In the cascade activation of glycogen catabolism leading to energy production in mammalian skeletal muscle, phosphorylase kinase (PbK) phosphorylate and activates glycogen phosphorylase, thus regulating carbohydrate metabolism. The activity of PbK, catalyzed by its gamma subunit, is controlled, in turn by neural, hormonal and metabolic stimuli, which it integrates through allosteric sites on its three regulatory subunits (alpha, Beta, and delta). Neural stimulation of PbK activity is effected by Ca2+ ions, which bind to the kinase's intrinsic calmodulin subunit delta, thus coupling muscle contraction with energy production to sustain contraction. Hormonal stimulation of PbK activity is mediated by Ca2+ and by cAMP, the latter signal causing activation through phosphorylation of PbK's B, and to a lesser extent a, high affinity nucleotide binding site on its Beta subunit. Reasoning that the prime metabolic feature of PbK is its regulatory role in integrating these diverse physiological signals, a long term objective of our laboratory is to determine how inter subunit interactions of PbK change in response to these signals, and as result, alter its catalytic activity. Although a great deal is known about the regulation of PbK's activity, much less is known about its structure, particularly its quaternary structure and the relationship of quaternary structure to activity. We have recently developed a three-dimensional structural model of the hexadecameric PbK holoenzyme in which four alpha/Beta/gamma/delta protomers are packed. This project has four interrelated specific aims concerning the enzyme's quaternary structure. (1) Defined regions of the four different subunits will be localized within the overall bridged, bilobal three-dimensional structure. (2) Specific adjacent regions of interacting subunit will be identified by subunit crosslinking, by functional effects of synthethic peptides corresponding to segments of subunits, and by two-hybrid genetic screening. Because the sequences of all the subunits are known, this information will further define subunit packing geometries and intersubunit communication. (3) The specific interactions between calmodulin (the delta subunit) and the catalytic subunit (gamma) will be studied by use of altered, recombinant calmodulins and by chemical crosslinking. This information will help determine which structural features of these two interacting subunits are necessary for binding and for activation. (4) Conformation changes associated with activation of the (alpha, beta, gamma, delta)4 holoenzyme will be studied by immunochemical techniques, differential chemical modification, and characterization of inhibitory peptides from the alpha/beta subunits. The knowledge gained from these specific aims will begin to define the relationships between quaternary structure and control of activity in his key 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 #
5R01DK032953-14
Application #
2138933
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1986-09-01
Project End
1999-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
14
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Tennessee Health Science Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
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
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
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
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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