Biochem 503 December 3, 2008Protein Ser/Thr Phosphatases (PPP family)

Controlling 99% of cellular phosphorylation

I. HistoryII. Type-1 Protein Phosphatase (PP1)III. Type-2A Protein phosphatase (PP2A)IV. Calcineurin (PP2B) Ca2+/CaM dependentV. PP2C or Mg2+-dependent PPase (MPP)

History of Ser/Thr Protein Phosphatases

1950’s 1. PR Enzyme Gerty Coriglycogen phosphorylase a to b conversion (phosphorylase phosphatase)

1960’s a lost decade? Ed Fischer students struggle

1970’s2. Glycogen synthase PPase - Tsuiki, Tamura, Kikuchi C’ville to Sendai connection…3 different enzymes separated later known as PP1, PP2A and PP2C (MPP)

3. The EtOH ppt method a grad student mistake becomes “standard” method - 35 kDa PP-ase (C subunit)

4. Isolation of inhibitor proteins (1976)..the C + I = CI idea of regulators

5. Same phosphatase for every substrate?…….histone, casein, myosin, tropomyosin, spectrin, ribosomes, enzymes……

“broad specificity” (i.e. non-specific) 35 kDa phosphatase

S. TamuraTohoku Univ.Sendai, Japan

E.Y.C. LeeU. Miami, now NYMC

History of Ser/Thr Protein Phosphatases

1980’s6. Separation of two phosphatases in 35 kDa fraction (Ingebritsen)

Classification (1983) Type 1 vs. type 2 A, B, C based on sensitivity to inhibitor proteins

7. Targeting hypothesis (Cohen, 1985) - glycogen-bound PP1 a dimer of [ PP1C plus glycogen-subunit]R subunits modulate activity and anchor C

8. PP1 binds to multiple different R subunits: GM and MYPT1myosin phosphatase PP1C plus MYPT1

9. PP2A binds 60 and 55 kDa subunits to form ABC trimercommon AC, different Bs (B55, B56, B72)NIH + Sendai + Hiroshima

Sir Phillip CohenDundee, Scotland

History

10. Cloning of C subunits of PP2A and PP1 unrelated to PTPs, but similar to purple acid PP-ase

11. Yeast-fungal genetics show functional conservation GLC7=PP112. Discovery of Okadaic Acid as selective PP1-PP2A inhibitor

1990’s13. Cloning of multiple PPP catalytic subunits PP4, PP5, PP6

Family Conservation & Diversity in Catalytic subunits (see PTW Cohen TIBS 22:245-251)

14. Many PP2A B subunits in Families- B, B’, B”15. Genomics - over 40 human genes, 24 in yeast

Marc MumbyUTSW

Tricia (PTW) Cohen(Lady Cohen)Dundee, Scotland

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.Edgar de Cruz e SilvaDundee, NYC, Portugal

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

David VirshupUniv. Utah, now SingaporeNUS-Duke Med Sch.

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.Anna DePaoli-RoachIndiana U Med.Sch.

B

The PPP Family of Protein Ser/Thr Phosphatases

Red = humanGreen = DrosophilaYellow = yeast

PP1

PP2APP4PP6

CaN (PP2B)

Toxin-sensitive PPPToxin-sensitive PPP

Natural Toxins from Diverse Sources Bind and Inhibit PPP Protein Phosphatases

DinoflagellatesProrocentrum lima

Blue-green Algae

Blister beetleColeoptera

Streptomyces (fostreus)

Protein Ser/Thr Phosphatases Are Dominant over Protein Kinases

most proteins are maintained in a de-phosphorylated state

Cell Signaling Technology 2002 catalogue (pg. 15): Western blot analysis of whole cell lysates of Jurkat cells,.untreated with 0.1M calyculin A for 20 minutes prior to lysis, using Phospho-Thr antibody.

