1. טירוזין קינאזות ציטופלסמטיות
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

4. 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).

5. What is pp60C-SRC ? csk catalytic
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). C
Y419
Y530 N
SH3
SH2
catalytic
PTP
csk
Y530-P
Y419 C
catalytic
SH3
SH2 N
Src transduces signals that are involved in control of a variety of cellular processes: proliferation, differentiation, motility and adhesion.

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

8. 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)

9. Src signaling mediates several cellular processes

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

11. The Bcr and Abl proteins

12. Bcr-Abl Fusion protein The 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.

13. Bcr-Abl substrates

14. Bcr-Abl signaling

15. Anafi, Ben-Neriah, Levitzki 1992-3
Age >50
AG AG 957
ATP Competitive Substrate competitive
Basel
1996
ATP competitive
STI 571/Gleevec/Glivec

16. 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.

17. 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

18. Mechanism of autoinhibition – not only in Src

19. Mechanisms of Abl autoinhibition

20. 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 that undergoes displacement upon Gleevec binding.
These mutants are 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.

21. 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 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.

22. Mechanisms of disease resistance