We will study the regulation and function of c-Src and its associated signal transduction system so as to understand how perturbations of this system can induce neoplastic transformation. We have shown that, for a short period beginning either just before or during mitosis, c-Src is phosphorylated by p34-cdc2 (a key cell cycle regulator), is partially dephoshorylated at Tyr 527 (an important control site), has increased specific kinase activity, has an increased ability to interact with peptides that bind its SH2 region, and induces transient tyrosine phosphorylation of specific protein substrates. These transient phenomena provide a unique physiological model for exploring the biochemical and physiological consequences of Src activation. We will explore the possibility that c-Src participates in entry into or progression through mitosis, explore mitotic regulation of and potential compensatory interactions among src-family members, and study mitotic and cell cycle- dependent regulation and activities of CSK, a src-family carboxyl terminal kinase, and PTPalpha, a tyrosine phosphatase that may regulate c-Src. In particular, we will determine if Src is activated before or during mitosis, determine whether reduced Tyr 527 phosphorylation in mitosis results from changes in intrinsic tyrosine phosphatase or kinase activities, conformationally-induced changes in the ability of Tyr 527 to act as a substrate, changes in association with cellular proteins, or changes in localization. We will identify the tyrosine kinase and/or phosphatase whose activity is transiently regulated and study their regulation in other parts of the cell cycle. Whether or not Src-mediated pathways play a critical role in mitotic control, it is likely that similar circuitry is used in other parts of the cell cycle. We will use techniques and reagents developed in our mitotic studies to look for transient changes in phosphorylation, activation and Src-binding of components of this circuitry at other times in the cell cycle. We will also study regulation of related signaling proteins known to be regulated at other points of the cell cycle, e.g., Raf, to see if they may play a role in mitotic Src-mediated signaling. Independently, molecular genetics approaches will be used to identify critical times for Src action in the cell cycle by using dominant negative and transiently regulated Src mutants. These studies with synchronized cell populations have the ability to identify transient biochemical phenomena that would be missed in studies with unsynchronized cell populations.
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