Steel factor induces tyrosine phosphorylation of CRKL and binding of CRKL to a complex containing c-kit, phosphatidylinositol 3-kinase, and p120(CBL).
Steel factor (SF) is a growth and survival factor for hematopoietic cells. The receptor for SF, c-Kit, contains intrinsic tyrosine kinase activity, and binding of SF induces rapid tyrosine phosphorylation of several cellular proteins, including c-Kit itself. Activation of c-Kit is shown here to induce tyrosine phosphorylation of CRKL, and CRKL coprecipitated with c-Kit through an interaction that required the CRKL SH3 domains and not the SH2 domain. CRKL associated with c-Kit indirectly as part of a larger complex of proteins. Two proteins in this complex were identified as the p85 regulatory subunit of phosphatidylinositol 3-kinase (p85(PI3K)) and the proto-oncoprotein p120(CBL). Because p85(PI3K) is known to bind to the activated c-Kit receptor, the possibility that CRKL interacted with c-Kit indirectly through p85(PI3K) was investigated. Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85(PI3K )in vitro. However, although a small amount of CRKL was preassociated with p85(PI3K), the interaction was increased after SF stimulation, suggesting that the interactions of these three proteins are complex. We conclude that SF induces the formation of a signaling complex potentially containing CRKL and p120(CBL), both of which bind to c-Kit through p85(PI3K). These data suggest that one function of CRKL in normal cells might be to recruit signaling molecules such as CBL into a complex with PI3K. Such complexes could be important in propagating signals involving PI3K such as gene expression and adhesion.
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