Biochem 503 December 1, 2008

Protein Tyr Phosphatases

assigned reading:

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

Tonks (2006) Nature Reviews MCB 7: 833-846

Phosphotyrosyl Protein Phosphatase PTPP (original name)

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

Swarup G, Cohen S, Garbers DL. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate.Biochem Biophys Res Commun. 1982 Aug;107(3):1104-9.

Swarup G, Speeg KV Jr, Cohen S, Garbers DL. Phosphotyrosyl-protein phosphatase of TCRC-2 cells.J Biol Chem. 1982 Jul 10;257(13):7298-301.

History of P-Tyr Phosphatases1981-1982 First description of P-Tyr specific phosphatase

activity in A431 cell membrane fractions. EGF-R 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 of membrane and cytosolic PTPs of 35 kDafrom various tissues and cell lines.

Reaction with EGFR, InsR, IGF-R, requirement for SH.

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

1988-1989 partial peptide sequence of PTP1B - Unique phosphatase (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 TonksCSHL

PTP1B

CD45, aka LCA, B220

40% 33%

1990’s cloning of multiple PTPs

Jack Dixon Purdue; U of Michigan; now at UCSD and CSO of HHMI(National Academy of Sciences USA)

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 Harvard Med. School, now at Ontario Cancer Inst, Toronto

ER localization of PTP1Bcloning and properties of SH2-PTPsmotheaten mouse (SHP hypomorph)PTP1B knockout mouse

1990’s 3D X-ray structures of PTPs - VH1, PTP1BKnockout mouse for PTP1B

PTP1B knockout mousecritical test of PTP as drug target for diabetes

David BarfordOxford England now ICR

Michel TremblayMcGill Univ., Montreal

Mark SaperUniv. of Michigan

Vanadate complex with VHR

mimics phospho-enzyme

PTP1B structure and discovery of

Cys Oxidation to cyclic sulfenamide

PTP-like Phosphatases in The Human Genome

Cell (2004) 117:699

transmembrane

cytoplasmic

assorted others

Crystal structures of six PTP domains show a conserved fold and C-backbone

N-terminal

Central -helix

Superimposition of PTP1B (magenta), RPTP (gray), RPTP (red), LAR (blue), SHP1 (green) and SHP2 (yellow).

Andersen et al Mol. Cell. Biol. 2001

Phe180Phe180

Asp181Asp181

Tyr46Tyr46

Gln262Gln262

Arg221Arg221

WPD-loop

pTyr-loop

Q-loop

PTP-loop

Cys215Cys215

Phosphotyrosine (Substrate)

The PTP1B active site

Protein Tyr Phosphatases (PTPs)

A. Common Enzyme Mechanism and Kinetics

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

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

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

3. Catalytic 2-step Mechanism of PTPs

fast

slow

pKa ~5.5

215

215

215

181

181

222

4. phospho-enzyme intermediate Cys-S-PO4

a. 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

5. reversible oxidation of Cys to regulate activity forms cyclic cysteinyl-sulfenyl-amide, can be reduced by thiols

OH S OH CH2 CH2

HN-CH-C-NH-CH-C-NH O O

ROS or H2O2

Cys-SH Cys-SOH

GSH

Red-Ox Regulation of PTPs

B. Sub-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? Oxidation? 5. activators of src kinases by Tyr527 dephosphorylation

b. differences

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

knockouts and trapping mutants3. inhibitors of active sites as pharmaceuticals

E14-16 LAR PTP-sigma PTP-delta

Receptor-like PTPsTransmembrane Proteins

Differential expression

Cys-SH

Cys-SH

Cys-SH

Cys-SNHROS

active inactive

Regulation of Transmembrane PTPs by Oxidation of D2 Domains

2. Cytosolic PTPs, the prototype PTP1B

a. common features 1. 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

Liver

Muscle

Muscle

Tyrosine Phosphorylation of Insulin ReceptorResistance to High Fat Diet

-/-,+/-

-/-,+/-

+/+

+/+

Co-Crystal of PTP1B with Chemical Inhibitor - Cmpd2

PTP catalytic domain

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

PTP

SH2

SH2PTP

SH2 SH2

P-Tyr P-Tyr

Y542

Y580

active

SHP2 phosphataseActivating mutations in Noonan’s Syndrome

PTP

SH2

SH2

Split open to expose interface

3. Dual Specificity Phosphatases, the prototype VH1

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

b. the MKPs (DUSPs), 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(??)

Dual-Specificity Phosphatase: DUSPs MAPK PPase Catalytic domain reacts with pTyr-X-pThr

Cys…Arg….Asp C R D

MAPK Phosphatases : Use of Docking + Catalysis

cdc25 - not really related to other PTPs

4. Other Cys-dependent Phosphatases

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