+ PPP inhibitor

Catalytic Subunit of Protein Phosphatase-1 (PP1)

Okadaic Acid Binds at the Active Site of PPP Protein Phosphatases

Fe O O

H O P O-Ser-substrate

Zn O H-His

Mechanism of Phospho-Ester Hydrolysis by PPP Phosphatases:in-line attack of metal-activated hydroxide,with triginal bipyramid intermediate and inversion of stereochemistryprotonation of the alcoholic leaving group by active site His

A. Catalytic subunit1. Bimetal center Fe::Zn, Mechanism of direct hydrolysis

Type-1 Protein Phosphatase (PP1)

1. Bi-metallic active site with Fe and Zn

2. 3D structure - beta sheet and alpha helix clusters

3. Isoforms differences mostly in C terminal, allow specific antibodies alpha NPGGRPITPPRN--SAKAKK gamma --ATRPVTPPRGMITKQAKK delta NSG-RPVTPPRTANPPK-KR

4. Phosphorylation in RPITPPR motif first found in yeast sds22cyclin-dep kinases (CDK) phosphorylate to inactivate; reversed by auto-dephosphorylation

5. Toxins - microcystin, okadaic acid, calyculin A bind at active site (3D structures)

B. Regulatory/targeting subunits for PP1C1. The pioneer…..glycogen-binding GM

2. The RVxF Motif as primary recognition site3. Myosin phosphatase MYPT1 as regulator & scaffold4. Dozens more, >200 total…..phospho regulation

C. Inhibitor phospho-proteins1. Inh1 and DARPP-322. Inh2, Inh43. CPI-174. Linking kinases to PP1 inhibition-

inhibitors target selective R-C complexes

D. Substrates1. abundant phosphatase,

handles large capacity, abundant substrates…myosin, lamins, glycogen enzymes, histones

Protein Ser(P)/Thr(P) Phosphatase - PP1many different regulatory-targeting subunits

with common catalytic subunit

GL GM

PTG

GADD34

RVSF

KVKF

VXF

C

C

C

CMYPT1

myosin - cytoskeleton

protein synthesis

glycogen metabolism

Neurabin

dendritic spines

PP1

C

Dozens of R/K-VxF subunits

VXF

P

PKC

CPI-17

Myosin Phosphatase: PP1PP1--MYPT1MYPT1

Myosin Phosphatase inhibition by phosphorylation-dependent inhibitor protein CPI-17.

MYPT1

First example of an inhibitor specific for a PP1 Holoenzyme

Myosin Phosphatase Inhibitor CPI-17single residue phosphorylation (not T to D mutation)

condenses conformation and increases potency >2000-fold

Selective Inhibition of Myosin Phosphatase by CPI-17

same C subunit isoform bound to different R subunits

0

50

100

Pho

spha

tase

Act

ivity

(%

)

[P-CPI-17] (-log M)

MYPT1•PP1

Glycogen-bound PP1

10 9 8 7 6 5

Myosin LC Phosphatase: A complex of PP1C with MYPT1 Subunit

C subunit bound to specific R subunit

Type-2A Protein phosphatase (PP2A)

A. Catalytic subunit

1. Bimetal center Fe::Zn and catalytic Mechanism same as PP1

2. 3D structure…known in complex with A and in ABC

3. Isoforms 10:1 ratio, essential for development

DYFLCOOH motif at C terminus conserved phosphorylation - PTKs, eg. Src, JAKmethylation - PMT and PME, alters subunit

association

5. Toxins - MCLR, OA bind at active site. Differences between PP1 and PP2A in12-13

loop

Protein Ser(P)/Thr(P) Phosphatase - PP2A

ABC

C

HEAT Helicalrepeats

Pph21Pph22

ATpd3

B

ABC = { [A] Scaffold + [C] Catalytic} + [B] Regulatory

Binding of C to A decreases Vmax and alters KM

A subunit

Protein Ser(P)/Thr(P) Phosphatase - PP2Acovalent modifications of the catalytic subunit

ABC

C

ATpd3

BRTPDYFLCOOH

Tyr kinasesSrc, JAK, RTK

Tyr307

RTPDYFLCOOMe

PMT

PME

S-AdoMet

RTPDYFLCOOXRTPDYFLCOOX

PTK

self

inactive

OO-P-O

B

B. Scaffold and Regulatory subunits- the ABC’s of PP2A.

1. The Scaffold "A" or PR65 (HEAT repeats) the AC dimer A and A 85:15, but A specific tumor suppressor2. Many Bs….are they for dedicated functions?

