Glycogen Synthase Kinase‐3 from Rabbit Skeletal Muscle

Abstract

1. A glycogen synthase kinase was partially purified from rabbit skeletal muscle by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and chromatography on hydroxyapatite.2. The enzyme was highly specific for glycogen synthase. In the standard assay, the relative rates of phosphorylation were : glycogen synthase (loo), phosvitin (2.5), phosphorylase kinase ( < l), casein (0.3), protein phosphatase inhibitor-I (< ().I), phosphorylase (< 0.01), mixed histones (< 0.01). The enzyme was separated from virtually all phosvitin kinase and casein kinase activity by the chromatography on DEAE-cellulose.3. The K , values for ATP and GTP were 0.02 mM and 0.5 mM respectively, and a similar maximum reaction velocity was obtained with each nucleoside triphosphate. The activity of the enzyme was unaffected by cyclic AMP, cyclic GMP, calcium ions, calcium ions plus calmodulin, and the specific protein inhibitor of cyclic-AMP-dependent protein kinase.4. The properties of the enzyme demonstrated that it was distinct from both cyclic-AMPdependent protein kinase and phosphorylase kinase, the two well characterized glycogen synthase kinases in skeletal muscle. This enzyme was therefore termed glycogen synthase kinase-3.5. The phosphorylation of glycogen synthase by glycogen synthase kinase-3 reached a pleateau near 1.5 molecule phosphate incorporated per subunit under optimal conditions. The activity of glycogen synthase measured in the absence of glucose 6-phosphate was decreased fivefold and the apparent K, for glucose 6-phosphate was increased 15-fold, when 1.2 molecule phosphate per subunit had been introduced into the enzyme. Phosphorylation to a similar extent with either cyclic-AMP-dependent protein kinase or phosphorylase kinase produced smaller changes in activity.6. Glycogen synthase was phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase and glycogen synthase kinase-3, using conditions where the phosphorylation by any one protein kinase reached a plateau near one molecule of phosphate incorporated per subunit. The different protein kinases were used separately and in combination to generate seven different phosphorylated species of glycogen synthase. The phosphorylation of glycogen synthase approached two molecules per subunit when any two protein kinases werc combined, and three molecules per subunit when all three protein kinases were combined, and the inactivation produced by the different protein kinases was essentially additive. The results imply that each protein kinase preferentially phosphorylates a different site(s) on glycogen synthase, and this is confirmed by the amino acid sequence analysis described in the following paper in this journal.

Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice

“…Nonphosphorylated GSK3␤ is constitutively active and represses glycogen synthase activity (28). Importantly, GW4064 treatment of db͞db mice increased the hepatic levels of phosphorylated GSK3␤ (at Ser-9) (Fig.…”

Glycogen Synthase Kinase 3 (GSK-3)-mediated Phosphorylation of Uracil N-Glycosylase 2 (UNG2) Facilitates the Repair of Floxuridine-induced DNA Lesions and Promotes Cell Survival

“…Notably, Thr 60 in UNG2 conforms to a consensus sequence for GSK-3 (Ser/Thr-XXX-Ser(P)/Thr(P)) when Ser 64 is phosphorylated. Originally identified in 1980 (29), GSK-3 has since been shown to phosphorylate nearly 100 substrates (28) and influence many distinct cellular processes, including metabolism, proliferation (30), differentiation (27), and cancer progression (31)(32)(33)(34)(35)(36).…”

Glycogen Synthase Kinase 3β Suppresses Myogenic Differentiation through Negative Regulation of NFATc3

“…The protein kinase GSK-3 2 is ubiquitously expressed, and, although it was originally identified as a suppressor of glycogen synthase (6), GSK-3 has been implicated in a myriad of metabolic and signaling pathways (7). Recent studies have identified GSK-3␤ as a negative regulator of both cardiac and skeletal muscle hypertrophy (8,9) as well as muscle differentiation (10).…”