lt t

o u

se t

his

com

pound t

o s

elec

tivel

y e

xam

ine

the

acti

ons

of

AC

K2 (

IC5

0

~80 p

M),

unle

ss e

xper

imen

ts w

ere

per

form

ed u

sing o

ther

inhib

itors

that

blo

ck o

nly

EG

F r

ecep

tor

acti

vit

y,

as a

mea

ns

of

dis

tinguis

hin

g t

he

conse

quen

ces

of

inhib

itin

g

107

EG

F r

ecep

tors

fro

m t

hose

res

ult

ing f

rom

the

inhib

itio

n o

f A

CK

2.

Futu

re s

tudie

s ar

e

bei

ng d

irec

ted t

ow

ard i

den

tify

ing m

ore

sp

ecif

ic i

nhib

itors

of

AC

K2 t

hat

do n

ot

affe

ct

the

EG

F r

ecep

tor.

108

Refe

ren

ces

1.

Sla

mon,

D.J

., e

t al

., H

um

an b

reast

cance

r: c

orr

elati

on o

f re

lapse

and s

urv

ival

wit

h a

mpli

fica

tion o

f th

e H

ER

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e. S

cien

ce,

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2.

Kas

prz

yk,

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t al

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her

apy

of

an a

nim

al

mo

del

of

hum

an g

ast

ric

cance

r

usi

ng a

com

bin

ati

on

of

anti

-erb

B-2

monocl

onal

anti

bodie

s. C

ance

r R

es,

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52(1

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2771-6

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3.

Yan

g,

W.,

et

al.,

Act

ivati

on o

f th

e C

dc4

2-a

ssoci

ate

d t

yrosi

ne k

inase

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AC

K-2

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by

cell

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ia i

nte

gri

n b

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J B

iol

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0.

4.

Yan

g,

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et

al., T

he

nonre

cepto

r ty

rosi

ne

kina

se A

CK

2,

a s

pec

ific

targ

et f

or

Cdc4

2 a

nd a

neg

ati

ve r

egula

tor

of

cell

gro

wth

and f

oca

l adhes

ion c

om

ple

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J

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l C

hem

, 2001. 276

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3.

5.

Worb

y,

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., e

t al

., D

roso

phil

a A

ck t

arg

ets

its

subst

rate

, th

e so

rtin

g n

exin

DSH

3P

X1,

to a

pro

tein

co

mple

x in

volv

ed i

n a

xonal

guid

ance

. J

Bio

l C

hem

,

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277(1

1):

p.

9422-8

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6.

Yan

g,

W.,

et

al., T

he

Cdc4

2 t

arg

et A

CK

2 d

irec

tly

inte

ract

s w

ith c

lath

rin a

nd

infl

uen

ces

clath

rin a

ssem

bly

. J

Bio

l C

hem

, 2001. 276(2

0):

p.

17468-7

3.

7.

Lundm

ark,

R.

and S

.R.

Car

lsso

n,

Sort

ing nexi

n 9 part

icip

ate

s in

cl

ath

rin-

med

iate

d e

ndocy

tosi

s th

rough i

nte

ract

ion

s w

ith

the

core

com

ponen

ts.

J B

iol

Chem

, 2003. 278(4

7):

p. 46772-8

1.

8.

Lundm

ark,

R.

and S

.R.

Car

lsso

n,

The

bet

a-a

ppen

dages

of

the

fou

r adapto

r-

pro

tein

(A

P)

com

ple

xes:

str

uct

ure

and b

indin

g p

roper

ties

, and i

den

tifi

cati

on o

f

sort

ing n

exin

9 a

s an

acc

ess

ory

pro

tein

to

AP

-2.

Bio

chem

J,

2002.

362(P

t 3):

p.

597-6

07.

109

9.

Soule

t,

F.,

et

al

.,

SN

X9

regula

tes

dyn

am

in

ass

embly

and

is

requir

ed

for

effi

cien

t cl

ath

rin

-med

iate

d e

ndocy

tosi

s. M

ol

Bio

l C

ell,

2005.

16(4

): p

. 20

58-

67.

10.

Lin

, Q

.,

et

al.,

The

Cdc4

2

targ

et

AC

K2

inte

ract

s w

ith

sort

ing

nex

in

9

(SH

3P

X1)

to r

egula

te e

pid

erm

al

gro

wth

fact

or

rece

pto

r deg

radati

on.

J B

iol

Chem

, 2002. 277(1

2):

p. 10134-8

.

11.

Kurt

en,

R.C

., D

.L.

Cad

ena,

an

d G

.N.

Gil

l, E

nhance

d deg

radati

on of

EG

F

rece

pto

rs b

y a s

ort

ing n

exin

, SN

X1.

Sci

ence

, 1996. 272(5

264):

p. 1008-1

0.

12.

Wan

g,

Y.,

et

al

.,

Dow

n-r

egula

tion

of

pro

tease

-act

ivate

d

rece

pto

r-1

is

regula

ted b

y so

rtin

g n

exin

1.

Mol

Bio

l C

ell,

2002. 13(6

): p

. 1965-7

6.

13.

Gull

apal

li,

A.,

et

al

.,

An

esse

nti

al

role

fo

r SN

X1

in

lyso

som

al

sort

ing

of

pro

tease

-act

ivate

d

recep

tor-

1:

evid

ence

fo

r re

trom

er-,

H

rs-,

and

Tsg

101-

indep

enden

t fu

nct

ions

of

sort

ing n

exi

ns.

Mol

Bio

l C

ell,

2006.

17(3

): p

. 122

8-

38.

14.

Burd

en,

J.J.

, et

al.

, So

rtin

g m

oti

fs i

n t

he

intr

ace

llula

r dom

ain

of

the

low

den

sity

lipopro

tein

rec

epto

r in

tera

ct w

ith a

nove

l do

main

of

sort

ing n

exin

-17.

J B

iol

Chem

, 2004. 279(1

6):

p. 16237-4

5.

15.

Haf

t, C

.R.,

et

al.,

Iden

tifi

cati

on o

f a f

am

ily

of

sort

ing n

exin

mole

cule

s and

chara

cter

izati

on

of

thei

r ass

oci

ati

on

wit

h

rece

pto

rs.

Mol

Cel

l B

iol,

1998.

18(1

2):

p.

7278-8

7.

16.

MaC

aula

y,

S.L

., e

t al

., I

nsu

lin s

tim

ula

tes

move

ment

of

sort

ing n

exin

9 b

etw

een

cell

ula

r co

mpart

men

ts:

a

puta

tive

ro

le

med

iati

ng

cell

su

rface

re

cepto

r

expre

ssio

n a

nd i

nsu

lin

act

ion. B

ioch

em J

, 2003.

376(P

t 1):

p.

123-3

4.

17.

DiF

iore

, P

.P., et

al

.,

Erb

B-2

is

a pote

nt

onco

gen

e w

hen

ove

rexp

ress

ed in

NIH

/3T

3 c

ells

. S

cien

ce, 1987. 237:

p.

178-1

82.

110

18.

Chil

dre

ss,

C.,

Q.

Lin

, an

d W

. Y

ang,

Dim

eri

zati

on i

s re

quir

ed f

or

SH

3P

X1

tyro

sine

phosp

ho

ryla

tion in

re

sponse

to

ep

ider

mal

gro

wth

fa

ctor

signall

ing

and i

nte

ract

ion w

ith A

CK

2.

Bio

chem

J,

2006.

394(P

t 3):

p. 693-8

.

19.

Rew

cast

le,

G.W

.,

et

al.,

T

yrosi

ne

kina

se

inhib

itors

. 14.

Str

uct

ure

-act

ivit

y

rela

tionsh

ips

for

met

hyl

am

ino-s

ub

stit

ute

d

der

ivati

ves

of

4-[

(3-

bro

mophen

yl)a

min

o]-

6-(

met

hyl

am

ino)-

pyr

ido[3

,4-d

]pyr

imid

ine

(PD

158780),

a p

ote

nt

and s

pec

ific

inhib

itor

of

the

tyro

sine

kin

ase

act

ivit

y of

rece

pto

rs f

or

the

EG

F f

am

ily

of

gro

wth

fact

ors

. J

Med

Chem

, 1998.

41(5

): p

. 742-5

1.

111

CH

AP

TE

R T

HR

EE

SH

3P

X1 I

S A

PH

OS

PH

O-S

UB

ST

RA

TE

FO

R B

OT

H T

HE

FO

CA

L A

DH

ES

ION

KIN

AS

E A

ND

SR

C

Ab

stra

ct

A

ro

le fo

r th

e so

rtin

g nex

in,

SH

3P

X1,

in th

e co

mple

x pro

cess

of

clat

hri

n-

med

iate

d e

ndocy

tosi

s h

as b

een

incr

easi

ngly

sugg

este

d i

n r

ecen

t yea

rs.

For

exam

ple

,

SH

3P

X1 b

een s

how

n t

o b

ind t

o k

ey e

ndocy

tic

pro

tein

s in

cludin

g a

dap

tor

pro

tein

-2

(AP

-2),

cl

athri

n,

and

dynam

in.

M

ore

over

, knock

ing

dow

n

endogen

ous

SH

3P

X1

expre

ssio

n i

n c

ells

usi

ng S

H3P

X1-R

NA

i, w

as

found t

o b

lock

dynam

in t

raff

ickin

g t

o

the

pla

sma

mem

bra

ne.

A

s a

resu

lts

of

thes

e an

d s

imil

ar f

indin

gs,

ther

e is

a g

row

ing

inte

rest

in e

luci

dat

ing e

xact

ly h

ow

SH

3P

X1 i

s re

gula

ted a

nd f

unct

ions

in e

ndocy

tic

pro

cess

es.

Tyro

sine

phosp

hory

lati

on a

ppea

rs t

o b

e one

mec

han

ism

by w

hic

h S

H3P

X1 i

s

regula

ted d

uri

ng t

he

endocy

tic

pro

cess

. O

ur

lab

ora

tory

and o

ther

s hav

e pre

vio

usl

y

dem

onst

rate

d t

hat

tyro

sine

ph

osp

hory

lati

on o

f S

H3P

X1 e

nhan

ces

dynam

in t

raff

ickin

g

to t

he

pla

sma

mem

bra

ne,

an

d s

ubse

quen

t en

docy

tosi

s an

d d

egra

dat

ion o

f th

e E

GF

rece

pto

r in

cel

ls.

Giv

en t

he

appar

ent

import

ant

of

SH

3P

X1 p

hosp

hory

lati

on,

we

wer

e

inte

rest

ed

in

iden

tify

ing

pro

tein

kin

ases

resp

onsi

ble

an

d

the

loca

tion

of

the

phosp

hory

lati

on si

tes.

O

ur

resu

lts

des

crib

ed in

ch

apte

r 2 id

enti

fy A

CK

2 as

on

e

pro

tein

ty

rosi

ne

kin

ase

that

ca

n

phosp

hory

late

S

H3P

X1,

wit

h

the

maj

or

site

of

phosp

hory

lati

on b

eing Y

287.

Her

e, w

e hav

e ex

amin

ed w

het

her

addit

ional

kin

ases

mig

ht

phosp

hory

late

SH

3P

X1,

nam

ely F

AK

and S

rc.

We

consi

der

ed f

oca

l ad

hes

ion

kin

ase

(FA

K)

to b

e a p

ote

nti

al c

andid

ate b

ased

on

the

fact

that

the

kin

ase d

om

ains

of

FA

K

and

AC

K2

shar

e a

hig

h

deg

ree

of

hom

olo

gy,

and

seco

ndly

, th

at

FA

K

phosp

hory

late

s en

dophil

in,

a pro

tein

wit

h s

imil

ar d

om

ain f

eatu

res

as

SH

3P

X1.

Src

112

was

ch

ose

n

as

an

addit

ional

ca

ndid

ate

giv

en

its

know

n

asso

ciat

ion

and

tandem

signal

ing w

ith F

AK

in c

ells

. H

ere,

we s

how

th

at F

AK

and S

rc c

an p

hosp

hory

late

SH

3P

X1 b

oth

in v

ivo a

nd i

n v

itro

. W

e d

emonst

rate

that

the

FA

K-

and

Src

-med

iate

d

phosp

hory

lati

on of

SH

3P

X1 is

m

uch

m

ore

pro

nounce

d th

an th

e A

CK

2-c

atal

yze

d

phosp

hory

lati

on.

M

ore

over

, w

e go

on to

dem

onst

rate

th

at

C-t

erm

inal

tr

unca

tion

muta

nts

of

SH

3P

X1 t

hat

are

def

ecti

ve

in t

hei

r ab

ilit

y t

o b

e phosp

hory

late

d b

y A

CK

2,

are

stil

l ca

pab

le

of

bei

ng

phosp

hory

late

d

by

FA

K

and

Src

.

Mas

s sp

ectr

om

etry

anal

ysi

s of

Src

-cat

alyze

d

pho

sphory

lati

on

of

SH

3P

X1

iden

tifi

ed

5

site

s of

phosp

hory

lati

on i

ncl

udin

g Y

177,

Y239,

Y269,

Y294,

and Y

561.

Y239 w

as s

how

n t

o

be

the

maj

or

phosp

hory

lati

on s

ite

by F

AK

and S

rc,

and m

uta

tion o

f al

l 5 t

yro

sine

resi

dues

resu

lted

in a

pho

sphory

lati

on-d

efec

tive

SH

3P

X1 m

uta

nt.

T

aken

toget

her

, our

resu

lts

suggest

that

SH

3P

X1 m

ay r

ecei

ve

spec

ific

sig

nal

s an

d/o

r par

tici

pat

e in

dis

tinct

signal

ing

pat

hw

ays

resu

ltin

g

from

A

CK

2-c

atal

yze

d,

and

FA

K-

and

Src

-cat

alyze

d

SH

3P

X1 p

hosp

hory

lati

on.

Intr

od

uct

ion

Sort

ing

nex

ins

are

a fa

mil

y

of

pro

tein

s th

at

hav

e bee

n

impli

cate

d

in

the

traf

fick

ing an

d pro

cess

ing of

cell

su

rfac

e re

cepto

rs.

T

he

hal

lmar

k of

this

div

erse

fam

ily o

f pro

tein

s is

the

PX

dom

ain,

iden

tifi

ed a

s a

conse

rved

moti

f in

the

p40

ph

ox a

nd

p47

ph

ox

subunit

s of

the

neu

trophil

N

AD

PH

oxid

ase

(phox)

super

oxid

e-gen

erat

ing

com

ple

x [

1].

