PMID- 10373549 OWN - NLM STAT- MEDLINE DCOM- 19990722 LR - 20191210 IS - 0270-7306 (Print) IS - 0270-7306 (Linking) VI - 19 IP - 7 DP - 1999 Jul TI - Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase. PG - 4989-5000 AB - Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, 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 alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked 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) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, 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-induced PKB activation. FAU - Filippa, N AU - Filippa N AD - Institut National de la Sante et de la Recherche Medicale Faculte de Medecine, 06107 Nice Cedex 2, France. FAU - Sable, C L AU - Sable CL FAU - Filloux, C AU - Filloux C FAU - Hemmings, B AU - Hemmings B FAU - Van Obberghen, E AU - Van Obberghen E LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - United States TA - Mol Cell Biol JT - Molecular and cellular biology JID - 8109087 RN - 0 (Androstadienes) RN - 0 (Enzyme Inhibitors) RN - 0 (Insulin) RN - 0 (Luminescent Proteins) RN - 0 (Phosphoinositide-3 Kinase Inhibitors) RN - 0 (Proto-Oncogene Proteins) RN - 0 (Recombinant Fusion Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - 1F7A44V6OU (Colforsin) RN - 452VLY9402 (Serine) RN - E0399OZS9N (Cyclic AMP) RN - EC 2.7.11.- (Glycogen Synthase Kinases) RN - EC 2.7.11.1 (Protein-Serine-Threonine Kinases) RN - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt) RN - EC 2.7.11.11 (Cyclic AMP-Dependent Protein Kinases) RN - EC 2.7.11.17 (Calcium-Calmodulin-Dependent Protein Kinases) RN - EC 2.7.11.26 (Glycogen Synthase Kinase 3) RN - XVA4O219QW (Wortmannin) SB - IM MH - Androstadienes/pharmacology MH - Animals MH - Biological Transport MH - COS Cells MH - Calcium-Calmodulin-Dependent Protein Kinases/metabolism MH - Cell Line, Transformed MH - Cell Membrane/metabolism MH - Colforsin/metabolism/pharmacology MH - Cyclic AMP/metabolism MH - Cyclic AMP-Dependent Protein Kinases/*metabolism MH - Enzyme Activation MH - Enzyme Inhibitors/pharmacology MH - Glycogen Synthase Kinase 3 MH - Glycogen Synthase Kinases MH - Green Fluorescent Proteins MH - Humans MH - Insulin/metabolism/pharmacology MH - Luminescent Proteins/genetics/metabolism MH - Phosphatidylinositol 3-Kinases/metabolism MH - Phosphoinositide-3 Kinase Inhibitors MH - Phosphorylation MH - *Protein-Serine-Threonine Kinases MH - Proto-Oncogene Proteins/*metabolism MH - Proto-Oncogene Proteins c-akt MH - Recombinant Fusion Proteins/genetics/metabolism MH - Serine/metabolism MH - Wortmannin MH - Xenopus PMC - PMC84322 EDAT- 1999/06/22 00:00 MHDA- 1999/06/22 00:01 CRDT- 1999/06/22 00:00 PHST- 1999/06/22 00:00 [pubmed] PHST- 1999/06/22 00:01 [medline] PHST- 1999/06/22 00:00 [entrez] AID - 10.1128/mcb.19.7.4989 [doi] PST - ppublish SO - Mol Cell Biol. 1999 Jul;19(7):4989-5000. doi: 10.1128/mcb.19.7.4989.