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Regulation of glycogen synthase. Dephosphorylation of the skeletal muscle enzyme


, : Regulation of glycogen synthase. Dephosphorylation of the skeletal muscle enzyme. Journal of Biological Chemistry 253(8): 2540-2545

The dephosphorylation of 32P-labeled glycogen synthase was studied using a phosphoprotein phosphatase purified 1000-fold from rabbit skeletal muscle by DEAE-cellulose column chromatography and Sephadex G-200 chromatography. The phosphatase activity, which was stimulated by MnCl2 (1-10 mM) but not by MgCl2, catalyzed dephosphorylation of 32P-glycogen synthase, 32P-phosphorylase and 32P-histones. By utilizing the differences in phosphorylation specificities displayed by the c[cyclic]AMP-dependent protein kinase catalytic subunit and the cAMP-independent synthase kinase, 3 species of 32P-synthase were prepared. The 1st 2 species contained 0.7 mol of 32P/synthase subunit (90,000 g) which were located predominantly (70-80%) in either the trypsin-sensitive or the trypsin-insensitive phosphorylation regions of glycogen synthase. These 2 species of 32P-synthase were slowly dephosphorylated at the same rate by the phosphoprotein phosphatase. The 3rd species of 32P-synthase was phosphorylated in both the trypsin-sensitive and trypsin-insensitive regions (1.5-1.9 mol of 32P/90,000 g) by the cAMP-dependent protein kinase. This 32P-synthase was initially dephosphorylated (from 1.9 down to 1.0 mol of 32P/90,000 g) at a 3- to 4-fold higher rate than either of the 32P-synthase species with only one region phosphorylated. Analysis of CNBr 32P-peptides derived from 32P-synthase showed that the phosphatase preferentially dephosphorylated the trypsin-insensitive region during the initial rapid phase of dephosphorylation. The rate of dephosphorylation of this 3rd species of 32P-synthase decreased markedly when the phosphate content was reduced by the phosphatase to approximately 1 mol of 32P/90,000 g. The phosphoprotein phosphatase has a specificity for the trypsin-insensitive phosphorylation region on glycogen synthase, which regulates the G-6-P dependency of synthase, but only if both the trypsin-sensitive and the trypsin-insensitive regions are phosphorylated. A role of the trypsin-sensitive region of phosphorylation may be to increase the rate of dephosphorylation of the trypsin-insensitive region by a synthase phosphatase, thereby enhancing the rate of activation of the enzyme.

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Accession: 006288182

PMID: 204655

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