Tumour necrosis factor-alpha activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser(473), but not Thr(308)

Tumour necrosis factor-alpha (TNF-alpha) may activate both cell survival an d cell death pathways. In the murine fibrosarcoma cell line WEHI-164, physi ological concentrations (1ng/ml) of TNF-alpha induced wortmannin-sensitive cell ruffling characteristic of the phosphoinositide 3-kinase (PI3-kinase) activation associated with cell survival. Wortmannin also enhanced cell dea th induced by TNF-alpha in the presence of actinomycin D, confirming that T NF-alpha activates a transcription-independent survival pathway requiring P I3-kinase activity. Both TNF-alpha and insulin-like growth factor 1 (IGF-1) caused a 6-10-fold wortmannin-sensitive increase in protein kinase B (PKB) activity within 5 min. For IGF-1, this was associated with an increase in phosphorylation of both Thr(308) and Ser(473) whereas for TNF-alpha only ph osphorylation of Ser(473) was increased. even in the presence of okadaic ac id to inhibit protein phosphatases 1 and 2A. TNF-alpha did not decrease the phosphorylation of Thr(308) induced by IGF-1, implying that TNF-alpha neit her inhibits phosphoinositide-dependent kinase 1 (PDK1) nor activates an op posing phosphatase. In WEHI cellsoverexpressing a form of PKB. IGF-1 increa sed phosphorylation of Ser(473) on PKB, but not its kinase activity, wherea s TNF-alpha failed to induce Ser(473) phosphorylation or kinase activation of either overexpressed T308A or wild-type PKB (where T308A is the mutant b earing the substitution Thr(308)-->A). IGF-1 caused translocation of green- fluorescent-protein-tagged ADP-ribosylation factor nucleotide-binding site opener (ARNO) to the plasma membrane of WEHI cells, but this was not detect ed with TNF-alpha. We conclude that, at physiological concentrations, TNF-a lpha activates endogenous PKB by stimulating PDK2 (increase in Ser(473) pho sphorylation) in a PI3-kinase-dependent (wortmannin-sensitive) manner, with out causing detectable stimulation of PDK1 (no increase in Thr(308) phospho rylation) or ARNO translocation. Possible explanations of these observation s are discussed.