C

om

par

ing t

he

PX

dom

ains

from

dif

fere

nt

pro

tein

s sh

ow

s th

at t

hey

hav

e

lim

ited

seq

uen

ce h

om

olo

gy a

side

from

a c

on

serv

ed p

roli

ne-

rich

moti

f, P

xxP

, w

hic

h

enab

les

PX

-dom

ain-c

onta

inin

g

pro

tein

s to

in

tera

ct

wit

h

SH

3

dom

ain-c

onta

inin

g

pro

tein

s.

Upst

ream

of

the

pro

line-

rich

dom

ain i

s a

phosp

holi

pid

-inte

ract

ion m

oti

f,

(R/K

)(R

/K)(

Y/F

)xxF

xxL

xxxL

and R

(R/K

)xxL

xx(Y

/F),

whic

h c

an i

nte

ract

wit

h a

wid

e

range

of

phophosp

holi

pid

s, th

ereb

y sp

ecif

ical

ly ta

rget

ing th

ese

pro

tein

s to

ce

llula

r

113

mem

bra

nes

enri

ched

in

phosp

hat

idyli

nosi

tols

[2,

3].

T

he

pho

spholi

pid

-inte

ract

ing

moti

fs ar

e oft

en co

nse

rved

am

ong P

X dom

ain-c

onta

inin

g pro

tein

s. W

ithin

th

ese

conse

nsu

s se

quen

ces,

th

e ly

sine

and ar

gin

ine

resi

dues

se

rve

to cr

eate

a

posi

tivel

y

char

ged

su

rfac

e on

the

pro

tein

, w

hic

h

is

attr

acte

d

to

neg

ativ

ely

char

ged

phosp

hat

idyli

nosi

tols

em

bed

ded

in t

he

pla

sma

mem

bra

ne.

In a

ddit

ion t

o t

he

sort

ing n

exin

s, P

X d

om

ains

are

also

found i

n s

ever

al o

ther

signal

ing

pro

tein

s su

ch

as

phosp

holi

pase

D

(P

LD

),

phosp

hat

idyli

nosi

tol

3-k

inas

e

(PI3

K),

and B

em1 a

nd B

em3,

two y

east

pro

tein

s in

volv

ed i

n b

ud e

mer

gen

ce a

nd c

ell

pola

rity

[4

, 5].

It

has

bee

n sh

ow

n th

at th

e P

X d

om

ains

of

the ab

ove m

enti

oned

pro

tein

s ar

e al

l in

volv

ed i

n b

indin

g t

o p

hosp

hat

idyli

nosi

tols

, as

wel

l as

med

iati

ng a

num

ber

of

pro

tein

-pro

tein

inte

ract

ions

thro

ugh t

hei

r pro

line-

rich

moti

fs {

Coll

ey,

1997

#179[6

, 7].

A

uniq

ue

role

for

the

PX

dom

ain

of

PL

D h

as a

lso b

een

rec

entl

y r

eport

ed.

It w

as d

emonst

rate

d t

hat

this

par

ticu

lar

PX

dom

ain

funct

ions

as a

GT

Pas

e-ac

tivat

ing

pro

tein

(G

AP

),

cata

lyzi

ng

the

hydro

lysi

s of

GT

P-b

ound

dynam

in

[8].

Arg

inin

e

resi

dues

128

an

d 197 of

PL

D hav

e bee

n im

pli

cate

d in

ca

taly

zing dynam

in G

TP

hydro

lysi

s, t

hus

funct

ionin

g a

s th

e ar

gin

ine

finger

of

this

puta

tive

GA

P.

The

sort

ing n

exin

s hav

e b

een c

lass

ifie

d i

nto

subfa

mil

ies

bas

ed o

n t

he

pre

sence

or

abse

nce

of

cert

ain s

ignal

ing

or

stru

ctura

l do

mai

ns

conta

ined

wit

hin

eac

h f

amil

y

mem

ber

. O

ne

subcl

ass,

com

pri

sed o

f S

NX

1-2

, S

NX

4-8

, S

NX

15,

and S

NX

16,

all

hav

e

long

carb

oxyl-

term

inal

ex

tensi

ons

conta

inin

g

1-3

ch

arac

teri

stic

co

iled

-coil

m

oti

fs

whic

h a

llow

for

hom

o-

or

het

ero-o

ligom

eriz

atio

n w

ith o

ther

sort

ing n

exin

pro

tein

s [9

].

The

seco

nd s

ub

-cla

ss,

mad

e u

p o

f S

NX

3,

SN

X10,

SN

X11,

SN

X12,

and S

NX

22-2

4,

is

char

acte

rize

d b

y t

he

abse

nce

of

addit

ional

sig

nal

ing d

om

ains

(ie.

SH

2,

SH

3,

pro

line-

rich

) outs

ide

of

the

PX

dom

ain.

The

final

sub

-cla

ss o

f so

rtin

g n

exin

s is

char

acte

rize

d

by t

he

pre

sence

of

var

ious

pro

tein

-pro

tein

inte

ract

ion m

oti

fs s

uch

as

SH

3 d

om

ains

[10,

11],

mem

bra

ne-

targ

etin

g d

om

ains

incl

udin

g h

ydro

phobic

seq

uen

ces

[12,

13],

and G

114

pro

tein

reg

ula

tory

seq

uen

ces

[14,

15].

S

H3P

X1 f

alls

into

this

last

sub

-cla

ss o

f so

rtin

g

nex

ins.

SH

3P

X1,

also

ref

erre

d t

o a

s so

rtin

g n

exin

9 (

SN

X9),

is

a 77-k

Da

pro

tein

, w

ith

an

amin

o-t

erm

inal

S

H3

dom

ain,

foll

ow

ed

by

its

PX

dom

ain,

and

a

Bin

/am

phip

hysi

n/R

vs

(BA

R)

moti

f at

the c

arboxy

l-te

rmin

us.

B

AR

moti

fs h

ave b

een

iden

tifi

ed i

n a

num

ber

of

scaf

fold

pro

tein

s an

d c

onsi

st o

f a

curv

ed d

imer

wit

h p

osi

tive

char

ges

dis

trib

ute

d al

ong th

e m

embra

ne-

inte

ract

ing co

nca

ve fa

ce.

T

hese

dom

ain

s

com

monly

show

up i

n t

andem

wit

h P

X o

r P

H d

om

ains,

and b

ecau

se t

hey

exis

t as

curv

ed,

alpha-

hel

ical

bundle

s, t

hey

are

sen

siti

ve

to m

embra

ne

curv

ature

, pre

ferr

ing

hig

hly

curv

ed m

embra

nes

[16,

17].

O

ne

exam

ple

of

this

pre

fere

nce

is

seen

in t

he

abil

ity o

f th

e G

TP

ase

acti

vat

ing p

rote

ins

(GA

Ps)

, oli

gophre

nin

and c

enta

uri

n"

2,

to

bin

d t

o l

iposo

mes

wit

h s

pec

ific

mem

bra

ne

curv

ature

thro

ugh t

hei

r re

spec

tive

BA

R

dom

ains

[16].

In

SH

3P

X1,

the

BA

R m

oti

f has

no

t only

bee

n s

ugges

ted t

o t

raff

ic t

his

pro

tein

to t

he

pla

sma

mem

bra

ne,

but

is a

lso t

hought

to p

rom

ote

the

dim

eriz

atio

n o

f

this

so

rtin

g nex

in w

ith it

self

an

d pote

nti

ally

oth

er m

ember

s of

the

sort

ing nex

in

fam

ily.

The

sort

ing a

nd t

raff

ickin

g o

f ce

ll s

urf

ace

rece

pto

rs f

rom

the

pla

sma

mem

bra

ne

is

a ro

le

that

m

ember

s of

the

sort

ing

nex

in

fam

ily

hav

e bee

n

impli

cate

d

in.

Tra

dit

ional

ly,

the

com

ple

x p

roce

ss o

f re

cepto

r en

docy

tosi

s in

volv

es

sever

al p

rote

ins,

whic

h w

ork

in c

on

cert

to d

eform

the m

embra

ne

into

a v

esi

cula

r bu

d t

hat

ult

imat

ely

pin

ches

off

to f

orm

a f

ree

clat

hri

n-c

oat

ed v

esi

cle.

T

he

core

pro

tein

s in

volv

ed i

n t

his

endocy

tic

pro

cess

in

clude

the

adap

tor

pro

tein

, A

P-2

, cl

athri

n,

and

the

GT

Pas

e,

dynam

in.

Upon r

ecru

itm

ent

to t

he

pla

sma

mem

bra

ne,

AP

-2 s

erves

to c

once

ntr

ate t

he

carg

o i

n t

he

emer

gin

g b

ud,

as w

ell

as t

o l

ink c

lath

rin t

o t

he

pla

sma

mem

bra

ne

[18-2

1].

Once

the

asse

mbly

of

clat

hri

n h

as b

een

init

iate

d a

t th

e em

ergin

g b

ud,

the

mem

bra

ne

115

acquir

es in

crea

sing

curv

ature

unti

l an

in

vag

inat

ed pit

fo

rms.

A

s th

e cl

athri

n pit

mat

ure

s, t

he

final

ste

p, fi

ssio

n, re

quir

es t

he

acti

on o

f th

e G

TP

ase,

dynam

in [

22].

Dynam

in,

a 100-k

Da

pro

tein

, is

a

rath

er

uniq

ue

mem

ber

of

the

GT

Pas

e

super

fam

ily d

ue

to i

ts l

ow

aff

init

y f

or

GT

P (

~30 µ

M)

and h

igh i

ntr

insi

c ra

te o

f G

TP

hydro

lysi

s.

Dynam

in i

s re

cruit

ed t

o t

he

cell

surf

ace

wher

e G

DP

-GT

P e

xch

ange

occ

urs

and s

om

ehow

tri

gger

s dynam

in a

ssem

bly

into

a h

elic

al c

oll

ar a

round t

he

nec

k o

f th

e

clat

hri

n-c

oat

ed p

it.

The

hydro

lysi

s of

GT

P-b

ound d

ynam

in t

ighte

ns

the

dynam

in-

asse

mb

led h

elic

al c

oll

ar u

nti

l a

free

cla

thri

n-c

oat

ed v

esic

le i

s gen

erat

ed.

Curr

entl

y,

the

mec

han

ism

for

dynam

in r

ecru

itm

ent

to t

he

nec

k o

f cl

athri

n-c

oat

ed p

its

is n

ot

wel

l

under

stood.

What

is

clea

r is

that

the

inte

ract

ion b

etw

een t

he

PH

dom

ain o

f dynam

in

and phosp

hat

idyli

nosi

tides

in

th

e pla

sma

mem

bra

ne

is not

suff

icie

nt

to re

cruit

th

e

GT

Pas

e to

the

cell

surf

ace

[23

].

Rat

her

, it

is

thought

that

oth

er c

yto

soli

c pro

tein

s m

ust

be

resp

onsi

ble

for

the

recr

uit

men

t of

dynam

in t

o t

he

site

s of

emer

gin

g b

uds.

SH

3P

X1 w

as r

ecen

tly i

den

tifi

ed a

s a

bin

din

g p

artn

er f

or

dynam

in-2

in K

562

hum

an er

yth

role

ukem

ia ce

lls

[24].

T

he

bin

din

g bet

wee

n dynam

in-2

an

d S

H3P

X1

occ

urs

thro

ugh t

he

SH

3 d

om

ain o

f th

e so

rtin

g n

exin

and t

he

pro

line-

rich

reg

ion o

f

dynam

in,

since

a

poin

t m

uta

tion

in

the

SH

3

dom

ain

of

SH

3P

X1

aboli

shes

the

inte

ract

ion

wit

h

dynam

in-2

.

Not

only

does

S

H3P

X1

bin

d

to

dynam

in-2

, but

incr

easi

ng

evid

ence

al

so

sugges

ts

that

dynam

in

may

be

traf

fick

ed

to

the

pla

sma

mem

bra

ne

by S

H3P

X1.

This

was

dem

onst

rate

d u

sing a

rec

onst

ituti

on a

ssay

in w

hic

h

liposo

mes

mim

ickin

g t

he

pla

sma

mem

bra

ne,

wer

e in

cubat

ed w

ith l

ysa

tes

from

cel

ls

that

wer

e dep

lete

d o

f S

H3P

X1 u

sing R

NA

i.

The

resu

lts

show

ed t

hat

dynam

in-2

co

uld

asso

ciat

e w

ith

the

liposo

mes

only

w

hen

S

H3

PX

1

was

pre

sent

in

the

cyto

sol.

How

ever

, if

rec

om

bin

ant

SH

3P

X1 w

as a

dded

back

to t

he

SH

3P

X1

-dep

lete

d c

yto

sol,

the

dynam

in-l

iposo

me

inte

ract

ion c

ould

be

rest

ore

d [

25].

A

noth

er l

ine

of

evid

ence

support

ing

a ro

le

for

SH

3P

X1

in

traf

fick

ing

dynam

in-2

co

mes

fr

om

116

imm

unofl

uore

scen

ce s

tudie

s th

at s

ho

wed

that

dy

nam

in-2

was

mis

loca

lized

fro

m t

he

pla

sma

mem

bra

ne

in H

eLa

cell

s ex

pre

ssin

g S

H3

PX

1-R

NA

i.

Tak

en t

oget

her

, th

ese

dat

a ar

gue

for

a cr

itic

al r

ole

for

SH

3P

X1 i

n m

edia

ting e

ndocy

tosi

s.

Tyro

sine

phosp

hory

lati

on of

SH

3P

X1 m

ay cr

uci

al in

th

is tr

affi

ckin

g ev

ent,

wher

e S

H3P

X1 an

d dynam

in-2

dem

onst

rate

th

e gre

ates

t bin

din

g to

pre

par

ed

cell

mem

bra

nes

in t

he

pre

sence

of

AT

P,

GT

P#S

, ort

hovan

adat

e at

37°C

. T

he

nec

essi

ty o

f

AT

P an

d ort

hovan

adat

e ar

gues

fo

r th

e si

gnif

ican

ce of

tyro

sine

phosp

hory

lati

on of

SH

3P

X1 i

n t

arget

ing d

ynam

in-2

to t

he

pla

sma

mem

bra

ne.

