Protein kinase B localization and activation differentially affect S6 kinase 1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation.
Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D008954:Models, Biological; D008957:Models, Genetic; D016296:Mutagenesis; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D000077191:Wortmannin