Regulation of glycogen synthase. Phosphorylation specifities of cAMP-dependent and cAMP-independent kinases for skeletal muscle synthase

T. R. Soderling, M. F. Jett, N. J. Hutson, B. S. Khatra

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    A cAMP-independent synthase kinase has been partially purified from rabbit skeletal muscle by ion exchange chromatography on DEAE-cellulose and P-cellulose. This kinase catalyzes the phosphorylation of glycogen synthase and the conversion of the I to the D form. The activity of this kinase is not affected by cyclic 3':5'-mononucleotides, Ca2+, ethylene glycol bis(β-aminoethyl ether) N,N'-tetraacetic acid, or the heat-stable inhibitory protein which inhibits the cAMP-dependent protein kinase. The cAMP-independent synthase kinase does not catalyze the phosphorylation of mixed histone or skeletal muscle phosphorylase b. Although the kinase preparation catalyzes phosphorylation of casein and phosvitin, heat stability studies indicate these activities primarily are due to a contaminating kinase(s). The cAMP-independent synthase kinase utilizes ATP but not GTP as the phosphoryl donor. Formation of synthase D correlates with incorporation of 1 mol of Pi/mol of synthase subunit (90,000 g) when the reaction is catalyzed by the cAMP-independent synthase kinase. With the cAMP-dependent protein kinase as catalyst, formation of synthase D requires incorporation of 2 mol of Pi/90,000 g. These differences in phosphorylation stoichiometries are due to different phosphorylation site specificities of the two kinases. This was deduced from studies of the trichloroacetic acid-soluble and -insoluble 32P-peptides from trypsinized 32P-synthase, and from analysis of CNBr 32P-peptides from 32P-synthase. It was concluded that the phosphorylation site which confers glucose-6-P dependency on synthase is not solubilized by trypsin ('trypsin-insensitive'). The trypsin-insensitive site corresponds to the lower molecular weight CNBr phosphopeptide. The site which is solubilized by trypsin ('trypsin-sensitive') is a larger molecular weight CNBr peptide and has little or no influence on glucose-6-P dependency of synthase. The first mole of Pi per subunit incorporated into synthase by the cAMP-independent kinase goes into the trypsin-insensitive site and thereby produces synthase D. There is evidence for positive cooperative synthase subunit interactions during this phosphorylation. The first mole of Pi per subunit incorporated into synthase by the cAMP-dependent kinase goes predominantly (70 to 80%) into the trypsin-sensitive site with only 20 to 30% into the trypsin-insensitive site resulting in a partially glucose-6-P dependent species. The second mole of Pi incorporated per synthase subunit by the cAMP-dependent kinase completes the phosphorylation of the trypsin-insensitive site which results in formation of synthase D.

    Original languageEnglish (US)
    Pages (from-to)7517-7524
    Number of pages8
    JournalJournal of Biological Chemistry
    Issue number21
    StatePublished - Dec 1 1977


    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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