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 fa ctors. The proto-oncogenic protein kinase B (PKB; also known as RAC pr otein 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, c auses activation of PKB. Stimulation of serum-starved PC12 rat pheochr omocytoma cells with NGF caused an increase of up to 14-fold in PKB ac tivity. This activation was detected within 1 min of stimulation and o ccurred at NGF concentrations that are consistent with TrkA-mediated s ignaling. PKB activation was accompanied by a decrease in electrophore tic mobility of the kinase, which is characteristic of phosphorylation . Both PKB activation and mobility changes were prevented by wortmanni n: indicating the upstream involvement of PtdIns 3-kinase in these eve nts. Analyses employing isoform-specific antibodies for immunoprecipit ation 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 n eurite retraction, neither stimulated PKB activity, nor affected NGF-i nduced 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 cytoske letal changes mediated by the small GTPase Rho.