The objective is to determine how the skeletal muscle cell controls the ability of glycogen synthase kinase-3 to catalyze BOTH the activation and inactivation of glycogen synthase. The studies center around the regulation of protein phosphatase-1 by the levels of glycogen in the skeletal muscle. As glycogen is degraded during exercise, the glycogen-protein complex dissociates and protein phosphatase-1 is susceptible to a modulator protein (inhibitor-2) which can inactivate this spontaneous active protein phosphatase and generate the ATP, Mg+2-dependent form. The way to reactivate this form of the phosphatase is to have glycogen synthase kinase-3 phosphorylate it, thereby causing dephosphorylation and activation of glycogen synthase. Since glycogen synthase kinase-3 is the most potent protein kinase to cause phosphorylation and inactivation of glycogen synthase, the skeletal muscle cell must control which of these effects of glycogen synthase kinase-3 are allowed to occur. The key factor in cellular control of these reactions may reside with the glycogen-protein complex. To study the regulation of thes enzymes under differing cellular glycogen levels, homogeneous pools of single skeletal muscle fibers will be analyzed. Pools are required since the assays are not sensitive enough to determine enzymes activities in single skeletal muscle fibers, but without separation the glycogen levels of adjacent fibers would vary too widely in an intact biopsy sample. This separation will also allow investigation of these regulatory mechanisms in fast-twitch and slow-twitch fibers, which differ in overall glycogenolytic ability but appear to otherwise have the same glycogenetic ability. Preliminary studies involving nonspecific assays of phosphatase and kinase activity suggest that the pattern of progressive inactivation of protein phosphatase-1 with falling glycogen levels and subsequent reactivation of the protein phosphatase due to elevated cyclic AMP-, calcium-independent glycogen synthase kinase activity (of which glycogen synthase kinase-3 is the major component) occurs in skeletal muscle during recovery from exercis-induced glycogenolysis. If the mechanism of regulation applies, the findings will be important physiologically because resynthesis of glycogen stores is critical to the functioning of skeletal muscle with subsequent exercise; biochemically it will be important because of the unique ability of glycogen synthase kinase-3 to catalyze two opposing reactions.
