Research Focus

We are studying how communication among subunits of the enzyme phosphorylase kinase (PhK) regulates its enzymatic activity. PhK, which functions in the cascade activation of glycogen breakdown, is a particularly attractive system to study regulatory mechanisms of this type because it is among the largest and most complex enzymes known. Of its 1.3 million Da mass, 90% has a regulatory role. Through allosteric sites on its three regulator subunits, PhK integrates metabolic (ADP), hormonal (cAMP and Ca2+) and neural (Ca2+) signals, resulting in large changes in its activity. This activity change in response to diverse physiological signals allows for the tight control of glycogenolysis and subsequent energy production, e.g., in skeletal muscle PhK activation by Ca2+ ions couples contraction with energy production to sustain contraction. We are determining, using a variety of approaches, the mechanisms for how these different signals alter intersubunit interactions and activity of PhK. Two-hybrid genetic screening, protein crosslinking and synthetic peptides are used to identify interacting regions of adjacent subunits. Immunoelectron microscopy with monoclonal antibodies is used to localize regions of subunits within PhK's overall tetrahedral structure. Immunochemistry and chemical modification are used to identify regions of the protein that are influenced by allosteric effectors. Finally, site-directed mutagenesis is used to define interacting residues between subunits and to introduce reporter groups. The results from these different experimental approaches coalesce to define the relationships between specific subunit interactions and the control of activity for this important regulatory enzyme of mammalian energy production.