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

AbstractThe 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 PKCα, PKCβI, PKCγ, PKCδ, PKCϵ, and PKC-related kinase-1 (PRK1) are vastly reduced compared to control ES cells (PDK1+/+ cells). The levels of PKCζ and PRK2 protein are only moderately reduced in the PDK1−/− ES cells. We demonstrate that in contrast to PKCζ expressed PDK1+/+ ES cells, PKCζ in ES cells lacking PDK1 is not phosphorylated at its T-loop residue. This provides the first genetic evidence that PKCζ 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

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2

AbstractBackground: Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown.Results: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBα, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBα to one that phosphorylates both Thr308 and Ser473 of PKBα in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBα in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies.Conclusions: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells

AbstractBackground: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the ‘T-loop’ of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown.Results: Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted were viable. In PDK1−/− ES cells, PKB, p70 S6 kinase and p90 Rsk were not activated by stimuli that induced strong activation in PDK1+/+ cells. Other AGC kinases — namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) — had normal activity or were activated normally in PDK1−/− cells. The insulin-like growth factor 1 (IGF1) induced PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1−/− cells. IGF1 did not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1−/− cells.Conclusions: PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1−/− cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase