Identification of novel tyrosine phosphorylation sites for Src family kinases within the Bcr-Abl SH3-SH2 region using mass spectrometry

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The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome, which arises from the reciprocal translocation of the c-abl proto-onogene on chromosome 9 and the bcr locus on chromosome 22. This translocation results in the expression of a 210 kDa fusion protein (Bcr-Abl) with constitutive tyrosine kinase activity that is responsible for CML pathogenesis. Recently, we demonstrated that selective inhibition of myeloid Src family kinase activity (Hck, Lyn, Fyn, and Fgr) induces growth arrest and apoptosis in Bcr-Abl-transformed cells. These and other data provide compelling evidence that oncogenic transformation by Bcr-Abl requires Src family kinase activity. In the present study, we investigated the biochemical interactions between myeloid Src family members and Bcr-Abl. We found that Hck, Lyn, Fyn and Fgr each bind the kinase domain, C-terminal tail, and SH3/SH2 region of Bcr-Abl. Unexpectedly, both Lyn and Hck strongly phosphorylated a recombinant, purified Abl GST-SH3/SH2 fusion protein in vitro. To identify the phosphorylation sites, the phosphorylated Abl GST-SH3/SH2 fusion protein was digested with trypsin and the fragements were analyzed by electrospray mass spectrometry. Two phosphorylated tyrosine residues were identified, both of which localize to the binding surface of the Abl SH3 domain. The position of these tyrosines in the recent crystal structure of c-Abl strongly suggests that phosphorylation by Src kinases may impact intramolecular linker binding and kinase regulation within Bcr-Abl. Surprisingly, specificity in terms of phosphorylation site selection was observed for Hck vs. Lyn: whereas Abl SH3 Tyr 134 was phosphorylated by both Hck and Lyn, Tyr 89 was phosphorylated exclusively by Hck. This result suggests that individual Src family members may have non-redundant effects on Bcr-Abl kinase activity and signaling in CML cells. We also examined the ability of Src kinases to transphosphorylate Abl Tyr 245 in the SH2-kinase linker and Tyr 412 in the activation loop. These tyrosines have been described previously as sites of autophosphorylation and kinase regulation in c-Abl. We found that Hck, Lyn and Fyn phosphorylate both of these sites in full-length Bcr-Abl in a defined expression system. Phosphorylation of the Bcr-Abl SH3 domain, linker, or activation loop by Src family kinases may affect Bcr-Abl kinase activity, sensitivity to Gleevec, and downstream signaling; these possibilities are currently under investigation.