Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase

Activation of protein kinase B (PKB) by growth factors and hormones has bee n demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). I n this report, we show that PKB can also be activated by PKA (cyclic AMP [c AMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Al though this activation required phosphorylation of PKB, PKB is not likely t o be a physiological substrate of PKA since a mutation in the sole PKA. con sensus phosphorylation site of PKB did not abolish PKA-induced activation o f PKB. In addition, mechanistically, this activation was different from tha t of growth factors since it did not require phosphorylation of the S473 re sidue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to al anine did not prevent it from being activated by forskolin. Moreover, phosp hopeptide maps of overexpressed PKB from COS cells showed differences betwe en insulin- and forskolin-stimulated cells that pointed to distinct activat ion mechanisms of PKB depending on whether insulin or cAMP was used. We loo ked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) a ctivity, a known in vivo substrate of PKB. Overexpression of a dominant neg ative PKB led to the loss of inhibition of GSK-3 in both insulin- and forsk olin-treated cells, demonstrating that PKB was responsible for this inhibit ion in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma m embrane. This process was inhibited by high concentrations of wortmannin (3 00 nM), suggesting that forskolin-induced PKB movement may require phosphol ipids, which are probably not generated by class I or class III PI3-kinase, However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-indu ced PKB activation.