The goal of the proposed research is to understand the molecular mechanisms underlying the control of cell proliferation, as a first step toward understanding the uncontrolled proliferation that occurs in tumor cells. Biochemical approaches will be used to study proteins involved in the fundamental regulation of cell proliferation at two critical control points in the cell cycle: the G1 stage (when the cell commits itself to DNA replication) and the late G2 stage (when the cell initiates mitosis). These studies will focus on two protein kinases already implicated in both of these control points: p60c-src, the cellular homologue of the viral transforming protein p60v-src; and p34cdc2, the catalytic component of maturation promoting factor and a key regulator of cell cycle events. During fibroblast mitosis, p60c-src is phosphorylated by p34cdc2 and its tyrosine kinase activity increases, suggesting that p60c-src may regulate mitotic events. This possibility will be explored by analyzing various aspects of p60c-src function in mitotic cells (kinase activity and subcellular location). The biological activity of mitotic p60c-src will be assessed by microinjecting the protein into interphase fibroblasts. Regulation of p60c-src function by p34cdc2 phosphorylation sites. Regulation of p60c-src by dephosphorylation will also be studied: cell extracts will be searched for phosphatase activity that removes the p34cdc2 phosphorylations on p60c-src (and other p34cdc2 subtrates). The role of p34cdc2 itself in cell growth control will also be investigated. In addition to its mitotic role, there is evidence that p34cdc2 regulates cell proliferation at the late G1 control points, and the proposed experiments will clarify this possibility. Several aspects of p34cdc2 function (phosphorylation state, kinase activity, and association with regulatory subunits) will be analyzed in detail during the G0/G1/S phases in mouse fibroblasts, and purification techniques will be developed to isolate regulatory proteins that phosphorylate or associate with p34cdc2 at these points. The importance of p34cdc2 in the control of proliferation will be investigated by testing the mitogenic effects of microinjecting the purified protein. These studies have an obvious medical significance: an understanding of the fundamental molecular processes underlying normal cell proliferation is clearly the first step toward understanding the abnormal processes that lead to uncontrolled proliferation of the cancer cell.
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