) Tyrosine phosphorylation provides a universal mechanism of signal transmission in response to extracellular cues that regulate proliferation and differentiation in normal cells. Uncontrolled tyrosine kinase activation is implicated in proliferation of cancerous cells and deficiencies of specific tyrosine kinases result in a number of pathological conditions such as developmental abnormalities or immuno-deficiencies. Therefore, understanding the biochemical basis of tyrosine kinase regulation is a major goal of research in cell and cancer biology. Little is known at present about how cellular tyrosine kinases are regulated. Biochemical studies from Dr. Band's laboratory and independent genetic studies in C. elegans have identified the c-cbl proto-oncogene product as a potential regulator of tyrosine kinase signaling. The N-terminal transforming region of Cbl (Cbl-N), which harbors a phosphotyrosine binding (PTB) domain is critical for this function. The principal investigator proposes the functional dissection of Cbl-N using ZAP70 and PDGF receptor-a, two well-characterized examples of receptor and non-receptor tyrosine kinases. This project will identify the Cbl PTB domain-binding motifs on PDGFRa and ZAP70 using in vitro binding assays. Mutants of PDGFRa and ZAP70 that are unable to interact with CBL's PTB domain will be transfected into PDGFRa-/-and Syk-/- cell lines, respectively, for assessment of their function. Analyses of tyrosine phosphorylation, kinase activity, signaling protein complexes, and sub-cellular localization of mutant proteins will be used to gain insights into the biochemical mechanisms of Cbl=s regulatory function. Truncation and mutational studies of Cbl-N, and its chimeras with tyrosine kinases will be used to distinguish the tethering versus the regulatory function of the Cbl PTB domain. Elucidation of a regulatory mechanism for cellular tyrosine kinases represents a major step forward in our understanding of how cell differentiation and proliferation are controlled, and should help in the future design of rational therapeutic strategies directed at tyrosine kinase machinery. Analyses of ZAP70 and PDGFRa should also provide insights directly relevant to immune deficiencies, auto-immunity and cancer.
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