Protein tyrosine kinases (PTKs) provide a pivotal mechanism of signal transmission in response to extracellular cues that regulate cell proliferation, differentiation and migration. Uncontrolled activation of PTKs is implicated in abnormal proliferation and migration of cancer cells and their deficiencies result in pathological conditions such as developmental abnormalities or immuno-deficiencies. Understanding the biochemical basis of PTK regulation is therefore a major goal in cell and cancer biology. The c-cbl protooncogene product has recently emerged as a negative regulator of PTKs. The applicant has shown that Cb functions as a negative regulator of Src-family tyrosine kinases (SFKs) which are known to play crucial roles in integrin-mediated cell migration. Based on these recent findings, the applicant will investigate the hypothesis that Cbl targets activated SFKs for ubiquitin-dependent degradation, by providing a scaffold for juxtaposition of a Cbl RING finger-bound ubiquitin conjugating enzyme (E2) and SFK-bound ubiquitin ligase (E3). Based on earlier findings on SFKs, and initial evidence using Cbl-/-cells, the applicant also hypothesizes that Cbl-dependent degradation of SFKs promotes focal adhesion disassembly, a process essential for directional cell migration. Here Dr. Band will test these hypotheses in the context of a well-characterized SFK, Fyn. The applicant will use Cbl-/-, Cbl-reconstituted and wildtype primary embryonic fibroblast (PEF) cells, and cells that are temperature-sensitive for ubiquitination, to demonstrate the role of Cbl in ubiquitination and degradation of SFKs. The applicants will use mutagenesis to define Cbl and Fyn domains required for their interactions with E2 and E3, respectively. The applicant will then use the mutants to reconstitute Cbl-/- or SFK-/- cells to define the requirement of Cbl in SFK-mediated cell migration. Together with analyses of the nature of focal adhesion signaling in reconstituted cells, these analyses aim to establish a novel paradigm of tyrosine kinase regulation and control of directed cell migration, a fundamental cell biological process. The insights gained through these studies are likely to be a significant relevance to a variety of conditions where tyrosine kinases are implicated, including immune deficiencies, autoimmunity and cancer. Given the role of cell migration in invasion and metastasis, the transforming ability of Src-family kinases when relieved of their negative regulatory mechanisms, and the oncogenic potential of Cbl, these studies are particularly relevant to cancer. These studies may also elucidate aspects of pathogenesis in Angelman's syndrome, caused by mutations in E6AP gene.
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