Characterization of PDK2 activity against protein kinase B gamma.
Protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase controlled by insulin, various growth factors, and phosphatidylinositol 3-kinase. Full activation of the PKB enzyme requires phosphorylation of a threonine in the activation loop and a serine in the C-terminal tail. PDK1 has clearly been shown to phosphorylate the threonine, but the mechanism leading to phosphorylation of the serine, the PDK2 site, is unclear. A yeast two-hybrid screen using full-length human PKBgamma identified protein kinase C (PKC) zeta, an atypical PKC, as an interactor with PKBgamma, an association requiring the pleckstrin homology domain of PKBgamma. Endogenous PKBgamma was shown to associate with endogenous PKCzeta both in cos-1 cells and in 3T3-L1 adipocytes, demonstrating a physiological interaction. Immunoprecipitates of PKCzeta, whether endogenous PKCzeta from insulin-stimulated 3T3-L1 adipocytes or overexpressed PKCzeta from cos-1 cells, phosphorylated S472 (the C-terminal serine phosphorylation site) of PKBgamma, in vitro. In vivo, overexpression of PKCzeta stimulated the phosphorylation of approximately 50% of the PKBgamma molecules, suggesting a physiologically meaningful effect. However, pure PKCzeta protein was incapable of phosphorylating S472 of PKBgamma. Antisense knockout studies and use of a PDK1 inhibitor showed that neither PKB autophosphorylation nor phosphorylation by PDK1 accounted for the S472 phosphorylation in PKCzeta immunoprecipitates. Staurosporine inhibited the PKCzeta activity but not the PDK2 activity in PKCzeta immunoprecipitates. Together these results indicate that an independent PDK2 activity exists that physically associates with PKCzeta and that PKCzeta, by binding PKBgamma, functions to deliver the PDK2 to a required location. PKCzeta thus functions as an adaptor, associating with a staurosporine-insensitive PDK2 enzyme that catalyzes the phosphorylation of S472 of PKBgamma. Because both PKCzeta and PKB have been proposed to be required for mediating a number of crucial insulin responses, formation of an active signaling complex containing PKCzeta, PKB, and PDK2 is an attractive mechanism for ensuring that all the critical sites on targets such as glycogen synthase kinase-3 are phosphorylated.
Hodgkinson
Conrad P
CP
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, United Kingdom.
Sale
Elizabeth M
EM
Sale
Graham J
GJ
D064413:3-Phosphoinositide-Dependent Protein Kinases; D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics; D011493:Protein Kinase C / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000099221:Protein Kinase C zeta
D064413:3-Phosphoinositide-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D015513:Oncogene Proteins; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine; D019311:Staurosporine; D000099221:Protein Kinase C zeta; C491754:AKT3 protein, human; C491753:Akt3 protein, mouse; C578562:PDPK1 protein, human; C578564:Pdpk1 protein, mouse