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J. Biol. Chem., Vol. 276, Issue 28, 25643-25646, July 13, 2001

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Insulin-stimulated Protein Kinase B Phosphorylation on Ser-473 Is Independent of Its Activity and Occurs through a Staurosporine-insensitive Kinase^*

Michelle M. Hill, Mirjana Andjelkovic, Derek P. Brazil, Stefano Ferrari^§, Doriano Fabbro^§, and Brian A. Hemmings^¶

From the Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel and the ^§ Department of Oncology, Novartis Pharma AG, CH-4057 Basel, Switzerland

Full activation of protein kinase B (PKB, also called Akt) requires phosphorylation on two regulatory sites, Thr-308 in the activation loop and Ser-473 in the hydrophobic C-terminal regulatory domain (numbering for PKB/Akt-1). Although 3'-phosphoinositide-dependent protein kinase 1 (PDK1) has now been identified as the Thr-308 kinase, the mechanism of the Ser-473 phosphorylation remains controversial. As a step to further characterize the Ser-473 kinase, we examined the effects of a range of protein kinase inhibitors on the activation and phosphorylation of PKB. We found that staurosporine, a broad-specificity kinase inhibitor and inducer of cell apoptosis, attenuated PKB activation exclusively through the inhibition of Thr-308 phosphorylation, with Ser-473 phosphorylation unaffected. The increase in Thr-308 phosphorylation because of overexpression of PDK1 was also inhibited by staurosporine. We further show that staurosporine (CGP 39360) potently inhibited PDK1 activity in vitro with an IC₅₀ of ~0.22 µM. These data indicate that agonist-induced phosphorylation of Ser-473 of PKB is independent of PDK1 or PKB activity and occurs through a distinct Ser-473 kinase that is not inhibited by staurosporine. Moreover, our results suggest that inhibition of PKB signaling is involved in the proapoptotic action of staurosporine.

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