Phosphorylase kinase plays a key role in the cascade regulation of glycogen breakdown in muscle when it activates glycogen phosphorylase. The prior activation of phosphorylase kinase, however, is less well understood. Besides being activated by protein kinase through the well-known cAMP-dependent mechanism, it can also be activated in vivo completely independently of cAMP through an unknown mechanism. Our overall objective will be to determine if the Ca 2 ion-dependent autophosphorylation and activation of phosphorylase kinase observed in vitro is responsible for the cAMP-independent stimulation of glycogenolysis in vivo. We will first characterize the autophosphorylation reaction by determining whether it occurs at the same catalytic site as that employed for the covalent conversion of phosphorylase, whether it occurs through an intermolecular or intramolecular process and how effectors of phosphorylase conversion influence the autophosphorylation reaction. Another goal will be to study the properties of the autophosphorylated enzyme. For instance, does autophosphorylation affect its Ca 2 ion-dependency or its reticulum? This last possibility will allow us to evaluate the role phosphorylase kinase may play in the regulation of cellular Ca 2 ion metabolism. A large portion of the proposed work involves detailed comparison of the sites in phosphorylase kinase that are phosphorylated by cAMP-dependent protein kinase as opposed to Ca 2 ion-dependent autophosphorylation. If unique autophosphorylation sites exist they will be used diagnostically to determine whether phosphorylase kinase is activated through a cAMP-dependent mechanism or a Ca 2 ion-dependent mechanism in response to any given stimulus.