GSK-3 beta regulation in skeletal muscles by adrenaline and insulin: Evidence that PKA and PKB regulate different pools of GSK-3

We have recently shown that while adrenaline alone has no effect on the activation of Protein Kinase B (PKB) in rat soleus muscle, it greatly potentiates the effects of insulin (Brennesvik et al., Cellular Signalling 17: 1551-1559, 2005). In the current study we went on to investigate whether this was paralleled by a similar effect on GSK-3, which is a major PKB target. Surprisingly adrenaline alone increased phosphorylation of GSK-3 beta Ser(9) and GSK-3 alpha Ser(21) and adrenaline's effects were additive with those of insulin but did not synergistically potentiate insulin action. Dibutyryl-cAMP (5 mM) and the PKA specific cAMP analogue N-6-Benzoyl-cAMP (2 mM) increased GSK-3 beta Ser(9) phosphorylation, whereas the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (1 mM) did not. Wortmannin (PI 3-kinase inhibitor; 1 mu M) blocked insulin-stimulated GSK-3 phosphorylation completely, but adrenaline increased GSK-3 beta Ser(9) phosphorylation in the presence of wortmannin. The PKA inhibitor H89 (50 mu M) reduced adrenaline-stimulated GSK-3 beta Ser(9) phosphorylation but did not influence the effects of insulin. Insulin-stimulated GSK-3 Ser9 phosphorylation was paralleled by decreased glycogen synthase phosphorylation at the sites phosphorylated by GSK-3 as expected. However, adrenaline-stimulated GSK-3 Ser9 phosphorylation was paralleled by increased glycogen synthase phosphorylation indicating this pool of GSK-3 may not be directly involved in phosphorylation of glycogen synthase. Our results indicate the existence of at least two distinct pools of GSK-3 beta in soleus muscle, one phosphorylated by PKA and another by PKB. Further, we hypothesise that each of these pools is involved in the control of different cellular processes. (c) 2006 Elsevier Inc. All rights reserved.