The covalent modification of enzymes and regulatory proteins through cyclic phosphorylation-dephosphorylation reactions is a crucial element in mediating the effects of many hormones and physiological stimuli. The long-term goal of this project is to identify phosphoprotein phosphatase (ptase) isozymes of cardiac muscle and other tissues, and to gain greater understanding of their physiological role and mechanism of regulation. This applicant has isolated and identified four different types of ptase from bovine heart and brain. Ptase-1 (major active form, Mr=75,000 or 75K) requires the action of a glycogen synthase kinase (50K, also termed GSK-3, FA or ptase-1 kinase) for activation and is sensitive to inhibition by Thr(P)-inhibitor-1 (P-I-1) phosphorylated by cAMP-dependent protein kinase. Ptase-2 (80K) is a Ca2+, calmodulin (CM)-dependent enzyme. The regulation of ptase-3 (including 3 forms: 156K, 161K, and 95K) and ptase-4 (45K, Mg2+/Mn2+-dependent) is not yet known. All four types exhibit broad substrate specificity and potentially can participate in the regulation of many cellular processes. In terms of glycogen metabolism, ptase-1, -3 and -4 represent the major phosphorylase, P-I-1 and glycogen synthase ptase activities, respectively, in cardiac muscle.
Specific aims i nclude: purification and characterization of the native form of ptase-1, F-A and ptase-3A; to elucidate the precise mechanism of regulation of ptase-1 catalyzed by F-A and that of ptase-2 by Ca2+, CM and Mg2+; the regulation of ptase-1 by P-I-1; the precise subunit composition and function of these 4 types of ptases; the structure-function relationship of the two subunits of the 75K ptase-1 and those of the 80K ptase-2; regulation of ptase-3 and -4; to prepare antibodies against purified ptases and their subunits; to study the structural inter-relationship among these 4 types of ptase; and to determine the role of Mg2+ as a physiological co-factor common to all 4 types of ptases. Current available techniques for studying enzymology and cellular regulation of enzymatic activity will be employed to achieve these aims.
