Fyn associates with Cbl and phosphorylates tyrosine 731 in Cbl, a binding site for phosphatidylinositol 3-kinase

We have investigated the interaction between Cbl and the Src-related tyrosi ne kinase Fyn. Fyn was observed to be constitutively associated with Cbl in lysates of several different cell types including the interleukin-3-depend ent murine myeloid cell line 32Dc13, and the prolactin-dependent rat thymom a cell line Nb2. Binding studies indicated that Cbl could bind to glutathio ne S-transferase (GST) fusion proteins encoding the unique, Src homology do main 3 (SH3), and SH2 domains of Fyn, Hck, or Lyn, Fusion proteins encoding either the SH3 or SH2 domains of Fyn bound to Cbl as effectively as the fu sion protein encoding the unique, SH3, and SH2 domains of Fyn. The Fyn SH2 domain bound to both tyrosine-phosphorylated and nonphosphorylated Cbl, imp lying that this interaction might be phosphotyrosine-independent. Binding o f the Fyn SH2 domain to Cbl was not disrupted by the addition of phosphotyr osine, phosphoserine, or phosphothreonine. A GST fusion protein encoding th e proline-rich region of Cbl bound to Fyn present in a total cell lysate. F ar Western blot analysis also indicated that the SH3 domain of Fyn bound pr eferentially to the proline-rich region of Cbl. The addition of [gamma-P-32 ]ATP to either anti-Cbl immunoprecipitates or anti-Fyn immunoprecipitates r esulted in the phosphorylation of both Cbl and Fyn as demonstrated by immun oprecipitation of the phosphorylated proteins with specific antisera. Fyn d irectly phosphorylated a GST fusion protein containing the C-terminal regio n of Cbl (GST-CBL-LZIP). In contrast, immunoprecipitated JAK2 was not able to phosphorylate this same region of Cbl. The GST-CBL-LZIP fusion protein c ontains a binding site for the SH2 domain of the p85 subunit of phosphatidy linositol 3-kinase, which mapped to Tyr(731), which is present in the seque nce YEAM. Mutation of Tyr(731) in GST-CBL-LZIP eliminated binding of the p8 5 subunit of phosphatidylinositol 3-kinase and substantially reduced the ph osphorylation of this fusion protein by Fyn, despite the presence of four o ther tyrosine residues in this fusion protein. These data are consistent wi th the hypothesis that Cbl represents a substrate for Src-like kinases that are activated in response to the engagement of cell surface receptors, and that Src-like kinases are responsible for the phosphorylation of a tyrosin e residue in Cbl that may regulate activation of phosphatidylinositol 3-kin ase.