Nonreceptor tyrosine kinases such as Src and Abl are involved in regulating the growth and differentiation of normal cells. Activated forms of these enzymes have been implicated in the development and progression of human cancer. The long-term objectives of this project are to understand how nonreceptor tyrosine kinases are regulated in a normal cell, and how the various domains of the enzymes contribute to substrate phosphorylation. There are two specific aims: 1. Many important Src substrates are processively phosphorylated at multiple tyrosine residues. The first hypothesis is that multisite phosphorylation of the focal adhesion protein p130Cas is critical for downstream signal transduction. This will be tested by constructing Cas mutants lacking phosporylation sites. These mutants will be tested as Src substrates in vitro and in fibroblasts derived from Cas-deficient mice. 2. The Src family kinase Hck plays an important role in hematopoietic cell physiology, but very few cellular substrates, activators, or effectors for Hck have been identified. The Pl's group recently identified the proteins WASP, WIP, and ELMO1 in a screen for Hck SH3 domain binding proteins in U937 monocytic cells. The second hypothesis is that these proteins are substrates for Hck, and that phosphorylation is important in Hck signaling. Phosphorylation of ELMO1 will be studied in activated U937 and THP-1 cells. The involvement of ELMO1 in phagocytosis and in Rac activation will also be investigated. A final component of this aim will be to investigate the general regulatory features of proteins that bind to the SH3 domain of Hck. These studies will provide new information on the regulation and substrate specificity of tyrosine kinases. This information could form the basis for the development of anticancer agents that disrupt substrate recognition and cellular transformation by oncogenic tyrosine kinases. ? ? ?
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