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Insulin-stimulated Phosphorylation of the Protein Phosphatase-1 Striated Muscle Glycogen-targeting Subunit and Activation of Glycogen Synthase*

Jun Liu,D. Brautigan

Published 2000 in Journal of Biological Chemistry

ABSTRACT

Protein phosphatase-1 (PP-1) in heart and skeletal muscle binds to a glycogen-targeting subunit (GM) in the sarcoplasmic reticulum. Phosphorylation of GM has been postulated to govern activity of PP1 in response to adrenaline and insulin. In this study, we used biochemical assays and GM expression in living cells to examine the effects of insulin on the phosphorylation of GM, and the binding of PP-1 to GM. We also assayed glycogen synthase activation in cells expressing wild type GM and GM mutated at the phosphorylation sites. In biochemical assays kinase(s) prepared from insulin-stimulated Chinese hamster ovary (CHO-IR) cells and C2C12 myotubes phosphorylated a glutathioneS-transferase (GST) fusion protein, GST-GM(1–240), at both site 1 (Ser48) and site 2 (Ser67). Phosphorylation of both sites was dependent on activation of the mitogen-activated protein kinase pathway, involving in particular ribosomal protein S6 kinase. Full-length GMwas expressed in CHO-IR cells and metabolic 32P labeling at sites 1 and 2 was increased by insulin treatment. The GMexpressed in CHO-IR cells or in C2C12 myotubes co-immunoprecipitated endogenous PP-1, and association was transiently lost following treatment of the cells with insulin. In contrast PP-1 binding to GM(S67T), a version of GM not phosphorylated at site 2, was unaffected by insulin treatment. Expression of GM increased basal activity of endogenous glycogen synthase in CHO-IR cells. Insulin stimulated glycogen synthase activity the same extent in cells expressing wild type GM or GMmutated to eliminate phosphorylation site 1 and/or site 2. Phosphorylation of GM is stimulated by insulin, but this phosphorylation is not involved in insulin control of glycogen metabolism. We speculate that other functions of GM at the sarcoplasmic reticulum membrane might be affected by insulin.

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