The broad, long-term objective of this proposal is to understand at the molecular level the mechanism by which phosphoinositide 3-kinase (PI 3- K) activates cellular signaling. PI 3-K is implicated in diverse cellular functions and deregulation of its biological signal results in the transformation of cells. Evidence is emerging that the lipid products of PI 3-K, PtdIns-3,4-P2 and PtdIns-3,4,5-P3 have multiple effectors in the cell. Presumably agonist-stimulated accumulation of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 on the inner leaflet of the plasma membrane recruits cytosolic proteins for activation. My published and preliminary studies suggest that PI 3-K lipid products can activate the protein kinase C (PKC) isoforms PKCepsilon and PKCzetu in vitro. Preliminary studies presented in this proposal also show that cells expressing an active PI 3-K and producing PtdIns-3,4-P2 and PtdIns-3,4,5-P3 have elevated PKCepsilon and PKCzetu activity. I also show that the protein kinase Akt/PKB associates with and activates PKCzetu in vivo. Finally, a direct binding of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 to PKC's is demonstrated. In the proposal, experiments are designed to determine the role of PtdIns-3,4,5-P3, Akt/PKB and PDK-1 in the regulation of the PKCepsilon, zetu and mu in vitro and in vivo. Activation of PKC's in vitro will be assessed using synthetic D3 phosphoinositides in the presence of Akt/PKB or PDK-1. Mutant PKC's will be made to demonstrate that PDK-1 phosphorylates a critical threonine residue, leading to activation. Mutant PKC's will be made in which the regulatory regions of PKCepsilon and PKCalpha will be switched to test the specificity of activation. Activation of PKC's in vivo will be investigated using constitutively active PI 3-K, Akt/PKB and PDK-1 mutants. Association of PKC's with Akt/PKB will also be investigated. The binding of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 to PKC's will also be measured. Finally, the lipid- binding site on PKC's will be determined using a labeled photoactivatable PtdIns-3,4,5-P3 which can be cross-linked to target proteins. The results of these studies will elucidate the role of PI 3-K products in cellular signaling. A hypothesis is proposed in which multiple mechanisms exist to regulate PKC's in the PI 3-K signaling pathway, including lipid binding, phosphorylation and association with other proteins. A better understanding of PI 3-K signaling with emphasis on its lipid products has implications for the understanding of cell function and may provide novel therapeutic approach for the treatment of human disease.
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