Growth Factors, Receptors, and Signal Transduction II

Tumor Biology: Cellular and Molecular Aspects of the Transformed Cell

Growth Factors, Receptors,

and Signal Transduction II

Overview

o Intracellular signaling “downstream” of receptors

- key pathways: Ras/MAPK, Src, PI3K

o Intracellular signaling defects in cancer

o Targeted therapies to intracellular signaling molecules

o TNF and TRAIL

Brief Review – Growth Factors and Receptors o most growth factors are secreted

o receptors are transmembrane proteins

o 3 major features:

o extracellular domain

o transmembrane region

o intracellular domain

o where, when, and

how they are expressed

determines their biological

function

Brief Review – Receptor Activity

o intracellular, or catalytic, domain has kinase activity

o kinases add phosphate groups to (phosphorylate)

specific amino acids

ATP-binding Phospho-transferase

ATP

ADP

P Amino Acid P

RAS P

RTK

P Grb2 SOS

P

Akt

PI3K

p85

p110

RAS

Raf

MEK

P

P

ERK

GROWTH

FACTOR

PIP3 PIP3

PDK1

PROLIFERATION

MDM2 BAD

P

NF-ĸB

P

FKHR

P

CELL SURVIVAL

p70S6K

P

GSK3

P P

PROTEIN SYNTHESIS

Consequences of RTK activation

. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

Intracellular Signaling

o begins with autophosphorylated or transphosphorylated

amino acids on the receptor

o Phosphorylation recruits other proteins to the receptor

o Amino acids surrounding the phosphorylation site

determine which proteins are bound…

Membrane

Kin

as

e D

om

ain

-Tyr

-Tyr

P

P

4 major protein interaction domains:

SH2, PTB, SH3, PH

Basics of Amino Acid Structure

Basics of Protein Structure

o Primary Structure =

“beads on a string”

o Quaternary Structure =

specific folding creates

domains, or “units” of the

protein

SH2 domains

o SH2 = src homology 2

o was first identified as a 100 amino acid region of

homology (“sameness”) in the src tyrosine kinase

o specifically recognizes phosphorylated Tyrosine

o 2 classes of SH2 domain-containing proteins…

- have enzymatic activity (like Src)

- don’t have enzymatic activity

o Those with no enzymatic activity bind other

proteins to the receptor…adaptor molecules

SH2 domain specificity

Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997

o specificity is determined by the amino acids C-terminal

to the phospho-Tyr

o in most cases, it is the amino acid at position +3 that

determines specificity

Hydrophobic amino acid

http://0-www.sciencemag.org.library.lausys.georgetown.edu/content/vol278/issue5346/images/large/se5276134002.jpeg

PTB domains


o phosphotyrosine binding domains recognize amino

acids N-terminal to the phospho-Tyr

o PTB-containing proteins also participate in hormone

signaling

any amino acid


SH3 domains


o src homology 3 domains recognize amino acid

sequences rich in Proline (Pro-rich)

o Is a more general protein-protein interaction motif…

many cytoskeletal proteins contain it

any amino acid


PH domains

o pleckstrin homology domains recognize phospholipids

(components of the plasma membrane)



Protein-Protein Interactions and Receptors

o Proteins with many different interaction domains

can bind to growth factor receptor family members

Figure 6.9 The Biology of Cancer (© Garland Science 2007)

o Protein-protein

interactions connect

receptors to their

intracellular signaling

networks

Receptors bind other Kinases,

and Adaptor Proteins

Figure 6.10a The Biology of Cancer (© Garland Science 2007)

EGFR and the Ras Pathway

P

EGFR

P Grb2 SOS

RAS

Raf

MEK

P

P

ERK

EGF

PROLIFERATION CELL SURVIVAL


Grb2: adaptor protein that binds to

phosphorylated Tyr on EGFR using

its SH2 domain

Grb2 has multiple protein interaction domains

Cell. 2004 Jan 23;116(2):191-203.

EGFR and the Ras pathway, cont’d.

