Biochem 503 Fall Protein Tyr Phosphatases

Transcription

1 Biochem 503 Fall 2005 Protein Tyr Phosphatases David Brautigan assigned reading: Stoker (2005) J. Endocrin. 185:19-33 History First description of P-Tyr specific phosphohydrolyase activity in cell membrane fractions. EGF-R as substrate Selective inhibition by Zn 2+ or vanadate Protein Phosphotyrosyl Phosphatase PubMed Items of :Swarup G, Cohen S, Garbers DL. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate.biochem Biophys Res Commun Aug;107(3): No abstract available. PMID: [PubMed - indexed for MEDLINE] 283:Swarup G, Speeg KV Jr, Cohen S, Garbers DL. Phosphotyrosyl-protein phosphatase of TCRC-2 cells.j Biol Chem Jul 10;257(13): PMID: [PubMed - indexed for MEDLINE 284:Brautigan DL, Bornstein P, Gallis B. Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn.J Biol Chem Jul 10;256(13): PMID: [PubMed - indexed for MEDLINE 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 forms size of 35 kda 1

2 purification of PTP from human placenta (Tonks and Fischer) partial peptide sequence of PTP1B (Charbonneau, Tonks, Walsh & Fischer) 1990 demonstration of CD45 as a PTPase Unexpected sequence similarity to existing protein CD-45 CD45 already known as abundant lymphocyte surface antigen Nick Tonks CSHL PTP1B 40% 33% CD45, aka LCA, B s sequence and cloning of other PTPs Ben Neel at Harvard Med. School ER localization of PTP1B cloning and properties of SH2-PTPs motheaten mouse (SHP hypomorph) Jack Dixon at Purdue; U of Michigan; UCSD showed Phospho-Cys intermediate in PTPs active site sequence motif used to find distant relatives VHR viral PTP related to VH1 DSP YOP51 Yersina virulence gene PTEN is a lipid phosphatase for PIP3 2

3 1990 s 3D X-ray structures of PTPs - VH1, PTP1B, etc Mark Saper Univ. of Michigan Vanadate complex that mimics phospho-enzyme Oxidation of Cys to cyclic David Barford Oxford England PTP1B knockout mouse critical test of PTP as drug target for diabetes Michel Tremblay McGill Univ., Montreal PTP Phosphatases in The Human Genome transmembrane cytoplasmic assorted others Cell (2004) 117:699 3

4 Protein Tyr Phosphatases (PTPs) A. Mechanism of action and Kinetics 1. specificity for P-Tyr, vs, P-Ser or P-Thr a large (long, aromatic) sidechain P-Tyr is a high energy phosphoester, used as intermediate in topoisomerases unusual K M < 1 um, implies special interactions - trapping mechanism 2. signature catalytic site sequence HCxxGxxR with essential Cys SH group, and R residue Catalytic Mechanism of Phosphoester Hydrolysis by PTPs pk a ~5.5 fast slow 4

5 3. phospho-enzyme intermediate Cys-S-PO 4 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 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 H 2 O 2 forms cyclic cysteinyl-sulfenyl-amide, can be reduced by thiols ROS or H 2 O 2 Cys-SH > Cys-SOH OH S OH CH 2 CH 2 HN-CH-C-NH-CH-C-NH O O 5

6 B. Families of Tyr Phosphatases (PTPs) 1.Transmembrane PTPs - the prototype CD-45 a. common features (most) 1. single TM helix to span membrane 2. double PTP domain, with activity in N terminal (D1) domain 3. large extracellular domains, related to cell-cell adhesion 4. inhibited by dimerization - the 'wedge' hypothesis 5. activators of src kinases by Tyr527 dephosphorylation b. differences 1. tissue and developmental expression 2. substrate specificity, but few targets known knockouts and trapping mutants 3. inhibitors of active sites as pharmaceuticals 6

7 Regulation of Transmembrane PTPs by Oxidation of D2 Domains Cys-SH Cys-SH ROS Cys-SH Cys-SNH active inactive 7

8 2. Cytosolic PTPs, the prototype PTP1B a. common features 1. single PTP domain, plus targeting sequences 2. specificity for P-Tyr vs. P-Ser 3. Phospho-Cys-enzyme intermediate 4. Substrate trap by conformational movement 5. Oxidation-reduction control mechanism b. differences 1. tissue expression 2. specificity for substrates 3. Inhibition by small molecules PTP catalytic domain 8

9 SHP2 phosphatase regulated by P-Tyr binding: either intrasteric or intermolecular P-Tyr P-Tyr SH2 SH2 SH2 PTP SH2 PTP Y542 Y580 9

10 SHP2 phosphatase Activating mutations in Noonan s Syndrome SH2 PTP 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, MAP kinase phosphatases binds to MAPK at site in N terminal domain and this activates the MKP C terminal catalytic domain several members : CL100, MKP1, 2, 3, 4, c. the cdc25 family of CDK phosphatases low activity phosphatase with extreme specificity large inhibitory domain, activated by phosphorylation not really a family member- it s like Rhodanase(??) 10

11 Dual-specificity Phosphatase - MAPK PP-ase Catalytic domain 11

12 cdc25 - not really related to other PTPs 12

13 4. Other Cys-dependent Phosphatases - prototype PTEN a. enzymes that share the catalytic site motif HCxxxR discovered by sequence searches b. PTEN mutated in many human tumors. lipid phosphatase, removes P from PIP3 to reverse action of PI3K results 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 13