PHOSPHORYLATION OF CBL FOLLOWING STIMULATION WITH INTERLEUKIN-3 AND ITS ASSOCIATION WITH GRB2, FYN, AND PHOSPHATIDYLINOSITOL 3-KINASE

We have demonstrated that a 120-kDa protein, identified as Cbl, become s rapidly phosphorylated on tyrosine residues following stimulation of factor-dependent cells with interleukin-3 (IL-3). Little or no phosph orylation of Cbl was observed in the absence of IL-3 stimulation and p hosphorylation is maximal by 20-30 min after IL-3 stimulation. Associa tion of Cbl with Grb2 was noted in unstimulated cells, and the amount of Cbl associated with Grb2 increased following IL-3 stimulation. The p85 subunit of phosphatidylinositol 3-kinase was constitutively associ ated with Cbl. Approximately 10% of the PI kinase activity present in anti-phosphotyrosine immunoprecipitates was present in anti-Cbl immuno precipitates of IL-3-stimulated cells. The constitutive association of Cbl with Fyn was also observed, Cbl was observed to bind to bacterial fusion proteins encoding the unique, SH3, and SH2 domains of Fyn, Hck , and Lyn. The SH2 domain of Fyn alone was able to bind Cbl to nearly the same extent as did the fusion protein encoding the unique, SH3, an d SH2 domains. This was not the case for the SH2 domain of Hck, howeve r, as binding of the Hck fusion protein to Cbl appeared to require mul tiple domains. The binding of the fusion proteins to Cbl occurred rega rdless of whether Cbl was tyrosine-phosphorylated or not, and the bind ing could not be disrupted by the addition of 30 mM free phosphotyrosi ne. These data suggest the unexpected conclusion that the Fyn SH2 doma in may bind to Cbl in a phosphotyrosine-independent manner.