University of Kansas Medical Center
913-588-7005
gcarlson@kumc.edu
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.
Nadeau, O.W., Wyckoff, G.J., Paschall, J.E., Artigues, A., Sage, J., Villar, M.T. and Carlson, G.M. (2008) CrossSearch, a User-friendly Search Engine for Detecting Chemically Cross-linked Peptides in Conjugated Proteins. Mol. Cell. Proteomics 7, 739-749.
Jeyasingham, M.D., Artigues, A., Nadeau, O.W. and Carlson, G.M. (2008) Structural Evidence for Co-evolution of the Regulation of Contraction and Energy Production in Skeletal Muscle. J. Mol. Biol. 377, 623-629.
Stiffin, R.S., Sullivan, S.M., Carlson, G.M. and Holyoak, T. (2008) Differential Inhibition of Cytosolic PEPCK by Substrate Analogues. Kinetic and Structural Characterization of Inhibitor Recognition. Biochemistry 47, 2099-2109.
Priddy, T.S., Price, E.S., Johnson, C.K. and Carlson, G.M. (2007) Single Molecule Analyses of the Conformational Substates of Calmodulin Bound to the Phosphorylase Kinase Complex. Protein Sci. 16, 1017-1023.
Priddy, T.S., MIddaugh, C.R. and Carlson, G.M. (2007) Electrostatic Changes in Phosphorylase Kinase Induced by Its Obligatory Allosteric Activator Ca2+. Protein Sci. 16, 517-527.
Nadeau, O.W., Anderson, D.W., Yang, Q., Artigues, A., Paschall, J.E., Wyckoff, G.J., McClintock, J.L. and Carlson, G.M. (2007) Evidence for the Location of the Allosteric Activation Switch in the Multisubunit Phosphorylase Kinase Complex from Mass Spectrometric Identification of Chemically Crosslinked Peptides. J. Mol. Biol. 365, 1429-1445.
Archila, S., King, M.A., Carlson, G.M. and Rice, N.A. (2006) The Cytoskeletal Organizing Protein Cdc42-interacting Protein 4 Associates with Phosphorylase Kinase in Skeletal Muscle. Biochem. Biophys. Res. Commun. 345, 1592-1599.
Hilder, T.L., Carlson, G.M., Haystead, T.A.J., Krebs, E.G. and Graves, L.M. (2005) Caspase-3 Dependent Cleavage and Activation of Skeletal Muscle Phosphorylase b Kinase. Molec. Cell. Biochem. 275, 233-242.
Nadeau, O.W. and Carlson, G.M. (2005) Protein Interactions Captured by Chemical Cross-linking, in Protein-protein Interactions: A Molecular Cloning Manual (E.A. Golemis, Ed., 2nd ed.), pp. 75-91, Cold Spring Harbor Laboratory Press, New York.
Priddy, T.S., MacDonald, B.A., Heller, W.T., Nadeau, O.W., Trewhella, J. and Carlson, G.M. (2005) Ca2+-Induced Structural Changes in Phosphorylase Kinase Detected by Small-angle X-ray Scattering. Protein Sci. 14, 1039-1048.
Nadeau, O.W., Gogol, E.P. and Carlson, G.M. (2005) Cryoelectron Microscopy Reveals New Features in the Three-dimensional Structure of Phosphorylase Kinase. Protein Sci. 14, 914-920.
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