BCR-ABL ONCOPROTEINS BIND DIRECTLY TO ACTIVATORS OF THE RAS SIGNALINGPATHWAY

The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play im portant roles in the development of Philadelphia chromosome positive ( Ph(+)) chronic myelogenous leukemia (CML) and acute lymphoblastic leuk emia (Ph(+) ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases invol ve stimulation of the Pas pathway, we analyzed Bcr-Abl oncoproteins fo r interactions with cytoplasmic proteins that mediate Pas activation. Such polypeptides include Grb2, which comprises a single Src homology 2 (SH2) domain flanked by two SH3 domains, and the 66, 52 and 46 kDa S he proteins which possess an SH2 domain in their carboxy-terminus. Grb 2 associates with tyrosine phosphorylated proteins through its SH2 dom ain, and with the Pas guanine nucleotide releasing protein mSos1 throu gh its SH3 domains. mSos1 stimulates conversion of the inactive GDP-bo und form of Pas to the active GTP-bound state. In bcr-abl-transformed cells, Grb2 and mSos1 formed a physical complex with Bcr-Abl. In vitro , the Grb2 SH2 domain bound Bcr-Abl through recognition of a tyrosine phosphorylation site within the amino-terminal bcr-encoded sequence (p .Tyr177-Val-Asn-Val), that is common to both Bcr-Abl proteins. These r esults suggest that autophosphorylation within the Bcr element of Bcr- Abl creates a direct physical link to Grb2-mSos1, and potentially to t he Pas pathway, and thereby modifies the target specificity of the Abl tyrosine kinase. Bcr-Abl tyrosine kinases also phosphorylate Shc prot eins on tyrosine, inducing the formation of an Shc-Grb2 complex that a lso has the potential to stimulate Pas. Bcr-Abl oncoproteins may there fore couple to the Pas pathway through the formation of multiple Grb2 complexes.