There is substantial evidence that the protooncogenic protein c-Cbl is involved in signal transduction in normal and transformed cells. c-Cbl becomes tyrosine phosphorylated in response to stimulation through a variety of receptors, as well as in the cells transformed by oncogenic forms of the protein tyrosine kinase Abl. Tyrosine phosphorylation of c-Cbl dramatically upregulates its binding to various crucial signaling molecules. However, the biological consequences of c-Cbl-mediated signal transduction are poorly understood. Recently, we have succeeded in mapping the tyrosine phosphorylation sites of c-Cbl. In order to analyze the biological functions of c-Cbl, we overexpressed wild-type c-Cbl or its mutant form lacking tyrosine phosphorylation sites in Abl-transformed fibroblasts and demonstrated that the wild-type c-Cbl suppresses transformation by oncogenic Abl, whereas the phosphorylation-defective c-Cbl enhances this transformation. The effect of wild-type c-Cbl is connected to the induction of cell adhesion and spreading on the extracellular matrix. These observations, taken together with the previous findings, led us to the hypothesis that c-Cbl recruits, in a tyrosine-phosphorylation dependent manner, PI-3 kinase and Crk-family proteins to the membrane and/or focal adhesion complexes. This, in turn, triggers signal transduction pathways facilitating the assembly of focal adhesion complexes and actin stress fibers. The tyrosine phosphorylation-dependent regulation of cell morphology and adhesion may constitute the major biological functions of c-Cbl. The overall objective of the current proposal is to test this hypothesis. Accordingly, the specific aims of our project are: 1) to determine the tyrosine phosphorylation sites of c-Cbl essential for its transformation-suppressing function; 2) to determine the relationship between the biological effects of c-Cbl tyrosine phosphorylation and the interactions of c-Cbl with the membrane, focal adhesion complexes and cytoskeleton in Abl-transformed fibroblasts; 3) to determine the relationship between PI-3 kinase- and Crk-dependent signal transduction pathways and the biological effects of c-Cbl in Abl-transformed fibroblasts; and 4) to assess the involvement of c-Cbl in biological responses to various types of extracellular stimulation mediated by protein tyrosine kinases in untransformed fibroblasts. The investigators are confident that this work will define the biological functions of c-Cbl and determine their molecular basis. This should substantially advance our understanding of normal cell activation and neoplastic transformation and may suggest novel therapeutic approaches that target signal transduction mechanisms.
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