Biochem 503 November 25, 2008 Protein Tyr Kinases Assigned reading (pdf are on-line) • Tony Pawson (2004) Cell 116: 196-203 • Joseph Schlessinger (2002) Cell 110: 660-672 • Blume-Jensen & Hunter (2001) Nature 411:355-365 Protein Kinases in the Human Genome Protein Tyr Kinases Cell Signaling Technologies <www.cellsignal.com> Protein Ser/Thr Kinases Protein Tyr Kinases - two classes “Receptor” i.e. transmembrane QuickTime™ and a TIFF (Uncompressed) decompressor are need ed to see this picture. 1. History of Protein Tyr Phosphorylation read assigned Pawson article in Cell, 2004 A. Viral oncogenes; SRC and Cancer Quic kT ime™ and a T IF F (Unc om pres sed) dec ompres s or are needed to see this pict ure. 1917 Peyton Rous - avian sarcoma virus, 1960’s the src virus gene as causative agent Peyton Rous 1976 Varmus & Bishop show a c-src gene in humans oncogene and proto-oncogene Qu i ckTi me ™ an d a - viral and endogenous TIFF (Un co mp re ss e d) de co mp re ss or a re ne ed ed to se e thi s p i ctu re . the virus as an agent to distort endogenous signaling that controls cell J. Michael Bishop and Harold Varmus proliferation UCSF; Nobel Prize 1978-1980 Ray Erikson (Univ. of Colorado) with postdoc Joan Brugge (now both at Harvard) antisera from rabbits injected with RSV QuickT ime™ and a IgG against the src protein: p60src T IF F (Uncompressed) decom pressor c n Qu i k Ti me ™ a d a are needed to see this pict ure. F re d e TIF (Un co m p s s e ) d e co m p r s s o r e t c a re n e e d d to s e e h i s p i tu re . Marc Collett and Erikson src protein immunoppt. + ATP-32P, get 32P-IgG HC (55 kDa) There’s a kinase activity in v-src! Tony Hunter (Salk Inst., LaJolla CA) QuickTime™ and a TIF F (U ncompr essed) decompr essor are needed to see this pictur e. recognizes P-Tyr in ppt of PYmTAg using 2D TLE method kinase activity, but unlike any known at the time. HN O CH-CH2 O P--O - O O=C Phospho-Tyrosine NH B. growth factor receptors Stanley Cohen 1980 EGF receptor, EGF found as hormone QuickT ime™ and a Vanderbilt T IF F (Uncompressed) decompressor are needed to see this picture. (Nobel Prize) action of EGF in A431 cells found 32P-labeling of 150 kDa band mistaken as P-Thr, then shown as P-Tyr Ora Rosen 1982 insulin receptor kinase activity Einstein Med. Col Tyr auto-phosphorylation-multiple P-Tyr deceased 1990 sequence reveals pre-pro-protein,cleaved and dimerized into a2b2 1983-85 technology of gas-phase peptide Jos. Schlessinger Qu i ckTi m e™ a nd a TIFF (Un co mp res se d) de c omp res so r micro sequencing a re ne ed ed to se e th is pi c tu re. Tel Aviv, NYU, Yale other GFRs shown to have PTK activity: IGF-1, PDGF, FGF v-erbB oncogene is cytoplasmic domain Q uic kT im e™ a n d a T IFF ( Un co m pr e ss e d) d ec o mp r es s or Tony Pawson of EGF receptor…..virus produces unregulated a re n ee d ed to s e e th is p ictu r e. Toronto version of growth regulator enzyme (PTK) GRB = growth factor binding proteins……contain SH2 domains that bind to P-Tyr 2. Analysis of Protein Phosphorylation Qu i ckTi me ™ an d a A. P-Tyr vs. P-Ser or P-Thr 2D method TIFF (Un co mp re ss e d) de co mp re ss or a re ne ed ed to se e thi s p i ctu re . a. requires 32P-phosphoamino acid analysis, done by HCl hydrolysis and HV-TLE in 2 dimensions, pH 1.9 & pH 3.5, now 1D at pH 2.5. Non-quantitative due to different rates of hydrolysis b. phospho-specific antibodies P-Tyr (4G10, PY20), c. site-specific P-Tyr antibodies Quic kT ime™ and a T IF F (Unc om press ed) dec om pres sor are needed t o see t his pict ure. (quantitation but no stoichiometry) 1D method B. Distribution of P-Tyr a. species - metazoan enzymes…. only in multicellular organisms P-Tyr in yeast, but no PTKs, all P-Tyr from dual specificity kinases b. subcellular -membranes >90%, in particulate cell fraction (B above), mostly receptors + cytoskeleton or signaling complexes c. P-Tyr proteins at UVA led to FAK (Parsons) and MAPK (Weber) v-src targets in cells are in signaling pathways 3. Protein Tyr Kinases A. Receptor Protein Tyr Kinases -(prototype EGFR) 1. Common Features a. single pass transmembrane domain (alpha-helix) b. single kinase domain - with or without 'insert’ (docking site) c. variable and often large extracellular domain (hormone binding) d. small but important juxta-(near) membrane segment (docking) e. similar mechanism of activation (dimerization + phosphorylation) 2. Diversity of cell-surface proteins a. many separate genes, wide repertoire (kinase and docking specificity) b. complex extracellular domains, Ig and FNII domains c. cell adhesion contacts-bidirectional signaling seen in Eph family Receptor Protein Tyr Kinases - resembling EGFR 3. Activation Mechanism for Receptor PTKs a. ligand-induced dimerization in membrane b. Tyr phosphorylation of P-loop residues -->activity c. phosphorylation of juxtamembrane and C terminal regions d. recruitment of substrates and effectors SH2 and PTB domains interact with P-Tyr e. phosphorylation of recruited substrates: PI3K, PLCg IRS f. internalization and other actions (nuclear) Activation Mechanism for Receptor PTKs Assignment: Explain how this model is not accurate, relative to new structural data (see Schlessenger paper) Mechanism for Receptor PTKs Downregulation EGFR1 internalized, ErbB2 is not. EGF induces degradation, TNFa gives recycling. Inhibitors of Receptor PTKs in Clinical Use Cetuximab (Erbitux) Gefitinib (Iressa) Erlotinib (Tarceva) Chemical Inhibitors of EGF Receptor Tyr Kinase Currently in Use in Clinical Oncology - Oral Agents Gefitinib = Iressa = ZD1839 Erlotinib = Tarceva = OSI774 Lapatinib = GW572016 Mutations in ATP binding site of EGF-R and ERB- B2 occur in tumors and produces resistance to inhibitors QuickTime™ and a TIFF (Uncompressed) decompressor are neede d to see this picture. Int. J. Cancer (2005) 118: 257-262 B. Non-Receptor Protein Tyr Kinases (PTKs) 1. Common Features- prototype is the src family a. kinase domain, (SH1) often near C terminus b. targeting domains, most often SH2 [P-Tyr] and SH3 [Pro-XX-Pro] c. auto-inhibited by multiple mechanisms (see below) d. often lipid modified to localize at membrane; palmitate, myristate 2. Diversity and Specificity a. different tissue and developmental expression b. different targeting because all SH3 and SH2 not equivalent c. FERM domains send some PTKs to integrins d. active site specificity revealed by selective inhibitors Non-Receptor Protein Tyr Kinases (PTKs) Development of Chronic Myeloid Leukemia Chromosome re-arrangement produces an activated KINASE BCL-ABL to drive cell proliferation. Sussessful Targeted Therapy Gleevec Cures CML Chronic Myeloid Leukemia ATP GleevecTM BCR-ABL kinase 3. Activation Mechanisms for src-family PTKs a. covalent - phosphorylation in P-loop; intrasteric effects 1. phosphorylation of P-loop to allow peptide substrate binding AND 2. dephosphorylation of P-Tyr to relieve intrasteric P-Tyr::SH2 b. allosteric - constraints on C helix and ATP site 1. SH3 engagement to release conformational restraint 2. SH2 binding to compete away intrasteric interaction Structure of src-family PTKs Explain src activation using this diagram (next slide) Activation Mechanisms for src-family PTKs Orientation of the C Helix is Critical for Kinase Activity positions the essential Glu91 for catalysis C. Dual-specificity Kinases [Tyr + Ser/Thr] 1. CDK-kinases that inactivate CDK (Myt1) a. dual specificity for Tyr-Thr in CDK N terminal domain b. regulators that prevent cell cycle progression c. conserved in early eucaryotes…source of P-Tyr where no PTKs 2. MAP kinase kinases (MEK, for MAP/ERK kinase) a. dual specificity for Tyr-X-Thr in activation loop, activate substrate kinase b. specificity for target kinase, MAPK, JNK, p38K, etc. c. inhibited by non-competitive compounds… ..novel structure and action and specificity Structure of Wee1 Kinase Complexed with Active Site Inhibitor QuickTime™ and a TIFF (Un compressed) decompressor are neede d to see this picture. Wee1 looks like a Ser/Thr kinase (in 1o and 3o structures) but phosphorylates only Y15 in CDK2. Structure of Dual-Specificity MEK1 Kinase with MgATP plus Non-Competitive Inhibitor QuickTime™ an d a TIFF (Uncompressed) decompressor are need ed to see this picture . MEK1 Kinase: Non-competitive Inhibitor Produces “Closed” Conformation with Displaced C Helix Comparison-Overlap of PKA and MEK1 As a result of the change in position of helix C, the side chain of the highly conserved QuickTime™ an d a TIFF (Uncompressed) decompressor glutamate residue Glu114 are need ed to see this p icture . (equivalent to Glu91 in PKA) is shifted away from the active site and is unable to form a critical ion pair with the conserved catalytic Lys97 (equivalent to Lys72 in PKA).