Yeast Cdc55 and Rts1 - distinct phenotypes, not complemented (conclusion?)

now 3 B families cloned - more than 15 genesB B55 B’ B56 B’’ B72(130)

3. Tumor antigens, sT, mT, E4orf replace B subunits to re-direct activity.

4. More and more Bs?, p107, p48, etc. proposed as alternates

Protein Ser(P)/Thr(P) Phosphatase - PP2Amultiple dedicated enzymes on one platform

C4

TAP42

B55CDC55

kinase inactivationG-like propeller structure

B’56RDS1

anti-apoptosis integrin signalingpolarity-Wnt signaling

P

B’’72 P retinoids

anti - apoptosis

ABC

CPPH21PPH22

ATpd3

B

PP2A can be > 80 different “enzymes”

sm-t tumor virustransformation

The B56 Family of PP2A Regulatory Subunits

D-weiderborst

Paxillin bindinganti-metastatic

Wnt-B56-Dishevelled

The 3D Structure of PP2A Trimer AB56CNature, Nov. 2006

Side view Top view

C. Inhibitor proteins for PP2A1. Inh1(PP2A) Damuni et al2. Inh2(PP2A) SET protein - important in solid tumors

ceramide “receptor” for PP2A activation

D. Alternative partners1. A binds HSF2 and PP52. C binds alpha4; in yeast part of TOR signaling

E. Substrates1. Signaling Kinases (MEK, PKB, PI3K, p70S6K, etc.)2. anti-apoptosis (PKB, Bad, Bcl)3. integrin signaling - Paxillin B56 deletions & increased invasion

Calcineurin (PP2B)(PPP3)Ca2+/CaM activated Phosphatase

A. Catalytic subunit1. C has Fe::Zn active site like PP1, PP2A2. Suppressor domain, CaM dependent

B. Regulatory subunits1. B subunit (Ca2+)2. additional CaM

3. FKBP + FK-506 or cyclosporin ($B product)C. Inhibitor Proteins

1. induced in response to signaling have reported activity as activator-inhibitor.

D. Substrates1. NFAT (co-transport into nucleus)2. Elk-1 (TCF transcription factor)3. DARPP-32 (NMDA receptor)

Calcineurin (CnA +CaB) Inhibited byCyclosporin (CsA) and its Binding protein (Cyp)

$B drug forimmunosuppressionfollowing organ transplants

PP2C = Mg2+-dependent PPase (MPP)

A. Catalytic subunit1. unrelated to PPP but bimetallic Mg:Mg active center 2. isoforms , etc.3. many new family members in genome

B. Regulatory subunits - none?C. Inhibitor Proteins - none?D. Substrates

1. CDKs2. the kinase activation loop3. PI3K4. Glycogen synthase

Maybe activated by small molecule second messenger? Lipids?

Protein Phosphatase 2C Mg2+-dependent Phosphatase (MPP)

Protein Phosphorylationrapid and reversible biochemical reactions

A molecular on/off switching mechanism.

TARGET

ATP ADP

KINASES

PHOSPHATASES

Pi H2O

TARGET P

Protein Tyr(P) Phosphatases

Yeast 5C. elegans 95Drosophila 22Human 56

Protein Phosphorylation: Kinases & Phosphatases

TARGET P-Tyr TARGET TARGET P-Ser/Thr-TARGET

Protein Tyr Kinases Protein Ser/Thr Kinases

Protein Ser(P)/Thr(P) Phosphatases

Yeast 13C. elegans 51Drosophila 19Human 15

KINASE Superfamily ~ 500 enzymes

Cys-SH Fe::Zn

ATP ATP

Mg MgPPP MPP

Protein Phosphorylation: Kinases & Phosphatases

KINASE Superfamily ~ 500 enzymes

Cys-SH

Fe::Zn

ATP ATP

Mg Mg MPP

ATP

ATPATPATP

ATP

ATP

ATP

Cys-SH

Cys-SH

ATP

ATP

Fe::Zn

Fe::Zn

Phosphatase families and ensembles ~ 500 enzymes

ATP

Protein Kinases and

Protein Phosphatases

ancient enzymes essential to cell signaling

and cellular regulation

New targets for Pharmaceuticals