Experiments described in this application focus on the characterization, regulation and physiological function of a relatively recently described group of kinases that act on phosphatidylinositosol. The enzymes are known as phosphoinositide 3-k (PI 3-K). Reactions involving these enzymes are monitored by radiolabeled product formation and Western blotting. Dr. Rittenhouse has carried out extensive preliminary studies with a specific inhibitor of PI 3-K activity is important for the signal cascade that culminates in the most important functional response of activated platelets-cohesion and aggregation. Inhibition of PI 3-K blocks agonist- stimulated exposure of activated glycoprotein Iib/IIIa, the integrin to which fibrinogen binds to platelets. This phenomenon has been detected by the GPIIb/IIIa antibody, known as PAC-1. Dr. Rittenhouse will study an extensive list of factors which regulate the activation and specificity of PI 3-Ks and the linkage of PI 3-Ks to the cytoskeleton. Among the factors to be studied are: Protein kinases, GTP-binding proteins, cytoskeletal components, and an important platelet protein-pleckstrin. Other preliminary experiments carried out by Dr. Rittenhouse have shown the product of PI 3-K activity, PI(3,4,5) P3, promotes protein phosphorylation when added to permeabilized platelets. The kinase(s) involved in this phosphorylation have not as yet been identified. The experiments on permeabilized platelets appear to be relevant to intact platelets, since wortmannin inhibits phosphorylation of specific proteins in activated platelets, and the addition of PI(3,4,5) P3 to permeabilized platelets overcomes the inhibitory effects of wortmannin on protein phosphorylation. Other experiment are designed to examine and characterize two new, additional PI 3-Ks:PI 3-K (theta) and PI 3-K(sigma). These experiments represent new research directions for Dr. Rittenhouse.
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