GSK-3β 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-3β Ser⁹ and GSK-3α Ser²¹ 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 N⁶-Benzoyl-cAMP (2 mM) increased GSK-3β Ser⁹ phosphorylation, whereas the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (1 mM) did not. Wortmannin (PI 3-kinase inhibitor; 1 μM) blocked insulin-stimulated GSK-3 phosphorylation completely, but adrenaline increased GSK-3β Ser⁹ phosphorylation in the presence of wortmannin. The PKA inhibitor H89 (50 μM) reduced adrenaline-stimulated GSK-3β Ser⁹ 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-3β 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.