Further evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms

The multi-site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T-loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1), In this study, we demonstrate that in embryonic stem (ES) cells lacking PDK1 (PDK1 -/- cells), the intracellular levels of endogenously expressed PKC alpha, PKC betaI, PKC gamma, PKC delta, PKC epsilon, and PKC-related kinase-1 (PRK1) are vastly reduced compared to control ES cells (PDK1 +/+ cells). The levels of PKC zeta and PRK2 protein are only moderately reduced in the PDK1 -/- ES cells. We demonstrate that in contrast to PKC zeta expressed PDK1 +/+ ES cells, PKC zeta in ES cells lacking PDK1 is not phosphorylated at its T-loop residue. This provides the first genetic evidence that PKC zeta is a physiological substrate for PDK1, In contrast, PRK2 is still partially phosphorylated at its T-loop in PDK1 -/- cells, indicating the existence of a PDK1-independent mechanism for the phosphorylation of PRK2 at this residue. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B,V, All rights reserved.