Signal transduction and protein kinase inhibitors. Feng Qian ( 钱峰 )

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1 Signal transduction and protein kinase inhibitors Feng Qian ( 钱峰 ) fengqian@sjtu.edu.cn

2 Protein Kinases in the Human Genome 518 kinases 1.7 % of human genome Lipid kinases Nucleotide kinases Cell Signaling Technologies <

3 Protein Kinases (518) The 478 conventional eukaryotic protein kinases (epks) plus 106 pseudogenes 388 Protein-serine/threonine kinases 90 Protein-tyrosine kinases 58 receptor PTKs 32 Non-receptor PTKs The 40 atypical protein kinases (e.g. EF2K/alpha kinases) = 518

4 History of protein kinase A. Viral oncogenes; SRC and Cancer 1917 Peyton Rous - avian sarcoma virus, 1976 Varmus & Bishop show a c-src gene in humans oncogene, endogenous signaling that controls cell proliferation, J. Michael Bishop and Harold Varmus, Nobel Prize There s a kinase activity in v-src! Tony Hunter (Salk Inst., LaJolla CA) recognizes P-Tyr, but unlike any known at the time.

5 History of protein kinase Stanley Cohen Vanderbilt (Nobel Prize) Ora Rosen Einstein Med. Col deceased 1990 B. growth factor receptors 1980 EGF receptor, EGF found as hormone action 1982 insulin receptor kinase activity Tyr auto-phosphorylation-multiple P-Tyr sequence other GFRs shown to have PTK activity: IGF-1, PDGF, FGF Jos. Schlessinger Tel Aviv, NYU, Yale Tony Pawson Toronto v-erbb oncogene is cytoplasmic domain of EGF receptor..virus produces unregulated version of growth regulator enzyme (PTK) GRB = growth factor binding proteins contain SH2 domains that bind to P-Tyr

6 Protein kinase superfamilies

7 General classes I ACG Group Protein kinase A; cyclic AMP-dependent protein kinase Protein kinase C Protein kinase G Basic amino acid-directed enzymes that phosphorylate serine/threonine

8 General classes II CaMK Calcium-calmodulin-dependent protein kinases I II III IV Type II is a broad specificity kinase

9 Calcium-Dependent Protein Kinases Protein Kinase C Requires calcium, diacylglycerol, and phospholipid for activity Diacylglycerol generated by the action of phospholipase C Activated by phorbol esters (tumor promoters) Many isozymes Calcium-calmodulin-Dependent Protein Kinases CAM Kinases I, II, III, IV Several other protein kinases activated by calmodulin including myosin-light chain kinase, phosphorylase kinase, some isoforms of adenylyl cyclase and some isoforms of phosphodiesterase

10 Protein Kinase C PKC C refers to calcium DAG and phospholipid were also described as necessary for the activation of this enzyme There are many isozymes that are products of different genes It is paradoxical to have PKCs that are independent of Ca 2+ and DAG

11 Selected Protein Kinase C Substrates PDGF receptor EGF receptor Insulin receptor Transferrin receptor Ribosomal protein S6 Raf

12 General classes III CMGC Cyclin-dependent protein kinases These are important regulators of the cell cycle MAP (Mitogen activated protein/microtubule associated protein) kinases Many of these promote cell division GSK3 (glycogen synthase kinase-3) Clk (Cyclin-dependent like kinase)

13 General classes IV PTK (Protein-tyrosine kinases) Receptor, e.g., epidermal growth factor receptor, insulin receptor Non-receptor, e.g., Src, Abl protein kinases Specifically phosphorylate protein-tyrosine (note they are not tyrosine kinases but protein-tyrosine kinases)

14 Protein Kinase Classifications Protein-Serine/threonine Protein-Tyrosine Receptor: ligand binding domain and catalytic site on the same polypeptide Non-receptor: catalytic domain separate from the receptor Dual Specificity (both serine/threonine and tyrosine) also occur Broad Specificity: have several substrates, e.g., PKA Narrow Specificity: have one or a few substrates, Classified by activator: PKA, PKG, PKC

15 Modulation of protein kinases The most important biochemicalregulatory system Reversible phosphorylation Transfer of -phosphate (ATP or GTP) Covalent modification Specialized binding domain Intitiating intracellular signaling

16 Protein kinase activation and inactivation

17 Phosphorylation of serine Phosphorylation of threonine Phosphorylation of tyrosine

18 Cellular functions of protein kinases Cell cycle (CDK, G0/1, S, and G2/M) Cell growth (Akt, proliferation) Cell apoptosis Cell shape and movement Metabolism Differentiation

19 Protein kinases in cancer cells

20 Protein tyrosine 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

21 Receptor protein Tyr kinases

22 Receptor protein Tyr kinases EGF receptorinsulin receptor

23 Activation mechanism for receptor PTKs Ligand-induced dimerization in membrane Tyr phosphorylation of P-loop residues -->activity Phosphorylation of juxtamembrane and C terminal regions Recruitment of substrates and effectors SH2 and PTB domains interact with P-Tyr Phosphorylation of recruited substrates: PI3K, PLC IRS Iinternalization and other actions (nuclear)