P

EGFR

P Grb2 SOS

RAS

Raf

MEK

P

P

ERK

EGF



SOS binds to the Grb2 SH3 domain

SOS activates Ras

DH PH CDC25

Proline-rich

Dbl-Homology

Membrane-

targeting

Catalytic Domain SH3-Binding

Rho GTPases (Rac)

Grb2

EGFR

o SOS is an exchange factor (it exchanges one

nucleotide for another)

A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).

What is Ras?

o Ras is an oncogene

o Ras is a small GTP-binding protein…it binds

guanine triphosphate

o Ras bound to GTP is active…Ras bound to GDP

is inactive

o Ras mutation is implicated in many kinds of cancer…



Lung carcinoma K-Ras codon 12 (GGTgly) >AGTser

Pancreatic Carcinoma K-Ras codon 12 (GGTgly) >GTTval

Bladder Carcinoma H-Ras codon 12 (GGCgly) >GTCval

Melanoma N-Ras codon 61 (CAAgln)>CGAarg

Ras Mutations display Tumor Specificity

How is Ras activated?


What does active Ras do?


Autocrine growth factor signaling

What else does active Ras do?

P

EGFR

P Grb2 SOS

RAS

Raf

MEK

P

P

ERK

EGF



Ras activates Raf and the Erk/MAPK

pathway

The Erk/MAPK pathway

o Erk = extracellular signal regulated kinase

o MAPK = mitogen activated protein kinase

o MAPK promotes cell growth and survival by

phosphorylating other proteins

o An immediate consequence of MAPK activation

is transcription (DNA → RNA)

o MAPK activation is a major

event following EGF

stimulation…

Nature Reviews Cancer 4, 937-947 (2004)

Inhibitors of the MAPK Pathway

Nature Reviews Cancer 4, 937-947 (2004)

Mechanisms of Ras/MAPK inhibitors

o inhibit Ras binding to the plasma membrane

o inhibit Raf

o inhibit MEK

o some of these have entered clinical studies…

P P Grb2 SOS

RAS

Raf

MEK

P

P

ERK

EGFR

EGF


P

RTK

P P

Akt

PI3K

p85

p110

GROWTH

FACTOR

PIP3 PIP3

PDK1

MDM2 BAD

P

NF-ĸB

P

FKHR

P

CELL SURVIVAL

P

GSK3

P

EGFR and the PI3K pathway


What is PI3K?

o PI3K = phosphatidyl inositol 3-kinase

o phosphatidyl inositol is a lipid, part of the plasma

membrane

o so, PI3K phosphorylates lipids (fats) instead of

other proteins

o There are 2 subunits of PI3K…

PI3K

p85

p110

p85 = regulatory subunit

p85 has an SH2 domain that binds

phospho-Tyrosine on the EGFR

p110 = catalytic subunit that

phosphorylates lipids Kin

as

e D

om

ain

-Tyr

-Tyr

P

P

PI3K Target(s)

o PI3K phosphorylates PIP2 to make PIP3

o PIP3 is now a binding site for Akt…


Akt – the real master regulator

P

RTK

P P

Akt

PI3K

p85

p110

GROWTH

FACTOR

PIP3

MDM2 BAD

P

NF-ĸB

P

FKHR

P P

GSK3

P

CELL SURVIVAL


o Akt is a Serine/Threonine

kinase

o Akt has targets in the

cytoplasm as well as the nucleus

o Akt inhibits the cell cycle

inhibitors

o inhibitor + inhibitor = GROWTH,

and SURVIVAL S

M

G1 G2 Cyclins,

Cyclin-dependent

kinases, and

inhibitors

Table 6.3 The Biology of Cancer (© Garland Science 2007)

PI3K/Akt Defects in Cancer

PI3-K

GF RTK

PIP3

Akt1/2

TSC1

RheB

mTOR

S6K 4EBP-1

Protein synthesis

Cell growth/size/survival

p

(Tuberous Sclerosis

Complex) TSC2

Tuberin Hamartin

Perelman (2004) Dematology Online Journal 10: 17. Kovich & Cohen (2004) Dematology Online Journal 10: 3.

