1 / 30

Biochem 503 December 1, 2008 Protein Tyr Phosphatases assigned reading: Stoker (2005) J. Endocrin. 185 :19-33 Tonks

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. History of P-Tyr Phosphatases 1981-1982 First description of P-Tyr specific phosphatase

fulbright
Download Presentation

Biochem 503 December 1, 2008 Protein Tyr Phosphatases assigned reading: Stoker (2005) J. Endocrin. 185 :19-33 Tonks

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Biochem 503 December 1, 2008Protein Tyr Phosphatasesassigned reading:Stoker (2005) J. Endocrin.185:19-33Tonks (2006) Nature Reviews MCB 7: 833-846

  2. History of P-Tyr Phosphatases 1981-1982 First description of P-Tyr specific phosphatase activity in A431 cell membrane fractions. EGF-R substrate Selective inhibition by Zn2+…… or vanadate 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. 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 kDa from various tissues and cell lines. Reaction with EGFR, InsR, IGF-R, requirement for SH.

  3. 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-45 CD45 already known as abundant lymphocyte surface antigen PTP1B Nick Tonks CSHL 33% 40% CD45, aka LCA, B220

  4. 1990’s cloning of multiple PTPs Ben Neel Harvard Med. School, now at Ontario Cancer Inst, Toronto ER localization of PTP1B cloning and properties of SH2-PTPs motheaten mouse (SHP hypomorph) PTP1B knockout mouse Jack Dixon Purdue; U of Michigan; now at UCSD and CSO of HHMI (National Academy of Sciences USA) 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

  5. 1990’s 3D X-ray structures of PTPs - VH1, PTP1B Knockout mouse for PTP1B Vanadate complex with VHR mimics phospho-enzyme PTP1B structure and discovery of Cys Oxidation to cyclic sulfenamide Mark Saper Univ. of Michigan David Barford Oxford England now ICR PTP1B knockout mouse critical test of PTP as drug target for diabetes Michel Tremblay McGill Univ., Montreal

  6. PTP-like Phosphatases in The Human Genome transmembrane cytoplasmic assorted others Cell (2004) 117:699

  7. N-terminal Central a3-helix Crystal structures of six PTP domains show a conserved fold and Ca-backbone Superimposition of PTP1B (magenta), RPTPa (gray), RPTPm (red), LAR (blue), SHP1 (green) and SHP2 (yellow). Andersen et al Mol. Cell. Biol. 2001

  8. The PTP1B active site Phosphotyrosine (Substrate) Phe180 Gln262 Q-loop pTyr-loop Tyr46 Asp181 WPD-loop PTP-loop Arg221 Cys215

  9. Protein Tyr Phosphatases (PTPs) A. Common Enzyme Mechanism and Kinetics1. 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 topoisomerases unusual KM < 1 uM, implies special interactions - trapping mechanism2. signature catalytic site sequence HCxxGxxRwith essential Cys SH group, and R residue

  10. 3. Catalytic 2-step Mechanism of PTPs pKa ~5.5 181 181 222 215 215 fast slow 215

  11. 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

  12. 5. reversible oxidation of Cys to regulate activityforms cyclic cysteinyl-sulfenyl-amide, can be reduced by thiols ROS or H2O2 Cys-SH Cys-SOH GSH OH S OH CH2 CH2 HN-CH-C-NH-CH-C-NH O O

  13. Red-Ox Regulation of PTPs

  14. B. Sub-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? Oxidation? 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

  15. Receptor-like PTPs Transmembrane Proteins Differential expression E14-16 LAR PTP-sigma PTP-delta

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

  17. 2. Cytosolic PTPs, the prototype PTP1Ba. 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

  18. PTP catalytic domain

  19. Tyrosine Phosphorylation of Insulin Receptor Resistance to High Fat Diet +/+ Liver -/-,+/- Muscle +/+ Muscle -/-,+/-

  20. Co-Crystal of PTP1B with Chemical Inhibitor - Cmpd2

  21. PTP catalytic domain

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

  23. SHP2 phosphatase Activating mutations in Noonan’s Syndrome SH2 PTP SH2 Split open to expose interface

  24. 3. Dual Specificity Phosphatases, the prototype VH1a. mechanism common with PTPs, i.e. Cys-Phosphate but shallow active site to accommodate P-Ser/P-Thrb. the MKPs (DUSPs), 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(??)

  25. Dual-Specificity Phosphatase: DUSPs MAPK PPase Catalytic domain reacts with pTyr-X-pThr Cys…Arg….Asp C R D

  26. MAPK Phosphatases : Use of Docking + Catalysis

  27. cdc25 - not really related to other PTPs

  28. 4. Other Cys-dependent Phosphatases 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

  29. Overview of the PTP Superfamily of Phosphatases

More Related