In

dee

d,

a pre

ceden

t has

alre

ady

bee

n

esta

bli

shed

fo

r th

e im

port

ance

of

SH

3P

X1

phosp

hory

lati

on

in

Dro

sophil

a,

resu

ltin

g i

n d

iver

gin

g s

ignal

ing p

ath

way

s.

Tyro

sine

phosp

hory

lati

on o

f

Dro

sophil

a S

H3P

X1 (

DS

H3

PX

1)

has

bee

n s

how

n t

o p

rom

ote

bin

din

g t

o t

he

adap

tor

pro

tein

, D

ock

, over

that

of

Wis

cott

-Ald

rich

syndro

me

pro

tein

(W

AS

P),

the

pre

ferr

ed

bin

din

g p

artn

er o

f unphosp

hory

late

d S

H3P

X1.

Whil

e, t

he

role

of

SH

3P

X1 i

n d

ynam

in t

raff

ickin

g i

s sl

ow

ly c

om

ing t

o l

ight,

ther

e re

mai

n

a num

ber

of

ques

tion

s to

ad

dre

ss.

N

amel

y,

iden

tify

ing

the

phosp

hory

lati

on s

ites

on t

he

sort

ing n

exin

res

ponsi

ble

for

the

regula

tion o

f dynam

in

and d

evel

opin

g a

syst

em i

n w

hic

h t

hes

e si

tes

can b

e m

uta

ted a

nd u

sed i

n e

xper

imen

ts

to

furt

her

el

uci

dat

e how

th

e phosp

hory

lati

on

infl

uen

ces

the

pro

cess

of

rece

pto

r

endocy

tosi

s.

As

a fi

rst

step

in d

evel

opin

g t

his

exper

imen

tal

syst

em,

we

set

out

to

iden

tify

kin

ases

that

are

resp

onsi

ble

for

the

pho

sphory

lati

on o

f S

H3P

X1.

To t

his

end,

we

hav

e id

enti

fied

tw

o n

ovel

kin

ases,

FA

K a

nd S

rc,

that

can

robust

ly p

hosp

hory

late

SH

3P

X1.

Conse

quen

tly,

we

focu

sed o

n c

har

acte

rizi

ng t

he

inte

rpla

y b

etw

een F

AK

,

Src

and S

H3P

X1,

map

pin

g t

he

tyro

sine

phosp

hory

lati

on s

ites

on t

he

sort

ing n

exin

, an

d

com

par

ing t

he

pat

tern

s of

ph

osp

hory

lati

on b

etw

een F

AK

and S

rc, an

d A

CK

2.

117

Mate

rials

an

d M

eth

od

s

Mate

rials

—A

nti

-HA

an

d

anti

-c-M

yc

anti

bodie

s w

ere

from

C

ovan

ce,

anti

-

phosp

hoty

rosi

ne

(4G

10)

anti

body

and

reco

mbin

ant

His

-Src

w

ere

from

U

pst

ate

Bio

tech

nolo

gy,

Inc.

, an

ti-H

is

poly

clonal

an

tibody

was

fr

om

C

ell

Sig

nal

ing

Tec

hnolo

gy,

and a

nti

-dynam

in a

nti

body w

as f

rom

BD

Tra

nsd

uct

ion L

abora

tori

esT

M.

T4 D

NA

lig

ase,

Lip

ofe

ctam

ine

Tra

nsf

ecti

on R

eagen

t, P

rote

in-G

agar

ose

bea

ds,

anti

-

V5

anti

body,

and

the

Bac

-to-B

ac

Bac

ulo

vir

us

Expre

ssio

n

Syst

em

wer

e fr

om

Invit

rogen

. T

he

Quik

Chan

ge

Sit

e-D

irec

ted M

uta

gen

esis

kit

was

fro

m S

trat

agen

e an

d

the

Qia

quic

k G

el E

xtr

acti

on k

it w

as f

rom

Qia

gen

. R

ecom

bin

ant

His

-FA

K w

as f

rom

Act

ive

Moti

f an

d [

32P

]-A

TP

was

fro

m P

erkin

Elm

er.

Con

stru

ctio

n

of

SH

3P

X1

Del

etio

n

Mu

tan

ts—

The

pcD

NA

3-S

H3P

X1

wil

d-t

ype

pla

smid

was

use

d a

s a t

empla

te t

o c

onst

ruct

the c

arboxyl-

term

inal

del

etio

n m

uta

nts

.

Bri

efly

, D

NA

pri

mer

s to

gen

erat

e ea

ch d

elet

ion

const

ruct

(S

H3P

X1

-!C

84, !

C197,

!C

339, !

C395, !

C547)

wer

e en

gin

eere

d w

ith B

amH

1 a

nd E

coR

1 r

estr

icti

on s

ites

,

and t

hen

thes

e D

NA

pri

mer

s w

ere

use

d i

n a

PC

R r

eact

ion (

PC

R S

pri

nt,

Hybai

d).

The

PC

R

pro

duct

s w

ere

ligat

ed

into

th

e pC

R2

-TO

PO

vec

tor,

fo

llow

ed

by

rest

rict

ion

dig

ests

wit

h B

amH

1 a

nd E

coR

1.

Thes

e D

NA

pro

duct

s w

ere

reso

lved

on a

1%

agar

ose

gel

conta

inin

g 8

0 n

g/m

L e

thid

ium

bro

mid

e, a

nd t

hen

the

DN

A i

nse

rts

wer

e ex

cise

d

from

the

gel

and p

uri

fied

wit

h t

he

Qia

quic

k G

el E

xtr

acti

on k

it.

The

resu

ltin

g D

NA

inse

rts

wer

e li

gat

ed i

nto

the

HA

-tag

ged

pcD

NA

3 e

xpre

ssio

n v

ecto

r usi

ng T

4 D

NA

ligas

e. C

onst

ruct

ion o

f S

H3P

X1 P

oin

t M

uta

nts

—S

H3P

X1 t

yro

sine-

to-p

hen

yla

lanin

e

poin

t m

uta

nts

w

ere

also

gen

erat

ed on a

pcD

NA

3-S

H3P

X1 w

ild-t

ype

bac

kgro

und.

Muta

nts

Y

177F

, Y

239F

, Y

269F

, Y

294F

, Y

561F

, as

w

ell

as

quin

tuple

m

uta

nt,

F177/F

239/F

269/F

294/F

561,

wer

e gen

erat

ed

by

PC

R

usi

ng

the

Quik

Chan

ge

Sit

e-

Dir

ecte

d M

uta

gen

esis

kit

fro

m S

trat

agen

e.

118

SH

3P

X1 w

ild-t

ype

and p

oin

t m

uta

nts

wer

e su

bcl

oned

into

the

V5-p

cDN

A 3

.1

vec

tor

usi

ng t

he

Dir

ecti

onal

TO

PO

Expre

ssio

n kit

fro

m I

nvit

rogen

. P

rim

ers

wer

e

des

igned

bas

ed

on

the

man

ufa

cture

r’s

inst

ruct

ions

and

SH

3P

X1

wil

d-t

ype

and

tyro

sine-

to-p

hen

yla

lanin

e m

uta

nt

DN

A i

nse

rts

were

gen

erat

ed b

y P

CR

usi

ng t

he H

A-

tagged

pcD

NA

3 S

H3P

X1

co

nst

ruct

s as

bac

kgro

und.

The

blu

nt-

end P

CR

pro

duct

s

wer

e re

solv

ed o

n a

1%

agar

ose

gel

conta

inin

g 8

0 n

g/m

L e

thid

ium

bro

mid

e, a

nd t

hen

the

DN

A

inse

rts

wer

e ex

cise

d

from

th

e gel

an

d

puri

fied

w

ith

the

Qia

quic

k

Gel

Extr

acti

on k

it.

The

resu

ltin

g D

NA

inse

rts

wer

e li

gat

ed i

nto

the

pcD

NA

3.1

/V5-H

is-

TO

PO

pla

smid

.

Cel

l C

ult

ure

, T

ran

sfec

tion

an

d P

rep

ara

tion

of

Lysa

tes—

CO

S-7

and H

EK

29

3 c

ells

wer

e cu

lture

d i

n D

ulb

ecco

’s m

odif

ied E

agle

’s m

ediu

m (

DM

EM

) co

nta

inin

g 1

0%

fet

al

bovin

e se

rum

. T

he

cel

l li

nes

wer

e m

ainta

ined

in

5%

CO

2 a

t 37°C

. T

o e

xpre

ss t

he

var

ious

form

s of

SH

3P

X1,

FA

K,

and S

rc i

n c

ells

, M

yc-

, H

A-,

or

V5-t

agged

const

ruct

s

enco

din

g t

he

pro

tein

s w

ere

tran

sfec

ted i

nto

cel

ls u

sing L

ipofe

ctam

ine

Tra

nsf

ecti

on

Rea

gen

t ac

cord

ing t

o t

he

man

ufa

cture

r’s

inst

ruct

ions

(Invit

rogen

).

Tw

enty

-four

hours

foll

ow

ing t

ransf

ecti

on,

the

cell

s w

ere

rinse

d w

ith p

hosp

hat

e-buff

ered

sal

ine

(PB

S)

and

lyse

d w

ith c

old

mam

mal

ian c

ell

lysi

s buff

er (

10 m

M T

ris

(pH

7.4

), 5

mM

MgC

l 2,

150

mM

N

aCl,

1%

T

rito

n

X-1

00,

1

mM

so

diu

m

ort

hovan

adat

e,

5

mM

bet

a-gly

cero

l

phosp

hat

e, 1

0 µ

g/m

L a

pro

tin

in,

10 µ

g/m

L l

eupep

tin)

foll

ow

ed b

y c

ell

scra

pin

g.

The

cell

lysa

tes

wer

e t

hen

cle

ared

by

cen

trif

ugat

ion a

t 16,1

00 r

pm

in a

mic

rofu

ge

for

12

min

ute

s.

Pro

tein

conce

ntr

atio

ns

wer

e det

erm

ined

by d

iluti

ng t

he

lysa

tes

1:2

50 w

ith 1

x

Bra

dfo

rd r

eagen

t.

Imm

un

op

reci

pit

ati

on

—C

ell

lysa

tes

wer

e in

cubate

d w

ith M

yc,

HA

, or

V5

anti

body

for

2-2

0 h

ours

, w

ith r

ota

tion a

t 4°C

. P

rote

in-G

agar

ose

bea

ds

wer

e ad

ded

, ro

tati

ng

for

an a

ddit

ional

hour.

T

he

bea

ds

wer

e th

en p

reci

pit

ated

in a

mic

rofu

ge

and w

ashed

3-4

119

tim

es w

ith c

old

mam

mal

ian c

ell

lysi

s buff

er.

They

wer

e th

en r

esusp

ended

in 5

x S

DS

load

ing b

uff

er a

nd t

he

asso

ciat

ed p

rote

ins

wer

e re

solv

ed b

y S

DS

-PA

GE

.

Mass

Sp

ectr

om

etry

An

aly

sis—

HE

K 2

93 c

ells

wer

e tr

ansf

ecte

d w

ith H

A-S

rc a

nd V

5-

SH

3P

X1.

The

cell

s w

ere

lyse

d a

fter

24 h

ours

in c

old

mam

mal

ian c

ell

lysi

s buff

er (

10

mM

Tri

s (p

H 7

.4),

5 m

M M

gC

l 2,

150 m

M N

aC

l, 1

% T

rito

n X

-100,

1 m

M s

odiu

m

ort

hovan

adat

e,

5

mM

bet

a-gly

cero

l phosp

hat

e,

10 µ

g/m

L

apro

tinin

, 10 µ

g/m

L

leupep

tin)

and V

5-S

H3P

X1 w

as

imm

unopre

cipit

ated

fro

m t

he

pre

par

ed l

ysa

tes

(~10

mg t

ota

l pro

tein

) w

ith a

nti

-V5

anti

body.

The

pre

cip

itat

ed s

ample

s w

ere

boil

ed,

run o

n

an S

DS

-PA

GE

gel

, st

ained

wit

h C

oom

assi

e, a

nd e

xci

sed.

Ali

quots

fro

m t

he

sam

ple

s

wer

e se

t as

ide a

nd t

yro

sine

phosp

hory

lati

on w

as

confi

rmed

by W

este

rn b

lott

ing.

The

sam

ple

s w

ere

then

su

bm

itte

d

to

the

Corn

ell

Bio

tech

nolo

gy

Reso

urc

e C

ente

r

Pro

teom

ics

and M

ass

Spec

trom

etry

Core

Fac

ilit

y.

Rec

om

bin

an

t P

rote

in E

xp

ress

ion

an

d P

uri

fica

tion

—E

xpre

ssio

n of

His

-SH

3P

X1

was

car

ried

out

in t

he

BL

21 E

. co

li s

trai

n.

The

cDN

A e

nco

din

g w

ild

-type

SH

3P

X1

was

clo

ned

in

to t

he

pE

T28A

vec

tor

for

reco

mbin

ant

expre

ssio

n.

Over

nig

ht

cult

ure

s

from

colo

nie

s of

BL

21 c

ells

(N

ovag

en)

tran

sform

ed w

ith t

he

expre

ssio

n v

ect

or

wer

e

gro

wn

at

37°C

. T

hes

e w

ere

use

d

to

inocu

late

one-

lite

r cu

lture

s of

super

bro

th,

enri

ched

bac

teri

al m

edia

. B

acte

rial

cult

ure

s w

ere

gro

wn t

o a

n O

D6

00 o

f 0.8

at

37°C

and i

nduce

d w

ith 2

00 µ

M I

PT

G o

ver

nig

ht

at r

oom

tem

per

ature

. C

ells

wer

e har

vest

ed

by c

entr

ifugat

ion a

t 4000 r

pm

for

10 m

inute

s an

d f

roze

n a

t !

80°C

. A

ll s

ubse

quen

t

puri

fica

tion s

teps

wer

e c

arri

ed o

ut

at 4

°C.

Bac

teri

al p

elle

ts w

ere

resu

spen

ded

in

Ni2

+

bin

din

g

buff

er

(20

mM

T

ris

(pH

7.9

),

500

mM

N

aCl,

20

mM

im

idaz

ole

, 10%

gly

cero

l) su

pple

men

ted w

ith pro

teas

e in

hib

itors

(1 m

M P

MS

F,

10 µ

g/m

L ea

ch of

apro

tinin

and l

eupep

tin,

10

µM

ben

zam

idin

e) a

nd l

yse

d b

y t

hre

e pas

sag

es t

hro

ugh a

Fre

nch

Pre

ssu

re C

ell.