24 Activation mechanism for receptor PTKs

25 Downstream signaling molecules with PTK

26 Inhibitors of receptor PTKs in Clinical Use Cetuximab (Erbitux) Gefitinib (Iressa) Erlotinib (Tarceva)

27 Chemical Inhibitors of EGF Receptor Tyr Kinase Currently in Use in Clinical Oncology - Oral Agents Gefitinib = Iressa = ZD1839 Erlotinib = Tarceva = OSI774

28 Protein Tyr kinases 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 d. often lipid modified to localize at membrane 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

29 Non-receptor protein Tyr kinases

30 Src, A Non-Receptor Protein Tyrosine Kinase v-src was discovered as an oncogene of Rous sarcoma virus, a chicken virus, and this was the first oncogene to be described c-src, the normal homolog, is activated by PDGF and CSF receptors, which are in turn activated by their ligands and protein-phosphorylated Src may be activated by transmembrane receptors that lack protein-tyrosine kinase activity c-src is also phosphorylated and activated during mitosis Myristoylation of the N-terminus is required for attachment to the plasma membrane and for activity (Src lacks a signal peptide and is found initially in the cytosol) The physiological substrates for c-src are unknown despite exhaustive experimentation High activity in brain, a non-dividing tissue The protein contains SH2 domains that bind to protein tyrosine phosphates and SH3 domains that bind to proline-containing regions

31 Activation mechanisms for src-family PTKs

32 Development of chronic myeloid leukemia (CML) Chromosome re-arrangement Activated kinase BCL-ABL Driving cell proliferation

33 Bcr-Abl Protein Kinase The Philadelphia chromosome occurs in the granulocytes in patients with chronic myelogenous leukemia (CML) The resulting Bcr-Abl protein-tyrosine kinase is constituitively activated (it is active all of the time) Gleevec is a specific inhibitor of the Bcr-Abl proteintyrosine kinase, and Gleevec is used therapeutically for chronic myelogenous leukemia This malignancy is unusual because it results from a single genetic alteration; most cancers result from multiple somatic genetic alterations

34 The Philadelphia Chromosome It results from the reciprocal translocation involving chromosomes 9 and 22 This fuses the Abl gene from chromosome 9 to the Bcr gene on chromosome 22 This results in the transcription of an mrna corresponding to a Bcr-Abl oncoprotein with protein-tyrosine kinase activity The oncoprotein produces chronic myelogenous leukemia Ph 1 is usually the paternally derived chromosome 9 that is translocated to the maternal chromosome 22; it is a shortened chromosome 22

35 Successful targeted theerapy (Gleevec) ATP Gleevec TM BCR-ABL kinase

36 Imatinib (Gleevec) The first Food and Drug Administrationapproved protein kinase inhibitor Targeting the mutated form of the Alb tyrosine (BCR-Abl) Treating the patients with chronic myeloid leukemia (CML)

37 Protein kinase antagonists Imatinib: KIT, PDGFR, and Bcr-Abl Erlotonib: VEGFR-2 and EGFR tyrosine kinases Sorafenib: Raf, VEGFR-2/3, PDGFR-B, KIT and FLT-3 Dasatinib: Multiple Src family tyrosine kinases Lestaurtinib: FLT3 and Trk CP : Jak3 (Rheumatoid arthritis Phase II)

38 Anti-cancer TKI

39 Dual-specificity protein kinases (Tyr Ser/Thr) 1. CDK-kinases that inactivate CDK 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.

40 Mitogen activated protein kinases (MAPK)

41 MAP kinase cascades activate transcription factors MAPK: mitogen-activated protein kinase

42 Protein kinase activation and inactivation

43 How Many Protein Phosphatases? Studies from the human genome indicate that there are the following number of P ases 32 serine/threonine phosphatases 42 protein-tyrosine phosphatases 46 dual specificity phosphatases It is surprising that there are so many proteintyrosine and dual specificity P ases

44 Protein-Serine/threonine Phosphatases Catalytic subunit PPP Family PP1 PP2A Regulatory Elements >15 that target and regulate the catalytic subunit B subunits target and regulate core enzyme Regulated Functions Glycogen metabolism, muscle contraction, cell cycle, mrna splicing MAP kinase pathway, metabolism, cell cycle PP2B Ca/CAM activates T-lymphocyte activation, brain NMDA receptor signaling PPM Family PP2C Integral N or C-terminal peptides Antagonism of stress activated kinases

45 Protein kinases in immune cells

46 TCR signal transduction pathways

47 Signal transduction of T cell activation

48 Cytokine Receptors

49

50 Cytokine receptor-mediated signal

51 Targeting IL-2R-mediated signal pathways

52 Sirolimus( 西罗莫司 ) 1. Pre-drug sirolimus binds FKBP, but the complex inhibits mtor kinase 2. mtor activates p70s6k 3. mtor inhibition prevents activity of p34cdc2 which complexes with cyclin E, thus preventing elimination of p27kip which is a negative regulator of cdks and eif-4f 4. Results in inhibition of cell cycle progression at G1 to S phase.

53 Akt signal promoting lymphocyte survival

54 Summary Protein tyrosine kinases Protein serine/threonine kinases Protein kinases in cancer development Protien kinases in immune responses