The long-term goal of this grant proposal is to elucidate the signal relay pathways that mediate growth factor-initiated cell proliferation. In the last grant period, we found that platelet-derived growth factor (PDGF) activates signaling enzymes such as phosphoinositide 3 kinase (PI3K) and protein kinase C (PKC) at two distinct time points. In addition to the early wave of activity that has been studied for many years, these enzymes are also activated 3-7 hrs post PDGF. Importantly, it is the late wave of activity that drives mitogenic signaling. The goal of this proposal is to study this novel and critical phase of signaling. 1. Investigate the mechanistic basis for how the second wave of PI3K activity is generated. The first wave of PI3K activity requires that the receptor be tyrosine phosphorylated at the appropriate sites and that Ras is activated. We will test whether these two variables also regulate the second wave of PI3K activity. 2. Determine how late activation of PI3K couples to events that drive cell cycle progression. Progression through the Gl phase of the cell cycle requires events such as synthesis of cyclins, a decrease in cyclin dependent kinase (cdk) inhibitors, activation of some of the cdks, and phosphorylation of Rb. We will determine which of these events are dependent on the second wave of PI3K activity. 3. Elucidate the DAG-directed pathway that promotes progression through GI and entry into S. The (3PDGFR is able to activate multiple signaling systems that lead to cell cycle progression; one is dependent on PI3K, whereas the other is independent of PI3K and is driven by DAG. We have made the surprising discovery that DAG-regulated members of the PKC family are activated mid to late G 1, and that these events drive cell cycle progression. As with the PI3K pathway, the novelty of this observation is that the DAG-directed pathway is required at times that have not been investigated. The goal of this specific alm is to investigate the mechanism underlying the late wave of PKC activity, and to identify PKC-dependent cell cycle events. These studies will significantly advance our understanding of how signal relay cascades regulate cell proliferation. This information will provide new opportunities to manage and prevent diseases that arise from unregulated cell proliferation such as cancer and atherosclerosis.
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