T

he

extr

acts

wer

e th

en s

onic

ated

for

5 m

inute

s an

d t

he

lysa

tes

wer

e cl

arif

ied by ult

race

ntr

ifugat

ion at

40,0

00 rp

m fo

r 45 m

inute

s. T

he

clar

ifie

d

120

lysa

tes

wer

e in

cubat

ed f

or

30

min

ute

s w

ith N

i2+ b

ead

s (A

mer

sham

) at

4°C

. T

he

bea

ds

wer

e th

en w

ash

ed w

ith

bin

din

g b

uff

er a

nd t

he p

rote

in r

ecover

ed w

ith e

luti

on b

uff

er

(20 m

M T

ris

(pH

7.9

), 5

00 m

M N

aCl,

200 m

M i

mid

azole

, 10%

gly

cero

l).

Sam

ple

s

from

th

e pro

tein

puri

fica

tion

w

ere

then

re

solv

ed by S

DS

-PA

GE

an

d st

ained

w

ith

Coom

assi

e blu

e.

In

Vit

ro

Tyro

sin

e K

inase

Ass

ay—

In v

itro

tyro

sine

phosp

hory

lati

on o

f S

H3P

X1 w

as

det

erm

ined

by

incu

bat

ing

50

ng

reco

mbin

ant

His

-Src

or

His

-FA

K

wit

h

500

ng

bac

teri

al p

uri

fied

His

-SH

3P

X1 i

n k

inas

e re

acti

on b

uff

er (

50 m

M T

ris-

HC

l (p

H 7

.4),

2

mM

MgC

l 2,

10 m

M M

nC

l 2"#,

1 #µ

M A

TP

, 10 µ

Ci

[32P

]-A

TP

).

Rea

ctio

ns

wer

e ca

rrie

d

out

at

room

te

mper

ature

fo

r var

ious

leng

ths

of

tim

e (5

-120

min

ute

s),

and

then

quen

ched

wit

h t

he

addit

ion o

f 5x S

DS

load

ing b

uff

er.

The

reac

tion s

ample

s w

ere

boil

ed,

reso

lved

by S

DS

-PA

GE

, an

d th

e gel

st

ain

ed w

ith C

oom

ass

ie.

T

he

ban

ds

corr

espondin

g t

o S

H3P

X1 w

ere

exci

sed a

nd t

he

exte

nt

of

phosp

hory

lati

on w

as r

ead

out

by

det

erm

inin

g

the

amo

unt

of

[32P

]-A

TP

in

corp

ora

ted

into

S

H3P

X1

usi

ng

a

scin

till

atio

n c

ounte

r.

Alt

ernat

ivel

y,

som

e of

the k

inas

e re

act

ions

wer

e r

esolv

ed b

y

SD

S-P

AG

E g

el a

nd t

ransf

erre

d t

o P

VD

F m

embra

nes

. T

he

exte

nt

of

phosp

hory

lati

on

was

det

erm

ined

by e

xposi

ng t

he

mem

bra

nes

to x

-ray

fil

m.

Res

ult

s Tyro

sine

phosp

hory

lati

on e

ven

ts a

re a

hal

lmar

k o

f ce

llula

r si

gnal

ing a

ctiv

itie

s

and p

lay a

n i

nte

gra

l ro

le i

n m

edia

ting n

earl

y a

ll c

ellu

lar

pro

cess

es.

SH

3P

X1 i

s a

pro

tein

that

has

bee

n l

inked

to t

he

endocy

tosi

s and s

ort

ing o

f ce

ll s

urf

ace

rece

pto

rs.

Stu

die

s hav

e dem

on

stra

ted t

hat

SH

3P

X1 i

s ty

rosi

ne

phosp

hory

late

d b

y A

CK

2 a

nd t

hat

this

phophory

lati

on

even

t is

im

port

ant

for

SH

3P

X1

to

med

iate

E

GF

re

cepto

r

endocy

tosi

s [2

9].

H

ere,

w

e hav

e se

t out

to det

erm

ine

whet

her

ad

dit

ional

pro

tein

121

kin

ases

ca

taly

ze

the

phosp

hory

lati

on

of

SH

3P

X1,

and

if

so,

do

thes

e kin

ases

phosp

hory

late

the

sam

e ty

rosi

ne

resi

dues

on S

H3P

X1 a

s A

CK

2 d

oes

.

We

chose

to

ex

amin

e w

het

her

F

AK

m

ight

phosp

hory

late

S

H3P

X1 fo

r tw

o

reas

ons.

F

irst

, F

AK

is

close

ly r

elat

ed t

o t

he

tyro

sin

e fa

mil

y o

f A

CK

s.

The

seco

nd

reas

on

is

base

d

on

pre

vio

us

findin

gs

whic

h

show

th

at

FA

K

can

pho

sphory

late

endophil

in,

a pro

tein

that

shar

es f

unct

ional

dom

ains

wit

h S

H3P

X1 a

nd p

lays

a ro

le i

n

endocy

tosi

s an

d t

raff

ickin

g o

f ce

ll s

urf

ace

rece

pto

rs.

Giv

en t

hat

Src

bin

ds

to F

AK

and

oft

en si

gnal

s in

ta

ndem

w

ith it

, w

e al

so ex

amin

ed th

e poss

ibil

ity th

at S

rc co

uld

phosp

hory

late

S

H3P

X1.

In

th

is

chap

ter,

w

e pro

vid

e ev

iden

ce

that

F

AK

an

d

its

signal

ing

par

tner

, S

rc,

can

phosp

hory

late

S

H3P

X1

on

tyro

sine

resi

dues

th

at

are

dis

tinct

fro

m t

hose

phosp

hory

late

d b

y A

CK

2.

FA

K P

hosp

hory

late

s S

H3P

X1 I

n v

ivo

In o

ur

pre

vio

us

work

, w

e hav

e sh

ow

n t

hat

AC

K2

can

phosp

hory

late

SH

3P

X1

both

in

vi

vo an

d in

vi

tro.

U

sing a

sim

ilar

ap

pro

ach,

we

exam

ined

w

het

her

F

AK

phosp

hory

late

s S

H3P

X1.

W

e fi

rst

co-t

ransf

ecte

d C

OS

-7 ce

lls

wit

h S

H3P

X1 an

d

eith

er w

ild-t

ype

FA

K,

the

kin

ase-

def

icie

nt

FA

K-Y

397F

muta

nt

or

wil

d-t

ype

AC

K2.

The

foll

ow

ing d

ay,

the

cell

s w

ere

pla

ced i

n m

ediu

m w

ithout

seru

m f

or

24 h

ours

, an

d

the

des

ignat

ed s

ample

s w

ere

stim

ula

ted w

ith c

om

ple

te m

edia

(co

nta

inin

g 1

0%

FB

S)

for

20

min

ute

s.

A

ll

of

the

cell

cu

lture

s w

ere

then

ly

sed

and

SH

3P

X1

was

imm

unopre

cipit

ated

fr

om

th

e w

hole

ce

ll

extr

acts

w

ith

anti

-V5

anti

body.

T

he

imm

unopre

cipit

ated

sa

mple

s w

ere

subje

cted

to

W

este

rn

blo

t an

alysi

s w

ith

an

ti-

phosp

hoty

rosi

ne

anti

body.

Fig

ure

3.1

show

s th

at w

hen

SH

3P

X1 w

as e

xpre

ssed

alo

ne

in C

OS

-7 c

ells

, no p

hosp

hory

lati

on w

as d

etec

ted (

Fig

ure

3.1

, la

ne

3).

C

onsi

sten

t w

ith

pre

vio

us

findin

gs,

SH

3P

X1 s

how

ed a

sli

ght,

but

consi

sten

t pho

sphory

lati

on w

hen

co-

expre

ssed

wit

h A

CK

2 (

Fig

ure

3.1

, la

ne

4).

T

his

phosp

hory

lati

on w

as n

ot

det

ecte

d

122

Fig

ure

3.1

F

AK

ph

osp

hory

late

s S

H3P

X1 i

n c

ells

.

CO

S-7

cel

ls w

ere

tran

sfec

ted w

ith t

he

var

ious

expre

ssio

n p

lasm

ids

indic

ated

. T

wen

ty-

four

to f

ort

y-e

ight

hours

aft

er t

ransf

ecti

on,

the

cell

s w

ere

pla

ced

in m

ediu

m c

onta

inin

g

1%

FB

S f

or

an a

ddit

ional

day

. J

ust

pri

or

to c

ell

lysi

s, s

om

e of

the

cell

cult

ure

s w

ere

stim

ula

ted w

ith m

ediu

m c

onta

inin

g 1

0%

FB

S f

or

20 m

inute

s.

V5-t

agged

SH

3P

X1

was

im

munopre

cipit

ated

w

ith an

ti-V

5 an

tibody an

d th

e pre

cipit

ated

sa

mple

s w

ere

reso

lved

by

SD

S-P

AG

E

and

then

su

bje

cted

to

W

este

rn

blo

ttin

g

wit

h

anti

-

phosp

hoty

rosi

ne

anti

body a

nd a

nti

-V5 a

nti

body.

123

124

when

SH

3P

X1 w

as e

xpre

ssed

wit

h a

kin

ase-

def

ecti

ve

form

of

AC

K2,

AC

K2-K

158R

(Fig

ure

3.1

, la

ne

5).

H

ow

ever

, w

hen

FA

K w

as c

o-e

xpre

ssed

wit

h S

H3P

X1,

a ro

bust

tyro

sine

phosp

hory

lati

on o

f S

H3P

X1 w

as d

etec

ted (

Fig

ure

3.1

, co

mpar

e la

ne

4 t

o l

ane

6).

W

hen

quan

tita

ted,

the

exte

nt

of

SH

3P

X1 p

ho

sphory

lati

on b

y F

AK

was

more

than

10 f

old

gre

ater

than

that

of

AC

K2.

Expre

ssio

n o

f th

e kin

ase-

def

ecti

ve

FA

K-Y

397F

muta

nt

did

not

resu

lt

in

the

tyro

sine

phosp

hory

lati

on

of

SH

3P

X1,

even

under

condit

ions

wher

e th

e se

rum

-sta

rved

sam

ple

s w

ere

stim

ula

ted w

ith m

ediu

m c

onta

inin

g

10%

FB

S (

Fig

ure

3.1

, la

nes

8 a

nd 9

).

Tak

en t

oget

her

, th

ese

findin

gs

indic

ate

that

incr

ease

s in

FA

K a

ctiv

ity i

n c

ells

lea

ds

to a

n e

xte

nt

of

SH

3P

X1 p

hosp

hory

lati

on t

hat

is s

ignif

ican

tly g

reat

er t

han

that

cat

alyze

d b

y A

CK

2.

We

nex

t as

ked

whet

her

FA

K c

an d

irec

tly p

hosp

hory

late

SH

3P

X1.

In o

rder

to

addre

ss t

his

ques

tion,

we

turn

ed t

o a

n i

n v

itro

kin

ase

assa

y.

His

-tag

ged

SH

3P

X1 w

as

expre

ssed

and p

uri

fied

fro

m b

acte

rial

cel

ls w

her

eas

His

-tag

ged

FA

K w

as

expre

ssed

and p

uri

fied

fro

m i

nse

ct c

ells

. K

inas

e re

acti

ons

wer

e ca

rrie

d o

ut

at r

oom

tem

per

ature

for

up t

o 2

hours

. F

igure

3.2

A s

how

s a

repre

sen

tati

ve

auto

radio

gra

ph o

f th

e kin

ase

assa

y.

The

FA

K-c

atal

yze

d p

hosp

hory

lati

on o

f S

H3P

X1 w

as d

etec

ted a

s ear

ly a

s 15

min

ute

s, w

ith m

axim

al p

hosp

hory

lati

on o

ccurr

ing w

ithin

45 m

inute

s.

The

resu

ltin

g

subst

rate

phosp

hory

lati

on w

as

also

quan

tifi

ed b

y e

xci

sing t

he

SH

3P

X1 b

and

s fr

om

the

gel

s an

d t

hen

dir

ectl

y m

easu

ring [

32P

]-phosp

hat

e in

corp

ora

tion u

sing a

sci

nti

llat

ion

counte

r.

Fig

ure

3.2

B s

how

s th

at F

AK

sti

mula

ted t

he

inco

rpora

tion o

f [3

2P

]-A

TP

into

SH

3P

X1

by

nea

rly

25-f

old

over

co

ntr

ol

cell

s.

T

hes

e fi

ndin

gs

concl

usi

vel

y

dem

onst

rate

that

FA

K c

an d

irec

tly p

hosp

hory

late

SH

3P

X1.

Src

Als

o A

cts

as

a K

inase

for

SH

3P

X1

FA

K a

nd S

rc o

ften

cooper

ate

in c

ells

as

a si

gnal

ing c

om

ple

x.

The

mec

han

ism

for

this

cooper

atio

n h

as b

een w

ell

esta

bli

shed

and i

s in

itia

ted w

ith t

he

125

Fig

ure

3.2

F

AK

ph

osp

hory

late

s S

H3P

X1 i

n v

itro.

Rec

om

bin

ant

FA

K (

8.3

ng

) an

d S

H3P

X1 (

500 n

g)

wer

e ad

ded

to

a k

inas

e r

eact

ion

mix

ture

conta

inin

g 5

0 m

M T

ris-

HC

l (p

H 7

.4),

2 m

M M

gC

l 2,

10 m

M M

nC

l 2,

1 µ

M

AT

P,

10 µ

Ci

[32P

]-A

TP

. T

he

kin

ase

reac

tion w

as

allo

wed

to p

roce

ed f

or

the

indic

ated

tim

e in

terv

als

and t

hen

the

reac

tion

was

quen

ched b

y t

he

addit

ion o

f 5x

SD

S s

ample

buff

er.

SD

S s

ample

buff

er (

5x)

was

added

to t

he

t=0 s

ample

bef

ore

the

addit

ion o

f

kin

ase.

E

ach

kin

ase

reac

tion

was

re

solv

ed

by

SD

S-P

AG

E,

tran

sfer

red

to

PV

DF

mem

bra

ne,

and e

xpose

d t

o x

-ray

fil

m.

The

sam

e b

lot

was

then

pro

bed

wit

h a

nti

-His

anti

body t

o s

how

that

the

amount

of

reco

mbin

ant

SH

3P

X1 w

as p

rese

nt

in e

ach k

inase

reac

tion (

A).

