Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells.
The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.
Andjelković
M
M
Friedrich Miescher-Institut, Basel, Switzerland.
Suidan
H S
HS
Meier
R
R
Frech
M
M
Alessi
D R
DR
Hemmings
B A
BA
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology; D007527:Isoenzymes; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D013917:Thrombin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D007328:Insulin; D007527:Isoenzymes; D008246:Lysophospholipids; D009414:Nerve Growth Factors; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D013917:Thrombin; D000077191:Wortmannin; C494920:Akt1 protein, rat