Tumour necrosis factor-α (TNF-α) may activate both cell survival and cell death pathways. In the murine fibrosarcoma cell line WEHI-164, physiological concentrations (1ng/ml) of TNF-α induced wortmannin-sensitive cell ruffling characteristic of the phosphoinositide 3-kinase (PI3-kinase) activation associated with cell survival. Wortmannin also enhanced cell death induced by TNF-α in the presence of actinomycin D, confirming that TNF-α activates a transcription-independent survival pathway requiring PI3-kinase activity. Both TNF-α and insulin-like growth factor 1 (IGF-1) caused a 6–10-fold wortmannin-sensitive increase in protein kinase B (PKB) activity within 5min. For IGF-1, this was associated with an increase in phosphorylation of both Thr308 and Ser473, whereas for TNF-α only phosphorylation of Ser473 was increased, even in the presence of okadaic acid to inhibit protein phosphatases 1 and 2A. TNF-α did not decrease the phosphorylation of Thr308 induced by IGF-1, implying that TNF-α neither inhibits phosphoinositide-dependent kinase 1 (PDK1) nor activates an opposing phosphatase. In WEHI cells overexpressing a form of PKB, IGF-1 increased phosphorylation of Ser473 on PKB, but not its kinase activity, whereas TNF-α failed to induce Ser473 phosphorylation or kinase activation of either overexpressed T308A or wild-type PKB (where T308A is the mutant bearing the substitution Thr308 → 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 detected with TNF-α. We conclude that, at physiological concentrations, TNF-α activates endogenous PKB by stimulating PDK2 (increase in Ser473 phosphorylation) in a PI3-kinase-dependent (wortmannin-sensitive) manner, without causing detectable stimulation of PDK1 (no increase in Thr308 phosphorylation) or ARNO translocation. Possible explanations of these observations are discussed.
Abbreviations used: ARNO, ADP-ribosylation factor nucleotide-binding site opener; CHO, Chinese hamster ovary; ECL, enhanced chemiluminesence; GFP, green fluorescent protein; GSK-3, glycogen synthase kinase 3; IGF-1, insulin-like growth factor 1; MAPKAPK2, mitogen- activated protein kinase-activated protein kinase 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NF-κB, nuclear factor-κB; PDK1/2, phosphoinositide-dependent kinase 1/2; PI3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; T308A, mutant bearing the substitution Thr308 → Ala, etc.; TNF-α, tumour necrosis factor-α.