A

lter

nat

ivel

y,

kin

ase

reac

tion m

ixtu

res

wer

e re

solv

ed b

y S

DS

-PA

GE

and s

tain

ed w

ith C

oom

ass

ie b

lue.

B

ands

of

SH

3P

X1 w

ere

exci

sed a

nd t

he e

xte

nt

of

phosp

hory

lati

on w

as r

ead o

ut

by

det

erm

inin

g t

he a

mount

of

[32P

]-A

TP

inco

rpora

ted

into

SH

3P

X1 u

sing a

sci

nti

llat

ion c

ounte

r (B

).

126

127

auto

phosp

hory

lati

on o

f F

AK

at

tyro

sine

397.

This

cre

ates

a b

indin

g s

ite

for

the

SH

2-

dom

ain o

f S

rc [

26].

T

he

bin

din

g o

f S

rc t

o F

AK

all

ow

s S

rc t

o f

urt

her

phosp

hory

late

FA

K a

t ad

dit

ional

sit

es i

ncl

udin

g r

esid

ues

407,

57

6,

577,

861,

and 9

25,

whic

h l

ead

s to

enhan

ced

FA

K

acti

vit

y

and

a

subse

quen

t in

crea

se

in

the

pho

sphory

lati

on

of

dow

nst

ream

eff

ecto

rs [

27]

[26,

28].

Thus,

w

e w

ere

inte

rest

ed

in

sein

g

whet

her

S

rc

mig

ht

also

ca

taly

ze

the

phosp

hory

lati

on o

f S

H3P

X1.

To t

est

this

poss

ibil

ity,

we

took t

he

sam

e ap

pro

ach a

s

we

did

to e

stab

lish

that

FA

K p

hosp

hory

late

s S

H3

PX

1.

HE

K 2

93 c

ells

expre

ssin

g S

rc

and S

H3P

X1 w

ere

lyse

d a

nd

SH

3P

X1 w

as i

mm

unopre

cipit

ated

fro

m t

he

whole

cel

l

lysa

tes

wit

h

anti

-V5

anti

body.

Im

munoblo

t an

alysi

s of

the

imm

unopre

cipit

ated

sam

ple

s w

ith a

nti

-phosp

hoty

rosi

ne

anti

body s

how

ed t

hat

Src

expre

ssio

n r

esult

ed i

n a

pote

nt

phosp

hory

lati

on

of

SH

3P

X1

(Fig

ure

3.3

).

T

he

phosp

hory

lati

on

signal

gen

erat

ed b

y S

rc w

as

signif

ican

tly a

nd

consi

sten

tly e

nhan

ced o

ver

that

obse

rved

wit

h

FA

K (F

igure

3.3

, co

mpar

e la

nes

2 an

d 4).

In

tere

stin

gly

, w

e did

not

obse

rve

an

incr

ease

in

S

H3P

X1

phosp

hory

lati

on

in

sam

ple

s w

her

e S

rc

and

FA

K

wer

e both

expre

ssed

wit

h S

H3P

X1 (

Fig

ure

3.3

, co

mpar

e la

nes

4 a

nd 5

), s

ugges

ting t

hat

Src

can

phosp

hory

late

eac

h o

f th

e si

tes

use

d b

y F

AK

. F

igure

3.3

(la

ne

6)

show

s th

at t

he

kin

ase-

def

icie

nt

FA

K-F

397

muta

nt,

w

as

unab

le

to

blo

ck

Src

-cat

alyze

d

phosp

hory

lati

on o

f S

H3P

X1.

W

e th

en e

xam

ined

whet

her

Src

can

dir

ectl

y p

hosp

hory

late

SH

3P

X1.

Fig

ure

3.4

show

s th

at t

he S

rc-c

atal

yze

d p

ho

sphory

lati

on o

f S

H3P

X1 w

as s

een

as

earl

y a

s 5

min

ute

s, a

nd t

hat

the

phosp

hory

lati

on s

ignal

conti

nued

to i

ncr

ease

thro

ugh 2

hours

.

Th

e P

hosp

hory

lati

on

of

SH

3P

X1 b

y F

AK

an

d S

rc D

iffe

rs f

rom

th

at

of

AC

K2

We

hav

e sh

ow

n

that

A

CK

2-c

atal

yzed

phosp

hory

lati

on

of

SH

3P

X1

is

elim

inat

ed w

hen

84 a

min

o a

cids

from

the

carb

oxyl-

term

inal

end w

ere

rem

oved

. T

his

128

Fig

ure

3.3

S

rc i

s a m

ore

eff

ecti

ve

kin

ase

for

SH

3P

X1 i

n c

ells

.

V5-t

agged

SH

3P

X1 w

as t

ransf

ecte

d a

long w

ith w

ith e

ither

HA

-tag

ged

Src

or

HA

-

tagged

FA

K i

n H

EK

293 c

ells

. A

ppro

xim

atel

y 2

4 h

ours

foll

ow

ing t

ransf

ecti

on,

the

cell

s w

ere

lyse

d.

SH

3P

X1 w

as i

mm

unopre

cipit

ated

wit

h a

nti

-V5 a

nti

body f

rom

cel

l

lysa

tes,

an

d

then

re

solv

ed

by

SD

S-P

AG

E.

T

he

gel

s w

ere

tran

sfer

red

to

PV

DF

mem

bra

ne

and s

uje

cted

to W

este

rn b

lott

ing a

nal

ysi

s w

ith a

nti

-phosp

hoty

rosi

ne

and

anti

-V5 a

nti

bodie

s.

129

130

Fig

ure

3. 4 S

rc p

hosp

hory

late

s S

H3P

X1 i

n v

itro.

Rec

om

bin

ant

Src

(5

0 ng)

and S

H3P

X1 (2

00 ng)

wer

e ad

ded

to

a

kin

ase

reac

tion

mix

ture

conta

inin

g 5

0 m

M T

ris-

HC

l (p

H 7

.4),

2 m

M M

gC

l 2,

10 m

M M

nC

l 2,

1 µ

M

AT

P,

10 µ

Ci

[32P

]-A

TP

. T

he

kin

ase

reac

tion w

as

allo

wed

to p

roce

ed f

or

the

indic

ated

tim

e in

terv

als

and t

hen

the

reac

tion

was

quen

ched b

y t

he

addit

ion o

f 5x

SD

S s

ample

buff

er.

SD

S s

ample

buff

er (

5x)

was

added

to t

he

t=0 s

ample

bef

ore

the

addit

ion o

f

kin

ase.

E

ach

kin

ase

reac

tion

was

re

solv

ed

by

SD

S-P

AG

E,

tran

sfer

red

to

PV

DF

mem

bra

ne,

and e

xpose

d t

o x

-ray

fil

m.

131

132

is l

ikel

y d

ue

to t

he

fact

that

AC

K2 i

s unab

le t

o b

ind t

o t

hes

e m

uta

nts

. A

fter

we

iden

tifi

ed F

AK

and S

rc a

s p

rote

ins

capab

le o

f phosp

hory

lati

ng S

H3P

X1,

we

wer

e

inte

rest

ed i

n c

om

par

ing t

hei

r ab

ilit

y t

o b

ind t

o S

H3P

X1.

Com

bin

atio

ns

of

FA

K,

Src

,

and S

H3P

X1 w

ere

tran

sfec

ted

into

HE

K 2

93 c

ells

, an

d t

hen

the

cell

s w

ere

lyse

d a

nd

FA

K

or

Src

w

as

imm

unopre

cipit

ated

w

ith

anti

-HA

an

tibody.

T

he

imm

unopre

cipit

atio

n s

ample

s w

ere

subje

cted

to W

este

rn b

lott

ing a

nal

ysi

s w

ith a

nti

-

V5 a

nti

body t

o d

etec

t as

soci

ated

SH

3P

X1.

Fig

ure

3.5

show

s th

at d

espit

e a

robust

expre

ssio

n o

f both

FA

K a

nd

Src

, S

H3P

X1 d

id n

ot

co-i

mm

unopre

cipit

ate

wit

h e

ither

pro

tein

. S

ince

w

e hav

e dem

onst

rate

d th

at both

F

AK

an

d S

rc ca

n phosp

hory

late

SH

3P

X1,

but

we

do n

ot

det

ect

an a

ssoci

atio

n b

etw

een F

AK

or

Src

and

SH

3P

X1,

we

can i

nfe

r th

at t

he

kin

ase-

sub

stra

te i

nte

ract

ions

bet

wee

n t

hes

e pro

tein

s ar

e tr

ansi

ent,

whic

h a

llow

for

rapid

enzy

mat

ic t

urn

over

. O

ne

way

of

test

ing t

his

hypoth

esis

would

be

to a

ttem

pt

to t

rap t

he

kin

ase

-sub

stra

te i

nte

ract

ion b

y u

sing k

inase

-def

ecti

ve

muta

nts

of

FA

K a

nd S

rc.

We

then

ask

ed w

het

her

FA

K a

nd S

rc p

hosp

hory

late

d t

he

sam

e si

tes

as A

CK

2.

We

took

advan

tage

of

the

C-t

erm

inal

del

etio

n

const

ruct

s of

SH

3P

X1

that

w

ere

pre

vio

usl

y

use

d

to

inves

tigat

e A

CK

2-c

atal

yze

d

phosp

hory

lati

on

of

SH

3P

X1.

A

schem

atic

of

thes

e c

onst

ruct

s is

show

n i

n F

igure

3.6

A.

All

of

thes

e c

onst

ruct

s, w

hen

co-e

xpre

ssed

alo

ng w

ith A

CK

2 i

n c

ells

, w

ere

able

to c

om

ple

tely

blo

ck t

he

abil

ity o

f

AC

K2 t

o b

ind t

o a

nd p

hosp

hory

late

SH

3P

X1 (

see

Fig

ure

2.4

in C

hap

ter

2).

T

hes

e

resu

lts

sugges

t th

at del

etin

g th

e ca

rboxyl-

term

inal

84 am

ino ac

ids

of

SH

3P

X1 is

suff

icie

nt

to e

lim

inat

e A

CK

2’s

abil

ity t

o p

hosp

hory

late

SH

3P

X1.

Fig

ure

s 3.6

and 3

.7

show

the

resu

lts

of

exper

imen

ts w

her

e F

AK

and S

rc,

resp

ecti

vel

y,

wer

e ex

amin

ed f

or

thei

r ab

ilit

ies

to p

hosp

hory

late

the

var

ious

C-t

erm

inal

tru

nca

tion m

uta

nts

of

SH

3P

X1.

Bri

efly

, th

ese

exper

imen

ts w

ere

carr

ied o

ut

by c

oex

pre

ssin

g F

AK

or

Src

wit

h e

ach

SH

3P

X1 d

elet

ion m

uta

nt,

SH

3P

X1-!

C84, !

C197, an

d !

C340 i

n H

EK

293 c

ells

. T

he

133

Fig

ure

3.5

F

AK

an

d S

rc d

o n

ot

bin

d t

o S

H3P

X1.

Pla

smid

s en

codin

g e

ither

HA

-FA

K o

r H

A-S

rc w

ere

co-t

ransf

ecte

d w

ith V

5-S

H3P

X1

in C

OS

-7 c

ells

. A

ppro

xim

atel

y 4

8 h

ours

foll

ow

ing t

he

tran

sfec

tion,

the

cell

s w

ere

lyse

d.

HA

-tag

ged

FA

K (

A)

or

Src

(B

) w

ere

imm

unopre

cipit

ated

fro

m w

hole

cel

l

lysa

tes

wit

h a

nti

-HA

anti

body

and r

esolv

ed b

y S

DS

-PA

GE

. T

he

gel

s w

ere

tran

sfer

red

to P

VD

F a

nd t

hen

the

blo

t w

as s

ubje

cted

to W

est

ern b

lott

ing a

nal

ysi

s usi

ng a

nti

-V5

anti

body t

o d

etec

t S

H3P

X1 b

indin

g.

134

135

Fig

ure

3.6

F

AK

ph

osp

hory

late

s th

e ca

rboxyl-

term

inal

SH

3P

X1 d

elet

ion

mu

tan

ts

!C

84, !

C197,

an

d !

C340.

A s

chem

atic

rep

rese

nta

tion o

f th

e S

H3P

X1 C

-ter

min

al

del

etio

n c

onst

ruct

s th

at w

ere

use

d i

n t

hese

stu

die

s (A

). T

he

HA

-FA

K p

lasm

id w

as c

o-

tran

sfec

ted w

ith t

he

var

ious

C-t

erm

inal

del

etio

n c

onst

ruct

s of

SH

3P

X1 o

utl

ined

ab

ove.

About

24 h

ours

foll

ow

ing t

ransf

ecti

on,

the

cell

s w

ere

lyse

d a

nd t

he d

elet

ion c

onst

ruct

s

of

SH

3P

X1 w

ere

imm

unop

reci

pit

ated

fro

m t

he

cell

extr

acts

usi

ng a

nti

-Myc

anti

body.

The

sam

ple

s w

ere

reso

lved

by S

DS

-PA

GE

, tr

ansf

erre

d t

o P

VD

F,

and i

mm

unoblo

ting

anal

ysi

s w

ith a

nti

-phosp

hoty

rosi

ne

and a

nti

-Myc

anti

bodie

s w

as p

erfo

rmed

(B

).

136

137

Fig

ure

3.7

S

rc p

hosp

hory

late

s th

e ca

rboxyl-

term

inal

SH

3P

X1 d

elet

ion

mu

tan

ts

!C

84, !

C197,

an

d !

C340.

A s

chem

atic

rep

rese

nta

tion o

f th

e S

H3

PX

1 C

-ter

min

al

del

etio

n c

onst

ruct

s gen

erat

ed (

A).

The

HA

-Src

pla

smid

was

co-t

ransf

ecte

d w

ith t

he

var

ious

C-t

erm

inal

del

etio

n

const

ruct

s of

SH

3P

X1.

A

ppro

xim

atel

y

24

hours

foll

ow

ing t

ransf

ecti

on,

the

cell

s w

ere

lyse

d a

nd

the

del

etio

n c

on

stru

cts

of

SH

3P

X1

wer

e im

munopre

cipit

ated

fr

om

th

e ce

ll

extr

acts

usi

ng

anti

-Myc

anti

body.

T

he

sam

ple

s w

ere

reso

lved

b

y

SD

S-P

AG

E,

tran

sfer

red

to

PV

DF

an

d

imm

unoblo

ting

anal

ysi

s w

ith a

nti

-phosp

hoty

rosi

ne

and a

nti

-Myc

anti

bodie

s w

as p

erfo

rmed

(B

).