(Ras-homology

enriched in brain)

(Target of rapamycin)

Lipid Kinase

Ser/Thr Kinase

GI, Br, Ov

Cancer Syndromes

PANC

Inhibitors to PI3K and/or

Akt are being developed

for patient use

pp60 c-Src

o “normal,” cellular Src is another protooncogene

o viral Src (v-src) is the transforming gene of the

avian Rous sarcoma virus

o Src is a tyrosine kinase, but NOT a receptor

o Cooperation/synergy with the EGFR in promoting

proliferation and tumorigenesis in breast cancer cells

Kin

as

e D

om

ain

-Tyr

-Tyr

P

P

Src

Cell Growth

Phosphorylation of transcription

factors

MAPK and PI3K pathways

c-Src phosphorylates many cellular proteins


Isolate protein and

load onto poly-acrylamide gel;

detect phospho-Tyrosine with

specific antibodies

c-Src and Cancer

o 30-70% of breast cancers overexpress Src, show

elevated activity…many of these also overexpress

EGFR

o Other cancers Src may contribute to are: colon,

lung, skin, endometrial, head/neck

o Src is usually not mutated…how is it activated?

Regulation of Src Activity

“Inactive” N

C

pY527

SH2

Kinase

SH3

Kinase domain has no

access to target

proteins

Y X

“Active”

N C

Kinase Kinase (p)Y527 SH2

SH3

Kinase domain is now

accessible

Viral Src has lost this

Tyrosine

How does Src become activated?

o Src can bind to EGFR or PDGFR, and the active

configuration is stabilized

o However, are other proteins that can bind to and

activate Src besides RTKs…

“Active”

N C

Kinase Kinase (p)Y527 SH2

SH3

Y X

Adaptor proteins with the right

binding sites for SH2 and SH3

domains could activate Src

The adaptor protein Cas can activate Src

RP640LPSPP

pY668DYV

N C

“Active” Kinase Kinase (p)Y527

SH2 SH3

Cas

o Cas is important for cell proliferation, cell migration,

and transformation by oncogenes (including viral Src)

Cas, Src, and Breast Cancer

o Cas, like Src, can also be overexpressed in breast

cancer

o Cas and Src bind to each other in breast cancer cells

o Cas and Src are localized to the same areas of

breast cancer cells

o Cas overexpression in breast cancer is associated

with resistance to a particular kind of drug

(Tamoxifen)…this involves Src activation

Cas Src Cas + Src

Inhibitors of Src in Cancer Therapy

o Inhibit protein-protein interactions (like those with

Cas)

o Inhibit kinase activity (preclinical/phase I trials)

o Inhibit protein stability (accelerate Src degradation)

o These will likely be used in combination with other

chemotherapy drugs, EGFR inhibitors, etc.

Adhesion Receptors

Figure 5.28b The Biology of Cancer (© Garland Science 2007)

Integrins

Figure 6.24b The Biology of Cancer (© Garland Science 2007)

Tyrosine Kinases and Adhesion Receptors:

Overlap in Cytoplasmic Signaling


G protein-coupled receptors:

More overlap

Tumor Necrosis Factor (TNF) Review o in some cells, TNF is mitogenic, but in

most it promotes cell death, or apoptosis

o this growth factor is not soluble, but is inserted into

the plasma membrane

o TNF receptors have no catalytic domain…rely on

intracellular proteins to signal

Juxtacrine

Intracrine

TNF family of growth factors

Schulze-Osthoff, Trends Cell Biol. 1994 Dec;4(12):421-426.

TNFR Signaling to Death

DD = death domain, another protein

interaction motif

DED = death effector domain

DD and DED form

dimers

CELL DEATH

Sci STKE. 2004 Jun 22;2004(239)

http://stke.sciencemag.org/content/sigtrans/vol2004/issue239/images/large/2392004re9F15.jpeg

TNFR Signaling to Survival

Other

proteins

CELL SURVIVAL

Sci STKE. 2004 Jun 22;2004(239)

TNF Signaling and cancer therapy:

Will this work?

http://stke.sciencemag.org/content/sigtrans/vol2004/issue239/images/large/2392004re9F15.jpeg

TRAIL Signaling to Survival and Death

TRAIL = TNF-related apoptosis-inducing ligand

Documents

Growth Factors, Receptors, and Signal Transduction II