PROTEIN KINASE C SUBSTRATES, REGULATORY PROTEINS, AND YEAST PROTEINS

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Protein Kinase C Substrates and Regulatory Proteins, and S-100-Like Protein of Baker's Yeast The long-term objectives are to elucidate the protein phosphorylation and limited proteolysis involved in yeast signal transduction process in comparison with that of mammalian cells. This specific aims are: (I) To prove the substrate nature of immunoprecipitated yeast constitutive nitric oxide synthase, m-calpain, mu-calpain, calpastatin, myelin-like protein, and integrin (alpha-6 subunit) by testing against immunoprecipitated yeast protein kinase C (PKC) alpha, beta, gamma: Protease-deficient Saccharomyces cerevisiae (baker's yeast) is used to prepare most enzymes and proteins. Phosphorylated substrates are subjected to SDS-PAGE and analyzed by autoradiography. (II) To differentiate immunoprecipitated yeast PKC (alpha, beta, gamma, delta, epsilon, theta, iota, lambda) with cofactor (Ca2+, phosphatidylserine, diacylglycerol) requirement, subcellular distribution, substrate specificity, isoelectric pH and molecular mass. (iii) To investigate the alteration of activity of yeast nitric oxide synthase after being phosphorylated by yeast PKC alpha; The change in activity is monitored by the production of cistrulline. (IV) To evaluate the alteration of activity of yeast calpains after being phosphorylated by yeast PKCalpha (using preautophosphorylated cAMP- dependent protein kinase labeled with 32p as a proteolytic substrate). (V) To investigate yeast heat-stable regulatory protein(s) of PKCalpha; The regulatory protein(s) are prepared by subjecting preboiled crude yeast extract to the DE-52 column eluted with linear NaC1 gradient. The inhibitory mechanism, molecular mass, and isoelectric pH will be determined. (VI) To identify the regulatory effect (a) of yeast S-100- like protein on the protein phosphorylation of yeast supernatant obtained after immunoprecipitations with anti=S=100 and anti-calmodulin. These studies may provide insight into the signal transduction process, thereby benefitting the treatments of certain pathological disorders, and yeast or fungal infections. It could also facilitate effective manipulation of the metabolic processes in baker's yeast for the production of valuable (genetic engineering) products, and for other industrial applications. Furthermore, three minority undergraduate students will receive training in enzymology-related biomedical research.