138

139

cell

s w

ere

then

lyse

d a

nd t

he

SH

3P

X1 m

uta

nts

wer

e im

munopre

cipit

ated

wit

h a

nti

-HA

anti

body

and

anal

yze

d

by

Wes

tern

blo

ttin

g

wit

h

anti

-phosp

hoty

rosi

ne

anti

body.

Contr

ary t

o w

hat

was

ob

serv

ed w

ith A

CK

2,

we

found t

hat

FA

K (

Fig

ure

3.6

B)

and S

rc

(Fig

ure

3.7

B)

not

only

phosp

hory

late

d t

he

SH

3P

X1-!

C84 c

onst

ruct

, but

they

ret

ained

the

abil

ity t

o p

hosp

hory

late

all

of

the S

H3P

X1 C

-ter

min

al d

elet

ion m

uta

nts

. T

hus,

the

dat

a su

ggest

th

at F

AK

an

d S

rc li

kel

y pho

sphory

late

dis

tinct

re

sidues

on

S

H3P

X1

rela

tive

to A

CK

2.

Tyro

sin

e R

esid

ues

177,

23

9,

269,

294,

an

d 5

61 a

re P

hosp

hory

late

d I

n v

ivo b

y S

rc

Our

labora

tory

an

d

oth

ers

hav

e sh

ow

n

that

ty

rosi

ne

phosp

hory

lati

on

of

SH

3P

X1

has

co

nse

quen

tial

ef

fect

s on

EG

F

rece

pto

r pro

cess

ing

and

dynam

in

traf

fick

ing [

25,

29].

In

par

ticu

lar,

AC

K2

-cat

alyze

d p

hosp

hory

lati

on o

f S

H3P

X1 i

s

crit

ical

fo

r th

e deg

radat

ion

of

EG

F

rece

pto

rs

in

cell

s [2

9],

an

d

SH

3P

X1

phosp

hory

lati

on i

s th

ought

to b

e im

port

ant

for

loca

lizi

ng d

ynam

in-2

to s

ites

on t

he

pla

sma

mem

bra

ne

wh

ere

endocy

tic

bud

s fo

rm.

[25].

G

iven

the

appar

ent

import

ance

of

SH

3P

X1 p

hosp

hory

lati

on o

n E

GF

rec

epto

r pro

cess

ing,

and t

he

fact

that

we

hav

e

iden

tifi

ed F

AK

and S

rc a

s novel

kin

ases

that

can

phosp

hory

late

SH

3P

X1,

we

wer

e

inte

rest

ed in

id

enti

fyin

g th

e in

div

idual

phosp

hory

lati

on si

tes

on S

H3P

X1 th

at ar

e

phosp

hory

late

d b

y F

AK

and S

rc.

We

turn

ed t

o m

ass

spec

trom

etry

as

a m

eans

to

iden

tify

the

site

s of

tyro

sine

phosp

hory

lati

on o

n S

H3P

X1.

In o

rder

to e

nsu

re a

via

ble

sam

ple

for

mas

s sp

ectr

om

etry

, w

e to

ok a

dvan

tage

of

Src

-cat

alyze

d p

hosp

hory

lati

on o

f

SH

3P

X1,

as i

t ex

hib

ited

the

most

act

ivit

y i

n c

ells

and i

n v

itro.

Bas

ed o

n o

ur

findin

gs

show

ing

that

co

-expre

ssio

n

of

SH

3P

X1

and

S

rc

in

cell

s re

sult

ed

in

robust

phosp

hory

lati

on of

SH

3P

X1 (s

ee F

igure

3.3

), w

e dec

ided

to

u

se th

is ap

pro

ach to

gen

erat

e a

sam

ple

fo

r m

ass

spec

trom

etry

an

alysi

s. In

bri

ef,

HE

K 293 ce

lls

wer

e

140

tran

sfec

ted

wit

h

HA

-Src

an

d

V5-S

H3P

X1.

T

wen

ty-f

our

hours

fo

llow

ing

the

tran

sfec

tion,

the

cell

s w

ere

lyse

d

and

V5-S

H3P

X1

was

im

munopre

cipit

ated

fr

om

appro

xim

atel

y

10

mg

of

tota

l cel

l ex

trac

t u

sing

an

anti

-V5

anti

body.

T

he

imm

unopre

cipit

atio

ns

wer

e div

ided

and

each

set

was

res

olv

ed o

n s

epar

ate S

DS

-PA

GE

gel

s.

One

gel

was

sta

ined

wit

h C

oom

ass

ie b

lue t

o v

isual

ize

all

pro

tein

s th

at c

ame

dow

n i

n t

he

imm

unopre

cipit

atio

n w

hil

e th

e se

cond g

el w

as t

ransf

erre

d t

o P

VD

F a

nd

then

subje

cted

to W

est

ern b

lot

anal

ysi

s w

ith a

n a

nti

-phosp

hoty

rosi

ne

anti

body (

Fig

ure

3.8

B).

The

imm

unoblo

t an

alysi

s co

nfi

rmed

th

at

the

SH

3P

X1

was

in

fact

phosp

hory

late

d b

y S

rc.

The

ban

d c

orr

espondin

g t

o p

hosp

hory

late

d S

H3P

X1 o

n t

he

Coom

assi

e-st

ained

gel

(F

igure

3.8

A)

was

then

exci

sed a

nd t

he

sam

ple

subm

itte

d t

o t

he

Corn

ell

Bio

tech

nolo

gy

Res

ourc

e C

ente

r P

rote

om

ics

and

Mas

s S

pec

trom

etry

C

ore

Fac

ilit

y.

The

resu

lts

of

the

mas

s sp

ectr

om

etry

anal

ysi

s id

enti

fied

tyro

sine r

esid

ues

177,

239,

269,

294,

and 5

61 a

s S

rc-c

atal

yze

d p

hosp

hory

lati

on s

ites

(T

able

3.1

).

The

resi

due

and p

epti

de

sequen

ce

show

n i

n t

he

firs

t tw

o c

olu

mns

of

the

table

ref

er t

o t

he

indiv

idual

phosp

ho-p

epti

des

iden

tifi

ed b

y l

iquid

chro

mat

ogra

phy (

LC

) M

S-M

S,

whil

e

M/Z

rat

io r

efer

s to

the

mas

s-to

-char

ge

rati

o o

f th

e pep

tides

. T

he

mascot

score r

efer

s to

an al

go

rith

m pro

gra

m use

d to

det

erm

ine

the

likel

ihood of

phosp

hory

lati

on (3

0 is

consi

der

ed t

he

bas

elin

e),

and %

Pi

refe

rs t

o t

he

per

centa

ge

each

tyro

sine

resi

due

was

phosp

hory

late

d in

our

sam

ple

. F

igure

3.9

is

an

ex

ample

of

a M

S/M

S sp

ectr

um

show

ing t

he

smal

ler

ions

dev

eloped

fro

m t

he

seco

nd r

ound o

f m

ass

spec

trom

etry

. I

t is

wort

h m

enti

onin

g t

hat

anoth

er s

ample

of

imm

unopre

cipit

ated

SH

3P

X1 w

as s

ubm

itte

d

for

mas

s sp

ectr

om

etry

an

alysi

s fr

om

ce

lls

in

whic

h

SH

3P

X1,

alone,

w

as

over

expre

ssed

in

HE

K 2

93 c

ells

. T

he

mas

s sp

ect

rom

etry

res

ult

s id

enti

fied

no t

yro

sin

e

phosp

hory

late

d

resi

dues

, th

us,

fu

rther

co

nfi

rmin

g

that

S

rc-c

atal

yze

d

the

phosp

hory

lati

on s

ites

on S

H3P

X1 i

den

tifi

ed b

y m

ass

spec

trom

etry

anal

ysi

s.

141

Fig

ure

3.8

Pre

para

tion

of

the

ph

osp

hory

late

d

SH

3P

X1

sam

ple

fo

r m

ass

spec

trom

etry

an

aly

sis.

H

A-S

rc a

nd V

5-S

H3P

X1 w

ere

co-e

xpre

ssed

in H

EK

293

cell

s.

V5-S

H3P

X1 w

as

imm

unopre

cipit

ated

fro

m t

he

whole

cel

l ly

sate

s w

ith a

nti

-V5

anti

body,

reso

lved

by S

DS

-PA

GE

, an

d s

tain

ed w

ith C

oom

assi

e.

The

ban

d c

onta

inin

g

SH

3P

X1 w

as e

xci

sed a

nd s

ubm

itte

d t

o t

he

Corn

ell

Mas

s S

pec

trom

etry

Core

Fac

ilit

y

(A).

A

liquots

fr

om

th

is sa

mple

w

ere

also

re

solv

ed by S

DS

-PA

GE

, tr

ansf

erre

d to

PV

DF

, an

d s

ubje

cted

to W

est

ern b

lott

ing a

nal

ysi

s w

ith a

nti

-phosp

hoty

rosi

ne

and a

nti

-

V5 a

nti

bodie

s (B

).

142

143

Tab

le 3

.1

Tyro

sin

e re

sid

ues

177,

239,

269,

294 a

nd

561 w

ere

iden

tifi

ed a

s S

rc-

cata

lyze

d

ph

osp

hory

lati

on

si

tes

on

S

H3P

X1

by

mass

sp

ectr

om

etry

. T

he

phosp

hory

late

d p

epti

des

are

lis

ted i

n t

he

far-

left

colu

mns.

M/Z

ref

ers

to t

he

mas

s-to

-

char

ge

rati

o o

f ea

ch p

epti

de,

mas

cot

score

ref

ers

to a

n a

lgori

thm

pro

gra

m u

sed t

o

det

erm

ine

the

likel

ihood o

f phosp

hory

lati

on (

30 i

s co

nsi

der

ed t

o b

e th

e base

line)

, an

d

%

phosp

hory

lati

on

refe

rs

to

the

per

centa

ge

of

each

ty

rosi

ne

resi

due

that

is

phosp

hory

late

d.

144

145

Fig

ure

3.9

D

epic

tion

of

the

MS

/MS

sp

ectr

um

fro

m t

he

pep

tid

e in

clu

din

g Y

294.

146

147

Foll

ow

ing t

he

iden

tifi

cati

on o

f th

e S

rc-c

atal

yze

d t

yro

sine

phosp

ho

ryla

tion s

ites

by

mas

s sp

ect

rom

etry

, w

e nex

t w

ante

d

to

confi

rm

thes

e re

sult

s usi

ng

a gen

etic

appro

ach.

S

ingle

an

d

mult

iple

ty

rosi

ne-

to-p

hen

yla

lanin

e poin

t m

uta

nts

w

ere

engin

eere

d

usi

ng

site

-dir

ecte

d

muta

gen

esi

s.

T

he

SH

3P

X1

muta

nts

gen

erat

ed

incl

uded

: Y

177F

, Y

239F

, Y

269F

, Y

294F

, Y

561F

, as

wel

l as

a m

uta

nt

in w

hic

h a

ll o

f

the

tyro

sine

resi

des

wer

e m

uta

ted,

Y177F

/Y239F

/Y269F

/Y294F

/Y561F

. C

ells

wer

e

co-t

ransf

ecte

d w

ith S

rc a

nd

eac

h o

f th

e S

H3P

X1

poin

t m

uta

nts

, an

d a

fter

24

hours

of

expre

ssio

n,

the

cell

s w

ere

lyse

d

and

the

vari

ous

poin

t m

uta

nts

of

SH

3P

X1

imm

unopre

cipit

ated

w

ith

anti

-V5

anti

body.

Im

munoblo

t an

alysi

s of

the

imm

unopre

cipit

ated

sam

ple

s w

ith a

nti

-phosp

hoty

rosi

ne

anti

body w

as p

erfo

rmed

and

the

resu

lts

are

show

n i

n F

igure

3.1

0.

The

blo

t in

dic

ates

that

only

the

SH

3P

X1-Y

239F

and S

H3P

X1-Y

177F

/Y239F

/Y269F

/Y29

4F

/Y561F

co

nst

ruct

s sh

ow

ed a

dim

inis

hed

capac

ity

to

be

phosp

hory

late

d

by

Src

.

In

the

SH

3P

X1-

Y177F

/Y239F

/Y269F

/Y294F

/Y561F

m

uta

nt,

nea

rly

all

of

the

Src

-med

iate

d

phosp

hory

lati

on o

f S

H3P

X1 w

as l

ost

, w

hil

e th

e 2

39 m

uta

nt

show

ed a

n a

ppro

xim

atel

y

60%

dec

reas

e in

the

amount

of

SH

3P

X1 p

hosp

hory

lati

on.

More

over

, w

e w

ent

on t

o

dem

onst

rate

that

FA

K-c

atal

yze

d t

he

phosp

hory

lati

on o

f S

H3P

X1 a

t th

e sa

me

site

s as

Src

(F

igure

3.1

1).

T

hes

e fi

ndin

gs

furt

her

support

the

idea

that

FA

K a

nd S

rc w

ork

in

tandem

to

phosp

hory

late

S

H3P

X1,

and

that

th

e m

ajor

site

of

FA

K

and

Src

phosp

hory

lati

on o

n S

H3P

X1 i

s Y

239.

Dis

cuss

ion

Endocy

tosi

s of

cell

surf

ace

rece

pto

rs i

s a

tightl

y r

egula

ted,

sequen

tial

pro

cess

in w

hic

h ce

ll su

rfac

e re

cepto

rs bec

om

e in

tern

aliz

ed in

to th

e cy

topla

sm.

C

lath

rin-

med

iate

d e

ndocy

tosi

s ca

n e

ither

occ

ur

const

ituti

vely

or

in r

esponse

to e

xte

rnal

sti

muli

.

Whil

e m

any p

rote

ins

hav

e bee

n i

mpli

cate

d i

n r

egula

ting e

ndocy

tosi

s, A

P-2

, cl

athri

n,

148

Fig

ure

3.1

0

SH

3P

X1

mu

tan

t Y

177F

/Y239F

/Y269F

/Y294F

/Y561

is

ph

osp

hory

lati

on

-def

ecti

ve,

wh

ere

tyro

sin

e 239 i

s th

e m

ajo

r si

te p

hosp

hory

late

d

by

Src

.

