Phosphorylation of rabbit skeletal muscle glycogen synthase 1 by the cAMP dependent protein kinase,the cAMP independent synthase kinase and the phosvitin kinase from human polymorphonuclear leukocytes

Summary cAMP dependent protein kinase and cAMP independent synthase kinase incorporated up to two P_i/subunit in rabbit skeletal muscle glycogen synthase I. The first P_i/subunit was incorporated much faster than the second. After incorporation of one Pi/subunit by the CAMP dependent protein kinase, the ratio of independence (RI) was 0.20 and the dissociation constant K_c for Glc-6-P was 0.3 mm, and quite different from the RI of 0.02 and K_c (Glc-6-P) of 1 mM, obtained when one P_i/subunit was incorporated by the cAMP independent synthase kinase. Within the first P_i/subunit, the cAMP dependent protein kinase predominantly phosphorylated in the trypsin sensitive region (60–70%), corresponding to two trichloro-acetic acid soluble tryptic phosphopeptides, termed site-1 and site-2. Site-2 was found to be phosphorylated prior to site-1. CNBr degradation resolved the phosphorylated regions in two phosphopeptides with M_r 28,000 and 10,000.The larger CNBr phosphopeptides were derived from the trypsin sensitive region. Within the first P_i/subunit, synthase kinase almost exclusively phosphorylated in the trypsin insensitive region (80%) corresponding to the smaller CNBr phosphopeptide. However, when two P_i/subunit were incorporated by either the cAMP dependent protein kinase or the synthase kinase the phosphates were almost equally distributed between the trypsin sensitive and insensitive regions and K_c (Glc-6-P) increased to 2 mm, Maximum phosphorylation (2.8–3.3 P_i/subunit and K_c (Glc-6-P) 9–11 mm) was only obtainable when both the cAMP dependent protein kinase and the synthase kinase were present.The phosvitin kinase very slowly incorporated one P_i/subunit.We suggest that within the first P₁subunit phosphorylation in the trypsin insensitive region determine the affinity for the allosteric activator, glucose-6-phosphate. Thereafter phosphorylation in the trypsin sensitive region is the major determinant. Purified glycogen-free rabbit skeletal muscle glycogen synthase binds glycogen with lower affinity than polymorphonuclear leukocyte glycogen synthase. Glycogen was found to increase the initial rate of phosphorylation and facilitate the phosphorylation of site-1.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - Glc-6-P glucose-6-phosphate - UDP-Glc uridine 5-diphosphoglucose - EGTA ethylene glycol-bis(-aminoethylether)-N,N-tetraacetic acid - EDTA ethylenediamine tetraacetic acid - CNBr cyanogen bromide - DTT dithiothreitol - SDS sodium dodecyl sulphate - RI ratio of independence