In the cascade activation of glycogen utilization leading 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 neural (Ca2+), hormonal (cAMP and Ca2+) and metabolic (ADP) stimuli, which are integrated through allosteric sites on its 3 regulatory subunits (alpha, beta and delta). This activation of PhK by diverse physiological signals allows for the tight control of glycogenolysis and subsequent energy production, e.g., in skeletal muscle the activation of PhK by Ca2+ couples contraction with energy production to sustain contraction. Given that the mass of the (alphabetagammadelta)4 PhK holoenzyme is 1.3 x 10[6] Da, with 90% involved in its regulation, PhK is among the largest and most complex regulatory enzymes known. Our long term goal is to determine how intersubunit interactions in PhK change in response to the different biological signals, and thus control its activity. The proposed project has 4 related aims directed toward this goal. (1) Adjacent regions of interacting subunits will be identified by chemical crosslinking of subunits and identification of the specific regions crosslinked, by the ability of synthetic peptides corresponding to specific regions of subunits to alter activity or structure, and by 2-hybrid genetic screening. (2) Defined regions of each subunit will be localized within the overall tetrahedral structural model of PhK, developed in the ending grant period, by immunoelectron microscopy using subunit specific antibodies against known epitopes and by visualization in scanning transmission electron microscopy of derivatized 3 subunits. This information will provide specific reference points for the arrangement of subunit polypeptide backbones within the overall tetrahedral structure. (3) High resolution structural information on PhK's topography will be gained through image averaging and three- dimensional reconstruction of images observed in cryoelectron microscopy, and higher resolution structural data will be sought through X-ray diffraction analyses of crystals of PhK (or of multimeric complexes or subunit fragments thereof). (4) Finally, expression systems will be developed to directly test the functions of the specific interacting regions of the various subunits identified by the above approaches. Knowledge gained from these aims will help define the relationship between quatemary structure and control of activity in this important regulatory enzyme of mammalian energy production.
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