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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK032953-19
Application #
6342440
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Sechi, Salvatore
Project Start
1986-09-01
Project End
2003-12-31
Budget Start
2001-01-01
Budget End
2001-12-31
Support Year
19
Fiscal Year
2001
Total Cost
$323,028
Indirect Cost
Name
University of Missouri Kansas City
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800772162
City
Kansas City
State
MO
Country
United States
Zip Code
64110
Thompson, Jackie A; Carlson, Gerald M (2017) The regulatory ? and ? subunits of phosphorylase kinase directly interact with its substrate, glycogen phosphorylase. Biochem Biophys Res Commun 482:221-225
Carlson, Gerald M; Fenton, Aron W (2016) What Mutagenesis Can and Cannot Reveal About Allostery. Biophys J 110:1912-23
Rimmer, Mary Ashley; Artigues, Antonio; Nadeau, Owen W et al. (2015) Mass Spectrometric Analysis of Surface-Exposed Regions in the Hexadecameric Phosphorylase Kinase Complex. Biochemistry 54:6887-95
Herrera, Julio E; Thompson, Jackie A; Rimmer, Mary Ashley et al. (2015) Activation of Phosphorylase Kinase by Physiological Temperature. Biochemistry 54:7524-30
Thompson, Jackie A; Nadeau, Owen W; Carlson, Gerald M (2015) A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero-length oxidative crosslinking. Protein Sci 24:1956-63
Liu, Weiya; Nadeau, Owen W; Sage, Jessica et al. (2013) Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg(2+). Protein Sci 22:444-54
Nadeau, Owen W; Lane, Laura A; Xu, Dong et al. (2012) Structure and location of the regulatory ? subunits in the (????)4 phosphorylase kinase complex. J Biol Chem 287:36651-61
Nadeau, Owen W; Carlson, Gerald M (2012) A review of methods used for identifying structural changes in a large protein complex. Methods Mol Biol 796:117-32
Lane, Laura A; Nadeau, Owen W; Carlson, Gerald M et al. (2012) Mass spectrometry reveals differences in stability and subunit interactions between activated and nonactivated conformers of the (????)4 phosphorylase kinase complex. Mol Cell Proteomics 11:1768-76
Nadeau, Owen W; Liu, Weiya; Boulatnikov, Igor G et al. (2010) The glucoamylase inhibitor acarbose is a direct activator of phosphorylase kinase. Biochemistry 49:6505-7

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