Glycogen synthase association with the striated muscle glycogen-targeting subunit of protein phosphatase-1: Synthase activation involves scaffolding regulated by β-adrenergic signaling

Glycogen-binding subunits for protein phosphatase-1 (PP1) target the PP1 catalytic subunit (PP1C) to glycogen particles, where the enzymes glycogen synthase and glycogen phosphorylase are concentrated. Here we identify sites within the striated muscle glycogen-binding subunit (G(M)) that mediate direct binding to glycogen synthase. Both PP1C and glycogen synthase were coimmunoprecipitated with a full-length FLAG-tagged G(M) transiently expressed in COS7 cells or C2C12 myotubes. Deletion and mutational analysis of a glutathione S-transferase (GST) fusion of the N-terminal domain of G(M) (residues 1-240) identified two putative sites for binding to glycogen synthase, one of which is the WXNXGXNYX(I/L) motif that is conserved among the family of PP1 glycogen-binding subunlts. Either deletion of this motif or Ala substitution of Asn-228 in this motif disrupted the binding of glycogen synthase. Expression of full-length FLAG-G(M) in cells increased the activity of endogenous glycogen synthase, but protein disabled in either PP1 binding or glycogen synthase binding did not produce synthase activation. The results show that efficient activation of glycogen synthase requires a scaffold function of G(M) that involves simultaneous binding of both PP1C and glycogen synthase. Isoproterenol and forskolin treatment of cells decreased glycogen synthase binding to FLAG-G(M), thereby limiting synthase activation by PP1. This response was insensitive to inhibition by H-89, therefore probably not involving cAMP-dependent protein kinase, but did require inclusion of microcystin-LR during cell lysis, implying that phosphorylation was modulating binding of glycogen synthase. Phosphorylation control of binding to a scaffold site on the G(M) subunit of PP1 offers a new mechanism for regulation of muscle glycogen synthase in response to β-adrenergic signals.