In the cascade activation of glycogen utilization that leads to energy production in mammalian skeletal muscle, phosphorylase kinase (PhK) phosphorylates and activates glycogen phosphorylase. In turn, the activity of PhK, catalyzed by its gamma subunit, is markedly enhanced by hormonal (cAMP and Ca2+), metabolic (ADP) and neural (Ca2+) stimuli, which it integrates through allosteric sites on its 3 regulatory subunits, alpha, beta and delta (the last being endogenous calmodulin). The (aa?d)4 PhK complex is among the largest and most complex enzymes known, with 90% of its 1.3 x 106 Da mass involved in its regulation (i.e., the integration of its activating signals). The simultaneous activation of PhK by these diverse biological signals allows for the tight control of glycogenolysis and subsequent energy production. For example, in skeletal muscle activation of PhK by Ca2+ couples contraction with energy production to sustain contraction. Moreover, the PhK complex shares high sequence similarity with a critical functional region of the troponin-actin Ca2+-dependent regulatory system of muscle contraction, suggesting co-evolution of the regulation of contraction and energy production from glycogen. Our long term goal is to elucidate the changes in intersubunit interactions that lead to activation of PhK in response to different biological signals, especially Ca2+ ions. The proposed project consists of 3 broad aims related to the effects of Ca on PhK. (1) We will characterize Ca2+-induced structural changes in PhK from the subunit level to the overall complex by employing chemical footprinting, spectroscopy, cryoelectron microscopy and X-ray crystallography. (2) Through baculovirus-mediated expression of recombinant complexes, we will evaluate the function of residues and regions that are thought to participate in PhK's Ca2+-sensitive a???d intersubunit communication network, which shares similarities with Ca2+ switching in the troponin-actin system. (3) We will seek to identify Ca2+-dependent targeting of PhK to proteins in muscle other than glycogen phosphorylase using two-hybrid screening and phosphoproteomics with PhK-deficient mice. Such targets would open the possibility of other pathways or processes being connected to the Ca2+-dependent contraction and energy production in muscle.