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Biochem 503 Fall 2005

Protein Tyr Phosphatases

David Brautigan

assigned reading:

Stoker (2005) J. Endocrin. 185:19-33

Protein Phosphotyrosyl Phosphatase

PubMed Items 282 - 284 of 284282:Swarup G, Cohen S, Garbers DL. Inhibition of membrane phosphotyrosyl-protein phosphatase activity byvanadate.Biochem Biophys Res Commun. 1982 Aug;107(3):1104-9. No abstract available. PMID: 6291515 [PubMed -indexed for MEDLINE]

283:Swarup G, Speeg KV Jr, Cohen S, Garbers DL. Phosphotyrosyl-protein phosphatase of TCRC-2 cells.J BiolChem. 1982 Jul 10;257(13):7298-301. PMID: 6282838 [PubMed - indexed for MEDLINE

284:Brautigan DL, Bornstein P, Gallis B. Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn.J Biol Chem.1981 Jul 10;256(13):6519-22. PMID: 6165721 [PubMed - indexed for MEDLINE

History 1981-1982 First description of P-Tyr specific phosphohydrolyase

activity in cell membrane fractions. EGF-R as substrateSelective inhibition by Zn2+ or vanadate

1983-1986 Development of assays using P-Tyr proteins: histone, poly[Glu:Tyr], denatured BSA and lysozyme.Biochemical fractionations and properties of PTPs from various tissues and cell lines. Reaction with GFRs, requirement for SH; both membrane and cytosolic formssize of 35 kDa

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1987-1988 purification of PTP from human placenta (Tonks and Fischer)

1988-1989 partial peptide sequence of PTP1B (Charbonneau, Tonks, Walsh & Fischer)

1990 demonstration of CD45 as a PTPase

Unexpected sequence similarity to existing protein CD-45CD45 already known as abundant lymphocyte surface antigen Nick Tonks

CSHL

PTP1B

CD45, aka LCA, B220

40% 33%

1990’s sequence and cloning of other PTPs

Jack Dixon at Purdue; U of Michigan; UCSD

showed Phospho-Cys intermediate in PTPsactive site sequence motif used to find distant relativesVHR viral PTP related to VH1 DSPYOP51 Yersina virulence genePTEN is a lipid phosphatase for PIP3

Ben Neel at Harvard Med. School

ER localization of PTP1Bcloning and properties of SH2-PTPsmotheaten mouse (SHP hypomorph)

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1990’s 3D X-ray structures of PTPs - VH1, PTP1B, etc

PTP1B knockout mousecritical test of PTP as drug target for diabetes

David BarfordOxford England

Michel TremblayMcGill Univ., Montreal

Mark SaperUniv. of Michigan

Vanadate complex that mimics phospho-enzyme

Oxidation of Cys to cyclic

PTP Phosphatases in The Human Genome

Cell (2004) 117:699

transmembrane

cytoplasmic

assorted others

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Protein Tyr Phosphatases (PTPs)A. Mechanism of action and Kinetics

1. specificity for P-Tyr, vs, P-Ser or P-Thra large (long, aromatic) sidechainP-Tyr is a high energy phosphoester, used as intermediate in

topoisomerasesunusual KM < 1 uM, implies special interactions - trapping mechanism

2. signature catalytic site sequence HCxxGxxRwith essential Cys SH group, and R residue

Catalytic Mechanism of Phosphoester Hydrolysis by PTPs

fastslow

pKa ~5.5

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3. phospho-enzyme intermediate Cys-S-PO4a. vanadate mimics the transition state of phosphate

b. proton transfer to leaving group TyrOH by D181 is essential c. hydrolysis and product (PO4) release is rate-limiting, d. 1000 sec-1 reduced to 0.02 sec-1 by D181A mutation e. either Cys to Ser or Asp to Ala mutations make inactive PTP

4. reversible oxidation mechanism of Cys by H2O2forms cyclic cysteinyl-sulfenyl-amide, can be reduced by thiols

OH S OH CH2 CH2HN-CH-C-NH-CH-C-NH O O

ROS or H2O2

Cys-SH ------> Cys-SOH

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B. Families of Tyr Phosphatases (PTPs)

1.Transmembrane PTPs - the prototype CD-45

a. common features (most)1. single TM helix to span membrane2. double PTP domain, with activity in N terminal (D1) domain3. large extracellular domains, related to cell-cell adhesion4. inhibited by dimerization - the 'wedge' hypothesis5. activators of src kinases by Tyr527 dephosphorylation

b. differences1. tissue and developmental expression2. substrate specificity, but few targets known

knockouts and trapping mutants3. inhibitors of active sites as pharmaceuticals

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Cys-SH

Cys-SH

Cys-SH

Cys-SNHROS

active inactive

Regulation of Transmembrane PTPs by Oxidation of D2 Domains

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2. Cytosolic PTPs, the prototype PTP1B

a. common features1. single PTP domain, plus targeting sequences2. specificity for P-Tyr vs. P-Ser3. Phospho-Cys-enzyme intermediate4. Substrate trap by conformational movement5. Oxidation-reduction control mechanism

b. differences1. tissue expression2. specificity for substrates3. Inhibition by small molecules

PTP catalytic domain

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SHP2 phosphatase regulated by P-Tyr binding:either intrasteric or intermolecular

PTP

SH2

SH2PTP

SH2 SH2

P-Tyr P-Tyr

Y542

Y580

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SHP2 phosphataseActivating mutationsin Noonan’s Syndrome

PTP

SH2

SH2

Split opento expose interface

3. Dual specificity Phosphatases, the prototype VH1

a. mechanism common with PTPs, i.e. Cys-Phosphatebut shallow active site to accommodate P-Ser/P-Thr

b. the MKPs, MAP kinase phosphatasesbinds to MAPK at site in N terminal domainand this activates the MKP C terminal catalytic domain

several members : CL100, MKP1, 2, 3, 4,

c. the cdc25 family of CDK phosphataseslow activity phosphatase with extreme specificitylarge inhibitory domain, activated by phosphorylationnot really a family member- it’s like Rhodanase(??)

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Dual-specificity Phosphatase - MAPK PP-aseCatalytic domain

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cdc25 - not really related to other PTPs

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4. Other Cys-dependent Phosphatases - prototype PTEN

a. enzymes that share the catalytic site motif HCxxxRdiscovered by sequence searches

b. PTEN mutated in many human tumors.lipid phosphatase, removes P from PIP3 to reverse action of PI3Kresults in elevated PIP3 and activation of Akt/PKB - survival signal

c. Myotubularin a family of proteins, other PIP3 phosphatases

d. cdc14 phosphatase dephosphorylates Thr in CDK activation loop

e. Slingshot dephosphorylates cofilin at Ser3 to regulate actin

Overview of the PTP Superfamily of Phosphatases