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תרגיל 3: טירוזין קינאזות ציטופלסמטיות

תרגיל 3: טירוזין קינאזות ציטופלסמטיות. Tyrosine Kinases. Non Receptors. Receptors. Membrane – spanning proteins with an extracellular ligand binding site. Cytosolic proteins. Mostly coupled to transmembrane proteins. Protein – protein interactions.

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תרגיל 3: טירוזין קינאזות ציטופלסמטיות

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  1. תרגיל 3: טירוזין קינאזות ציטופלסמטיות

  2. Tyrosine Kinases Non Receptors Receptors Membrane – spanning proteins with an extracellular ligand binding site Cytosolic proteins. Mostly coupled to transmembrane proteins Protein – protein interactions Growth and Differentiation Factors, Hormones

  3. The Road to Src: 1910 – Peyton Rous discovers a “filterable agent” that can transmit tumors in chickens. Later it was identified as the Rous Sarcoma Virus. Late 1950s – Some strains could induce tumors in mammalian hosts. 1958 – The development of the foci assay (Temin and Rubin) made it possible to study the interaction of a single cell with single virus particle 1960s- The ability of RSV to transform cells is independent of its ability to replicate. Isolation of mutant of RSV with a transformation-specific defect. Determination of the RSV sequence – it’s a retrovirus. Late 1970s- Brugge and Erikson identified a 60kDa phosphoprotein from RSV transformed cells using antibodies from tumor bearing rabbits. Early 1980s- Tony Hunter accidentally realized that Src is a tyrosin kinase and it was itself phosphorylated at tyrosine. Bishop and Varmus identified the cellular src gene (c-src).

  4. Y530 catalytic N C SH3 SH2 csk PTP Y419 N SH2 SH3 Y530-P catalytic C Y419 What is pp60C-SRC ? c-Src is the cellular homologue of v-src, the first oncogene found (1910, Rous). Src contains SH2 and SH3 domains, a C-terminal kinase domain and an N-terminal signal for myristoylation. c-Src has 2 regulatory phosphorylation sites: Y530 (negative) and Y419 (positive). Src transduces signals that are involved in control of a variety of cellular processes: proliferation, differentiation, motility and adhesion.

  5. c-Src ו – v-Src . ההבדלים... ל- c-SRC שני אתרי בקרה העוברים זירחון: Y527 (negative) Y416 (positive) c-Src v-Src

  6. How can Src be constitutively active without an activating mutation? • Dephosphorylation of Y530: • Upresgulation of phosphatases (PTP, CD45) • Downregulation of Csk Src Activation by SH2 from other sources (PDGFR, EGFR, Integrins)

  7. Src signaling mediates several cellular processes

  8. The development of chronic myelogenous leukaemia. Incidence: ~ 1:100,000 Age >50 Therapy: IFNa,Bone marrow transplantation Cure rate 20-30%

  9. The Bcr and Abl proteins

  10. Bcr-Abl Fusion proteinThe Philadelphia Chromosome הטרנסלוקציה מופיעה ב-95% מחולי Chronic Myelogenous Leukimia (CML). לחלבון הכימרי פעילות TK גבוהה בסדרי גודל מהחלבון c-ABL והוא מזרחן סובסטרטים שונים מה-wt.

  11. Bcr-Abl substrates

  12. Bcr-Abl signaling

  13. Anafi, Ben-Neriah, Levitzki 1992-3 Age >50 AG 1112AG 957 ATP CompetitiveSubstrate competitive Basel 1996 ATP competitive STI 571/Gleevec/Glivec

  14. The story of Gleevec Gleevec is ATP competitive (Ki- 85nM in c-Abl). Gleevec binds to the kinase inactive conformation. This mode of binding is critical to its high selectivity for Abl.

  15. Gleevec’s concentraions causing 50% reduction in kinase activity (IC50) IC50s for c-Abl, v-Abl and Bcr-Abl in most cell lines range between 0.1 to 0.5mM. It is specific for Abl tyrosine kinases (EGF, insulin, IGF-1 FGF mediated signaling are insensitive to it). PDGF-R lignad stimulated autophosphorylation is inhibited at an IC50 of 0.1-1mM

  16. Mechanism of autoinhibition – not only in Src

  17. Mechanisms of Abl autoinhibition

  18. Mutation in Bcr-Abl can confer resistance to Gleevec Mutation of the Bcr-Abl kinase domain are found in 50-90% of patients with secondary resistance to Gleevec. There are four distinguishable clusters: ATP binding loop (P-loop), T315, M251, A-loop. P-loop mutations (Y253) The P-loop is a stretch of amino acids 244-255 that undergoes displacement upon Gleevec binding. These mutants are 70-100 fold less sensitive to Gleevec. Other P-loop mutations (besides Y253) may shift the equilibrium towards an active state. Mutations of T315 T315 forms a hydrogen bond with Gleevc. T315I disrupts this bond and interferes with drug binding.

  19. Mutations of M351 M351 contacts the Abl SH2 and helps to stabilize the autoinibited comformation of Abl. A-loop mutations The A –loop comprises residues 381-402 of Abl. Its position regulates the kinase activity. It is likely that mutations in the A-loop prevent the kinase from adopting its inactive conformation. Mutations in other regions A mutagenesis screen discovered many additional mutants, also in the SH2 and SH3 domains. Their relevance for clinical resistance is still unknown. In addition, mutants were detected in samples from patients before Gleevec treatment.

  20. Mechanisms of disease resistance

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