Pla

smid

s en

codin

g

HA

-Src

an

d

V5-S

H3P

X1

poin

t m

uta

nts

w

ere

co-

tran

sfec

ted i

nto

HE

K 2

93 c

ells

. A

ppro

xim

atel

y 2

4 h

ours

aft

er t

ransf

ecti

on,

cell

s w

ere

lyse

d a

nd t

he S

H3P

X1 p

oin

t m

uta

nts

wer

e im

mun

opre

cipit

ated

wit

h a

nti

-V5 a

nti

body.

The

pre

cipit

ated

sa

mple

s w

ere

reso

lved

by

SD

S-P

AG

E,

afte

r w

hic

h

they

w

ere

tran

sfer

red t

o P

VD

F a

nd s

ubje

cted

to W

est

ern b

lott

ing w

ith a

nti

-phosp

hoty

rosi

ne

and

anti

-V5 a

nti

bodie

s.

149

150

Fig

ure

3.1

1

F

AK

is

u

nab

le

to

ph

osp

hory

late

th

e S

H3P

X1

mu

tan

t

Y177F

/Y239F

/Y269F

/Y294F

/Y561,

wh

ere

the

majo

r si

te

of

ph

osp

hory

lati

on

ap

pea

rs t

o b

e ty

rosi

ne

239.

Pla

smid

s en

codin

g H

A-F

AK

and V

5-S

H3P

X1 p

oin

t

muta

nts

w

ere

co-t

ransf

ecte

d

into

H

EK

293

ce

lls.

Appro

xim

atel

y

24

hours

af

ter

tran

sfec

tion,

cell

s w

ere

lyse

d a

nd t

he

SH

3P

X1 p

oin

t m

uta

nts

wer

e im

munopre

cipit

ated

wit

h a

nti

-V5 a

nti

body.

The

pre

cipit

ated

sam

ple

s w

ere

reso

lved

by S

DS

-PA

GE

, af

ter

whic

h t

hey

wer

e tr

ansf

erre

d t

o P

VD

F a

nd s

ubje

cted

to W

este

rn b

lott

ing w

ith a

nti

-

phosp

hoty

rosi

ne

and a

nti

-V5 a

nti

bodie

s.

151

152

and d

ynam

in h

ave

bee

n s

how

n t

o h

ave

esse

nti

al r

ole

s in

this

cel

lula

r pro

cess

. B

rief

ly,

the

acti

on o

f A

P-2

ser

ves

to i

nit

iate

the

endocy

tic

pro

cess

by c

once

ntr

atin

g t

he

carg

o

in t

he

emer

gin

g e

ndocy

tic b

ud a

s w

ell

as r

ecru

itin

g c

lath

rin t

o t

he

pla

sma

mem

bra

ne

[18-2

1].

The

tris

kel

ion

stru

cture

of

clat

hri

n

pro

vid

es

the

geo

met

ric

const

rain

ts

nec

essa

ry f

or

the

mem

bra

ne

to a

cquir

e as

incr

easi

ng c

urv

ature

form

ing a

n i

nvag

inat

ed

pit

. A

s th

e cl

athri

n p

it c

onti

nues

to m

ature

, th

e fi

nal

ste

p i

nvolv

es f

issi

on t

hro

ugh t

he

acti

on o

f th

e G

TP

ase,

dynam

in [

22].

SH

3P

X1 i

s a r

ecen

tly i

den

tifi

ed p

rote

in t

hought

to f

unct

ion i

n e

ndocy

tosi

s.

It

has

bee

n s

how

n t

o b

ind t

o d

ynam

in i

n K

562 h

um

an e

ryth

role

ukem

ia c

ells

[24]

and t

o

stim

ula

te d

ynam

in’s

bas

al G

TP

ase

acti

vit

y i

n v

itro

[30].

More

over

, a

convin

cing r

ole

for

SH

3P

X1 i

n m

edia

ting e

ndocy

tosi

s w

as d

emonst

rate

d b

y t

he

mis

loca

liza

tion o

f

dynam

in fr

om

th

e pla

sma

mem

bra

ne of

cell

s d

eple

ted of

SH

3P

X1 ex

pre

ssio

n by

SH

3P

X1-R

NA

i [2

5].

Our

labora

tory

an

d

oth

ers

hav

e re

port

ed

that

S

H3P

X1

is

tyro

sine

phosp

hory

late

d,

how

ever

the

effe

cts

of

this

tyro

sine

pho

sphory

lati

on e

ven

t on

the

funct

ion o

f S

H3P

X1 h

ave

yet

to b

e fu

lly c

har

acte

rize

d.

In th

is st

udy,

we

hav

e ch

ose

n to

fo

cus

on

es

tabli

shin

g w

het

her

ad

dit

ional

pro

tein

kin

ases

asi

de f

rom

AC

K2 c

an p

hosp

hory

late

SH

3P

X1.

To t

his

end,

we h

ave

iden

tifi

ed

two

pre

vio

usl

y

unre

port

ed

kin

ases

fo

r S

H3P

X1,

FA

K

and

Src

.

We

dem

onst

rate

d t

hat

over

expre

ssio

n o

f ei

ther

of

thes

e kin

ases

resu

lted

in

robust

SH

3P

X1

phosp

hory

lati

on.

F

AK

w

as

init

iall

y

iden

tifi

ed as

a

pote

nti

al

candid

ate

bas

ed

on

sim

ilar

itie

s bet

wee

n t

he

kin

ase

dom

ains

of

AC

K2 a

nd

FA

K,

note

d i

n a

BL

AS

T s

earc

h

of

the

Nat

ional

C

ente

r of

Bio

tech

nolo

gy

Info

rmat

ion

(NC

BI)

dat

abas

e [3

1].

Its

addit

ional

role

in e

ndophil

in p

hosp

hory

lati

on f

urt

her

hin

ted a

t it

s pote

nti

al r

ole

, bas

ed

on t

he

sim

ilar

itie

s in

dom

ain s

truct

ure

and f

unct

ion o

f S

H3P

X1 a

nd e

ndophil

in [

32].

Src

th

en bec

ame

an ad

dit

ional

ca

ndid

ate

giv

en it

s know

n as

soci

atio

n an

d ta

ndem

signal

ing w

ith F

AK

in c

ells

.

153

When

we

firs

t in

itia

ted t

hes

e ex

per

imen

ts,

we

exp

ecte

d F

AK

and S

rc t

o e

xhib

it

sim

ilar

bin

din

g a

nd p

ho

sphory

lati

on p

atte

rns

as t

hose

ob

serv

ed w

ith A

CK

2.

How

ever

,

we

hav

e fo

und

that

FA

K a

nd S

rc a

ppear

to s

ignal

in a

dis

tinct

man

ner

. T

he

FA

K-

and

Src

-cat

alyze

d S

H3P

X1 p

hosp

hory

lati

on i

s al

most

10 f

old

gre

ater

than

that

of

AC

K2 i

n

cell

s.

This

obse

rvat

ion i

s li

kel

y d

ue

in p

art

to t

he

incr

ease

d c

atal

yti

c ac

tivit

ies

of

FA

K

and S

rc.

Of

the

two

kin

ases,

Src

-cat

alyze

d p

ho

sphory

lati

on o

f S

H3P

X1 w

as t

he m

ost

inte

nse

, a

pro

per

ty

we

too

k

advan

tage

of

in

the

iden

tifi

cati

on

of

five

pri

mar

y

phosp

hory

lati

on s

ites

on S

H3P

X1

by m

ass

spec

trom

etry

, ty

rosi

ne

resi

dues

177,

239,

269,

294,

and 5

61.

A

side

from

tyro

sine

561,

four

of

the

tyro

sine

resi

dues

, Y

177,

Y239,

Y269 a

nd Y

294 a

re j

ust

N-t

erm

inal

of

the

PX

dom

ain.

This

is

intr

iguin

g g

iven

rece

nt

report

s th

at t

yro

sine

phosp

hory

lati

on o

f th

is r

egio

n m

ay b

e cr

itic

al i

n t

arget

ing

the

endocy

tic

pro

tein

, dynam

in,

to th

e ce

ll su

rfac

e [2

5].

N

one

of

the

FA

K/S

rc-

cata

lyze

d p

hosp

hory

lati

on s

ites

appea

r to

be

shar

ed w

ith t

hose

sit

es p

ho

sphory

late

d b

y

AC

K2.

This

sugges

ts t

hat

these

dif

fere

nt

phosp

hory

lati

on e

ven

ts a

re l

ikel

y t

o h

ave

dis

tinct

fu

nct

ional

co

nse

quen

ces.

The

gen

erat

ion

of

SH

3P

X1

muta

nts

th

at

are

def

ecti

ve

for

FA

K/S

rc-c

atal

yze

d p

hosp

hory

lati

on, ver

sus

muta

nts

def

ecti

ve

for

AC

K2-

pro

mote

d phosp

hory

lati

on,

should

be

inval

uab

le in

es

tabli

shin

g th

e ro

le fo

r th

ese

dif

fere

nt

phosp

hory

lati

on e

ven

ts i

n e

ndocy

tosi

s an

d E

GF

rec

epto

r deg

radat

ion.

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ove

l dom

ain

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nd

Ptd

Ins

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RK

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Src

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AC

K2

inte

ract

s w

ith

sort

ing

nex

in

9

(SH

3P

X1)

to r

egula

te e

pid

erm

al

gro

wth

fact

or

rece

pto

r deg

radati

on.

J B

iol

Chem

, 2002. 277(1

2):

p. 10134-8

.

30.

Soule

t,

F.,

et

al

.,

SN

X9

regula

tes

dyn

am

in

ass

embly

and

is

requir

ed

for

effi

cien

t cl

ath

rin

-med

iate

d e

ndocy

tosi

s. M

ol

Bio

l C

ell,

2005.

16(4

): p

. 20

58-

67.

31.

Yan

g,

W.

and R

.A.

Cer

ione,

Clo

nin

g a

nd c

hara

cter

izati

on o

f a n

ove

l C

dc4

2-

ass

oci

ate

d ty

rosi

ne

kinase

, A

CK

-2,

from

bovi

ne

bra

in.

J B

iol

Chem

, 1997.

272(4

0):

p.

24819-2

4.

157

32.

Wu,

X., e

t al

., F

AK

-med

iate

d s

rc p

hosp

hory

lati

on o

f en

dophil

in A

2 i

nhib

its

endocy

tosi

s of

MT

1-M

MP

and p

rom

ote

s E

CM

deg

radati

on.

Dev

Cel

l, 2

005.

9(2

): p

. 185-9

6.

158

CH

AP

TE

R F

OU

R

CO

NC

LU

SIO

N

The

regula

tion o

f gro

wth

fac

tor

rece

pto

r le

vel

s and k

inas

e a

ctiv

ity a

t th

e cel

l

surf

ace

is c

riti

cal

in m

edia

ting s

ignal

ing p

athw

ays

involv

ed i

n m

itogen

esis

. A

lter

ed

expre

ssio

n le

vel

s an

d m

uta

tions

resu

ltin

g in

unre

gula

ted kin

ase

acti

vit

y ar

e oft

en

suff

icie

nt

to c

ause

abnorm

al c

ell

gro

wth

and

pro

life

rati

on.

Of

the

sever

al m

echan

ism

s

uti

lize

d

by

the

cell

to

m

ainta

in

norm

al

pro

life

rati

on,

rece

pto

r en

docy

tosi

s an

d

deg

radat

ion p

lay k

ey r

ole

s by e

nsu

ring t

he

pro

per

hom

eost

asis

of

rece

pto

r ty

rosi

ne

kin

ases

. SH

3P

X1 h

as r

ecen

tly b

een i

mpli

cate

d i

n t

he

endo

cyti

c pro

cess

. A

mem

ber

of

the

sort

ing n

exin

fam

ily,

SH

3P

X1 h

as n

ot

only

been

show

n t

o b

ind

to A

P-2

, cl

athri

n,

and d

ynam

in,

but

when

tyro

sine-

phosp

hory

late

d,

it h

as a

lso b

een s

how

n t

o r

ecru

it

dynam

in t

o t

he

pla

sma

mem

bra

ne.

A

s a

resu

lt,

we

wer

e in

tere

sted

in c

har

acte

rizi

ng

this

phosp

hory

lati

on e

ven

t, a

nd d

evel

opin

g p

ote

nti

al d

om

inan

t-neg

ativ

e m

uta

nts

that

mig

ht

be

use

ful

for

furt

her

exam

inin

g t

he

role

of

SH

3P

X1 i

n e

ndocy

tosi

s.

In c

hap

ter

2,

I ch

arac

teri

ze t

he

inte

ract

ion

s bet

wee

n A

CK

2 a

nd S

H3P

X1 a

nd

the

non-r

ecep

tor

tyro

sine

kin

ase,

AC

K2,

a C

dc4

2-s

pec

ific

eff

ecto

r pro

tein

. A

ddit

ional

ly,

I des

crib

e in

vitr

o k

inas

e ass

ays

use

d t

o i

den

tify

pyri

dopyri

mid

ine,

PD

158780,

as a

pote

nt

inhib

itor

of

AC

K2 k

inas

e ac

tivit

y.

In c

hap

ter

3,

I fo

cus

on t

he

iden

tifi

cati

on o

f F

AK

and S

rc a

s

novel

kin

ases

for

SH

3P

X1.

I s

how

that

FA

K a

nd S

rc a

re m

uch

more

eff

ecti

ve

at

phosp

hory

lati

ng S

H3P

X1 c

om

par

ed t

o A

CK

2

It i

s in

tere

stin

g t

hat

wit

h t

he

exce

pti

on o

f Y

561,

all

of

the

phosp

hory

lati

on s

ites

are

loca

ted w

ithin

100 a

min

o a

cids

of

the P

X d

om

ain,

whic

h h

as b

een

im

pli

cate

d i

n

targ

etin

g p

rote

ins

to t

he

pla

sma

mem

bra

ne

[1].

O

ther

lab

ora

tori

es h

ave

report

ed t

hat

the

phosp

hory

lati

on o

f S

H3P

X1 w

ithin

this

reg

ion (

resi

dues

151

-185)

is c

riti

cal

for

159

med

iati

ng t

he

traf

fick

ing o

f dynam

in-2

[2].

In

thes

e st

udie

s, t

yro

sine

phosp

hory

lati

on

elim

inat

es b

indin

g o

f th

e gly

coly

tic e

nzy

me,

ald

ola

se.

