:3תרגיל טירוזין קינאזות ציטופלסמטיות

Tyrosine Kinases

ReceptorsNon

Receptors

Membrane – spanning proteins with an

extracellular ligand binding site

Cytosolic proteins. Mostly coupled to

transmembrane proteins

Growth and Differentiation

Factors, Hormones

Protein – protein interactions

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).

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.

Y530-P

Y419

Ccatalytic

SH3SH2 N

cskPTP

C

Y419

Y530

N SH3 SH2 catalytic

c-Src – ו v-Src...ההבדלים .

שני אתרי בקרה c-SRCל- העוברים זירחון:

Y527 (negative)Y416 (positive)

c-Src

v-Src

How can Src be constitutively active without an activating

mutation ?

Src

Dephosphorylation of Y530: 1. Upresgulation of phosphatases (PTP, CD45)2. Downregulation of Csk

Activation by SH2 from other sources (PDGFR, EGFR, Integrins)

Src signaling mediates several cellular processes

The development of chronic myelogenous leukaemia.

Incidence: ~ 1:100,000 Age >50Therapy: IFNa,Bone marrow transplantation Cure rate 20-30%

The Bcr and Abl proteins

Bcr-Abl Fusion proteinThe Philadelphia Chromosome

.Chronic Myelogenous Leukimia (CML) מחולי 95%ב-הטרנסלוקציה מופיעה והוא מזרחן סובסטרטיםc-ABL גבוהה בסדרי גודל מהחלבון TKלחלבון הכימרי פעילות

. wtשונים מה-

Bcr-Abl substrates

Bcr-Abl signaling

Age >50N

N

N

HN

H3C

HN

O

N

N

NH

CN

NHN

NH2

C N

OH

OH

NH

COOCH3

AG 1112 AG 957ATP Competitive Substrate competitive

1992-3

1996

ATP competitive

Anafi, Ben-Neriah, Levitzki

Basel

STI 571/Gleevec/Glivec

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.

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 0.5M.

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 0.1-1M

Mechanism of autoinhibition – not only in Src

Mechanisms of Abl autoinhibition

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 T315T315 forms a hydrogen bond with Gleevc. T315I disrupts this bond and interferes with drug binding.

Mutations of M351M351 contacts the Abl SH2 and helps to stabilize the autoinibited comformation of Abl.

A-loop mutationsThe 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 regionsA 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.

Mechanisms of disease resistance