Glycogen Synthase from Rabbit Skeletal Muscle; Effect of Insulin on the State of phosphorylation of the Seven Phosphoserine Residues in vivo

Abstract

Rabbits were starved for 24 h and injected with propranolol ( 2 mg/kg) with or without insulin (33 pg/kg) 15 min prior to sacrifice. In muscle extracts prepared from propranolol-treated animals the activity ratio (F glucose 6-phosphate) of glycogen synthase was 0.18 0.02 and the K, for glucose 6-phosphate was 1.2 0.1 mM. In (propranolol + insuIin)-treated animals the activity ratio was 0.35 & 0.02 and the I(, for glucose 6-phosphate was 0.60 & 0.05 mM.0.09 niol phosphate/mol subunit, whereas this value was 2.33 0.09 mol phosphate/mol subunit from (propranolol + insulin)-treated animals.The distribution of phosphate between the seven phosphorylation sites was elucidated. In (propranolol + insulin)-treated animals there was a decrease of 0.4-0.45 mol phosphate/mol subunit in sites (3a + 3b + 3c), and no significant changes in any other site (la, lb, 2 and 5). The results demonstrate that dephosphorylation of sites (3a + 3b + 3c) is responsible for the activation of glycogen synthase observed after acute administration of insulin.Neither protein phosphatase 1 nor protein phosphatase 2A, which account for virtually all the glycogen synthase phosphatase activity in skeletal muscle extracts, were able to mimic the specific dephosphorylation of sites (3a + 3b + 3c) produced by insulin. Both enzymes dephosphorylated site 2 and sites (3a + 3b + 3c) in v i m at comparable rates. The action of insulin may therefore involve a decrease in the activity of glycogen synthase kinase 3 and the regulation of this enzyme is discussed.The failure of insulin to produce a significant dephosphorylation of the sites phosphorylated by cyclic-AMPdependent protein kinase (la, I b and 2) demonstrates that inhibition of this protein kinase does not underlie the activation of glycogen synthase by this hormone. This conclusion is supported by measurements of the phosphorylation states of inhibitor 1, phosphorylase kinase and phosphorylase.Neither 24-h starvation, nor alloxon-induced diabetes, produced significant changes in the kinetic properties or phosphorylation state of glycogen synthase. Possible reasons for these findings are discussed.Glycogen synthase purified to homogeneity from propranolol-treated animals contained 2.74Glycogen synthase is the best documented and one of the most complex examples of 'multisite phosphorylation', a phenomenon that is being encountered with increasing frequency [I]. The enzyme is phosphorylated on seven serine residues by at least five different protein kinases [2] [7].In general, phosphorylation increases the K , for UDPglucose, decreases the Ki for inhibitors such as Pi and ADP, and increases the K, for the activator glucose-6-P [S]. Phosphorylation of sites (3a + 3b + 3c) or site 2 influences the kinetic parameters to a greater extent than site l a , but the ~ Abbreviations. SDS, sodium dodecyl sulphate; HEDTA, N '-(2-hydroxyethyl)-ethylenedianiine-N,N',N-triacetate; HPLC, high-performance liquid chromatography ; glucose-6-P, glucose 6-phosphate.Enzymes. Phosphorylase (EC 2.4.1.1); phosphorylase kina...

Oxidative stress-induced insulin resistance in rat skeletal muscle: role of glycogen synthase kinase-3

“…3), inasmuch as Akt can phosphorylate GSK-3 on serine residues and inhibit its activity (4). The decreased insulin-dependent activation of Akt observed under these conditions would therefore allow GSK-3 to attain an overactive state and inhibit glycogen synthase activity via serine phosphorylation (22,25,34). In support of this concept is our observation of a significant correlation between phosphorylation (Ser 473 ) of Akt and the glycogen synthase activity ratio in control and oxidant-treated soleus muscle (r ϭ 0.453, P ϭ 0.045).…”

Glycogen synthase kinase-3: cryoprotection and glycogen metabolism in the freeze-tolerant wood frog

“…With GSK-3 inhibited, protein substrates including GS could be dephosphorylated and activated by protein phosphatases. This mechanism is integral to the insulin-induced activation of glycogen synthesis and other pathways, such as protein synthesis (Ingebritsen and Cohen, 1983;Parker et al, 1983;Ali et al, 2001). In the absence of insulin and other stimuli, GSK-3 is constitutively active (Cohen and Frame, 2001;Doble and Woodget, 2003) and free to phosphorylate its downstream targets, maintaining GS in an inactive state and glucose in a free, nonpolymerized form.…”

GSK-3 – at the crossroads of cell death and survival

“…A few years later, insulin was described to have the opposite effect on glycogen synthase; by inducing the dephosphorylation of the sites that are targeted by GSK-3, it mediates the activation of glycogen synthase (Parker et al, 1983).…”