In t

he

abse

nce

of

aldola

se

bin

din

g,

SH

3P

X1 re

gai

ns

its

abil

ity to

as

soci

ate

wit

h th

e pla

sma

mem

bra

ne,

th

us

traf

fick

ing d

ynam

in t

o i

ts r

egio

n o

f ac

tion a

t th

e nec

k o

f cl

athri

n-c

oat

ed p

its.

In

a

more

gen

eral

exam

ple

, phosp

hory

lati

on o

f se

rine

412 o

f !

-arr

esti

n h

as b

een s

how

n t

o

loca

lize

the

endocy

tic

pro

tein

to t

he

cell

mem

bra

ne

wher

e it

is

involv

ed i

n i

nit

iati

ng

endocy

tosi

s of

the !

-adre

ner

gic

re

cepto

r [3

],

thus

esta

bli

shin

g

a

pre

ceden

t fo

r

phosp

hory

lati

on a

s a

mea

ns

of

regula

ting p

rote

in t

raff

ickin

g.

Sev

eral

re

port

s hav

e su

gges

ted

that

S

H3P

X1

has

th

e ab

ilit

y

to

dim

eriz

e

thro

ugh i

ts C

-ter

min

al B

AR

dom

ain,

a co

iled

-coil

moti

f m

ade

up o

f th

e la

st 2

0 a

min

o

acid

s.

In c

hap

ter

2,

I des

crib

e th

e in

abil

ity o

f A

CK

2 t

o b

ind t

o a

nd p

hosp

hory

late

SH

3P

X1 m

ole

cule

s th

at l

ack t

his

C-t

erm

inal

dom

ain.

How

ever

, F

AK

and S

rc a

re a

ble

to p

hosp

hory

late

thes

e sa

me

const

ruct

s.

This

rai

ses

the

poss

ibil

ity t

hat

the

monom

er-

dim

er

equil

ibri

um

of

SH

3P

X1

det

erm

ines

w

hic

h

site

s bec

om

e

avai

lable

fo

r

phosp

hory

lati

on b

y t

hes

e d

iffe

rent

kin

ases

. F

or

exam

ple

, w

e bel

ieve

that

SH

3P

X1

dim

eriz

atio

n c

ould

pote

nti

ally

med

iate

its

phosp

hory

lati

on b

y m

ult

iple

pro

tein

kin

ases

.

Dim

eriz

atio

n o

f S

H3P

X1 m

ight

allo

w f

or

AC

K2

-cat

alyze

d p

hosp

hory

lati

on,

wher

eas,

FA

K a

nd S

rc m

ight

pre

fere

nti

ally

phosp

hory

late

th

e m

onom

eric

form

. T

his

could

pro

vid

e a

mec

han

ism

by w

hic

h S

H3P

X1 a

ccom

odat

es d

iver

se,

upst

ream

sig

nal

s su

ch

that

it

can p

arti

cipat

e in

FA

K/S

rc-p

rom

ote

d r

ecru

itm

ent

of

dynam

in t

o e

ndocy

tic

site

s

at

the

cell

su

rfac

e an

d

AC

K2-m

edia

ted

EG

F

rece

pto

r so

rtin

g

and

deg

radat

ion.

How

ever

, th

e sp

ecif

ic m

ech

anis

ms

by w

hic

h p

hosp

hory

late

d S

H3P

X1 m

edia

tes

the

recr

uit

men

t of

dynam

in o

r th

e so

rtin

g o

f E

GF

rec

epto

rs a

re l

ikel

y t

o b

e co

mple

x a

nd

wil

l be

the

focu

s of

futu

re s

tudie

s in

our

labora

tory

. O

ne

such

lin

e of

study w

ill

involv

e fu

rther

char

acte

riza

tion o

f th

e in

tera

ctio

ns

of

SH

3P

X1 w

ith d

ynam

in.

We

hav

e beg

un t

o e

xam

ine

this

an

d a

s sh

ow

n i

n F

igure

4.1

, w

e hav

e fo

und t

hat

muta

ting

160

Fig

ure

4.1

A

CK

2 a

nd

Dyn

am

in b

ind

to S

H3P

X1 t

hro

ugh

th

e S

H3 d

om

ain

of

SH

3P

X1.

C

OS

-7

cell

s w

ere

tran

sfec

ted

wit

h

the

var

ious

expre

ssio

n

pla

smid

s

indic

ated

. T

wen

ty-f

our

to s

even

ty-t

wo h

ours

aft

er

tran

sfec

tion,

cell

s w

ere

lyse

d a

nd

AC

K2 (

A)

and d

ynam

in-2

(B

) w

ere

imm

unopre

cipit

ated

wit

h a

nti

-Myc

and a

nti

-HA

anti

bodie

s,

resp

ecti

vel

y.

P

reci

pit

ated

sa

mple

s w

ere

reso

lved

by

SD

S-P

AG

E

and

tran

sfer

red t

o P

VD

F.

The

PV

DF

mem

bra

ne

was

then

pro

bed

wit

h a

nti

-Myc,

anti

-HA

,

and a

nti

-V5 a

nti

bodie

s.

161

162

the

conse

rved

try

pto

phan

res

idue

in t

he

SH

3 d

om

ain o

f S

H3P

X1,

whic

h i

s es

senti

al

for

bin

din

g

to

pro

line-

rich

dom

ains

on

oth

er

pro

tein

s,

elim

inat

es

the

abil

ity

of

SH

3P

X1 t

o b

ind t

o e

ither

AC

K2 o

r dynam

in-2

(F

igure

4.1

, la

ne

2 o

f A

and

B).

G

iven

that

the

bin

din

g p

artn

ers

report

ed f

or

SH

3P

X1 h

ave

each

bee

n s

how

n t

o b

ind t

hro

ugh

the

SH

3 d

om

ain o

f th

e so

rtin

g n

exin

, it

is

likel

y t

hat

ther

e ar

e co

mpet

ing i

nte

ract

ions.

Our

resu

lts

also

su

gges

t co

ndit

ions

that

al

low

A

CK

2-c

atal

yze

d phosp

hory

lati

on of

SH

3P

X1 m

ay c

om

pro

mis

e it

s ab

ilit

y t

o b

ind t

o d

ynam

in (

Fig

ure

4.2

B,

com

par

e la

nes

1 a

nd 2

), a

s en

han

ced

co

-im

munopre

cipit

atio

n o

f dynam

in w

ith S

H3P

X1 i

s obse

rved

in

cell

s w

her

e S

H3P

X1

has

been

co

-expre

ssed

w

ith

kin

ase

-def

ecti

ve

AC

K2.

How

ever

, w

e hope

to u

se p

hosp

hory

lati

on-d

efec

tive

muta

nts

of

SH

3P

X1 i

n o

rder

to

ver

ify t

hes

e pre

lim

inar

y o

bse

rvat

ions.

S

pec

ific

ally

, w

e ar

e in

tere

sted

in c

arry

ing o

ut

imm

unofl

uore

scen

ce s

tudie

s w

ith c

ells

expre

ssin

g p

hosp

hory

lati

on

-def

ecti

ve

SH

3P

X1

muta

nts

and e

xam

inin

g h

ow

this

infl

uen

ces

the

loca

liza

tion o

f en

dogen

ous

dyn

amin

.

It w

ill

be

inte

rest

ing t

o s

ee w

het

her

the

SH

3P

X1 m

uta

nt

that

is

def

ecti

ve

for

AC

K2

enhan

ces

the

inte

ract

ions

bet

ween

dynam

in a

nd S

H3P

X1.

We

would

als

o l

ike

to s

ee

whet

her

S

H3P

X1 m

uta

nts

th

at ar

e def

ecti

ve

for

phosp

hory

lati

on by F

AK

an

d S

rc

affe

ct d

ynam

in l

oca

liza

tion.

In p

arti

cula

r, w

e w

ould

lik

e to

know

whet

her

FA

K/S

rc-

cata

lyze

d

phosp

hory

lati

on

of

SH

3P

X1

repre

sen

ts

the

regula

tory

cu

e th

at

recr

uit

s

dynam

in t

o t

he

cell

surf

ace.

If

so,

it w

ould

sugges

t th

at t

he

dif

fere

nt

phosp

hory

lati

on

even

ts b

y A

CK

2 v

ersu

s F

AK

and S

rc h

ave o

pp

osi

ng e

ffec

ts o

n S

H3P

X1-d

ynam

in

inte

ract

ions.

L

ikew

ise,

it

w

ill

be

inte

rest

ing to

se

e if

th

e S

H3P

X1 m

uta

nt

that

is

def

ecti

ve

for

AC

K2-c

atal

yze

d

phosp

hory

lati

on

blo

cks

EG

F

rece

pto

r so

rtin

g

and

deg

radat

ion a

s w

ould

be

sug

ges

ted f

rom

our

earl

ier

work

show

ing a

lin

k b

etw

een

AC

K2-m

edia

ted p

hosp

hory

lati

on o

f S

H3P

X1 o

n E

GF

rec

epto

r deg

radat

ion [

4].

A l

egit

imat

e ro

le f

or

SH

3P

X1 i

n c

lath

rin

-med

iate

d e

ndocy

tosi

s an

d r

ecep

tor

pro

cess

ing h

as b

een i

ncr

easi

ngly

est

abli

shed

. T

o d

ate,

SH

3P

X1 h

as b

een i

mpli

cate

d

163

Fig

ure

4.2

S

H3P

X1 b

ind

ing t

o d

yn

am

in i

s n

ot

med

iate

d b

y A

CK

2.

In

ad

dit

ion

,

dyn

am

in-2

bin

ds

to u

np

ho

sph

ory

late

d S

H3P

X1.

HA

-dynam

in-2

was

tra

nsf

ecte

d

wit

h e

ither

Myc-

AC

K2 o

r V

5-S

H3P

X1 i

n C

OS

-7 c

ells

. T

wen

ty-f

our

to s

even

ty-t

wo

hours

af

ter

tran

sfec

tion,

AC

K2

and

SH

3P

X1

wer

e im

munopre

cipit

ated

fr

om

ce

ll

lysa

tes

wit

h a

nti

-Myc

and a

nti

-V5 a

nti

bodie

s, r

espec

tivel

y.

Pre

cipit

ated

sam

ple

s w

ere

reso

lved

by S

DS

-PA

GE

and

tra

nsf

erre

d t

o P

VD

F.

The

PV

DF

mem

bra

ne

was

then

subje

cted

to W

est

ern b

lott

ing a

nal

ysi

s w

ith a

nti

-HA

to m

easu

re d

ynam

in-2

bin

din

g

(A).

C

OS

-7 c

ells

wer

e tr

ansf

ecte

d w

ith V

5-S

H3P

X1,

HA

-dynam

in-2

, an

d e

ither

Myc-

AC

K2

or

Myc-

AC

K2

-K158R

.

Appro

xim

atel

y

24-7

2

hours

af

ter

tran

sfec

tion,

SH

3P

X1

was

im

munopre

cip

itat

ed

from

ce

ll

lysa

tes

wit

h

anti

-V5

anti

body.

T

he

sam

ple

s w

ere

reso

lved

by S

DS

-PA

GE

, an

d t

ransf

erre

d t

o P

VD

F m

embra

ne.

L

ater

, th

e

sam

ple

s w

ere

subje

cted

to

W

este

rn

blo

ttin

g

wit

h

anti

-HA

an

tibody

to

mea

sure

dynam

in b

indin

g i

n t

he

pre

sence

of

AC

K2 a

nd t

he

kin

ase-

def

ecti

ve

muta

nt

AC

K2-

K158R

(B

).

164

165

in t

he

traf

fick

ing o

f se

ver

al c

ell

surf

ace

rece

pto

rs i

ncl

udin

g t

he

insu

lin [

5],

EG

F [

4],

and

tran

sfer

rin

rece

pto

rs

[6].

Giv

en

the

import

ance

of

med

iati

ng

the

signal

ing

acti

vit

ies

and p

roper

dow

n-r

egula

tion o

f th

ese

rece

pto

rs,

we

bel

ieve

that

elu

cidat

ing

the

role

of

SH

3P

X1 a

nd i

ts p

hosp

hory

lati

on i

n r

ecep

tor

endocy

tosi

s w

ill

ult

imat

ely

lead

to

a

bet

ter

under

stan

din

g

of

the

bio

logic

al

pat

hw

ays

under

lyin

g

abnorm

al

pro

life

rati

ve

dis

ease

.

166

Refe

ren

ces

1.

Xu,

Y., et

al

., SN

X3 re

gula

tes

endoso

mal

funct

ion th

rough it

s P

X-d

om

ain

-

med

iate

d i

nte

ract

ion w

ith P

tdIn

s(3)P

. N

at C

ell

Bio

l, 2

001.

3(7

): p

. 658-6

6.

2.

Lundm

ark,

R.

and

S.R

. C

arls

son,

Reg

ula

ted

mem

bra

ne

recr

uit

men

t of

dyn

am

in-2

med

iate

d b

y so

rtin

g n

exi

n 9

. J

Bio

l C

hem

, 2004.

279(4

1):

p.

42694-

702.

3.

Shen

oy,

S.K

. an

d R

.J.

Lef

kow

itz,

Mult

iface

ted r

ole

s of

bet

a-a

rres

tin

s in

the

regula

tion of

seve

n-m

em

bra

ne-

spannin

g re

cepto

r tr

aff

icki

ng and si

gnall

ing.

Bio

chem

J,

2003.

375(P

t 3):

p. 503-1

5.

4.

Lin

, Q

.,

et

al.,

The

Cdc4

2

targ

et

AC

K2

inte

ract

s w

ith

sort

ing

nex

in

9

(SH

3P

X1)

to r

egula

te e

pid

erm

al

gro

wth

fact

or

rece

pto

r deg

radati

on.

J B

iol

Chem

, 2002. 277(1

2):

p. 10134-8

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5.

MaC

aula

y,

S.L

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t al

., I

nsu

lin s

tim

ula

tes

move

ment

of

sort

ing n

exin

9 b

etw

een

cell

ula

r co

mpart

men

ts:

a

puta

tive

ro

le

med

iati

ng

cell

su

rfa

ce

rece

pto

r

expre

ssio

n a

nd i

nsu

lin

act

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ioch

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p.

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4.

6.

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. an

d S

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lsso

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Sort

ing n

exin

9 p

art

icip

ate

s in

cla

thri

n-

med

iate

d e

ndocy

tosi

s th

rough i

nte

ract

ions

wit

h t

he

core

com

ponen

ts.

J B

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Chem

, 2003. 278(4

7):

p. 46772-8

1.