Tumour necrosis factor-alpha activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308.
Tumour necrosis factor-alpha (TNF-alpha) may activate both cell survival and cell death pathways. In the murine fibrosarcoma cell line WEHI-164, physiological concentrations (1 ng/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 death induced by TNF-alpha in the presence of actinomycin D, confirming that TNF-alpha activates a transcription-independent survival pathway requiring PI3-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 phosphorylation of Ser(473) was increased, even in the presence of okadaic acid 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 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 Ser(473) on PKB, but not its kinase activity, whereas TNF-alpha failed to induce Ser(473) phosphorylation or kinase activation of either overexpressed T308A or wild-type PKB (where T308A is the mutant bearing 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 detected with TNF-alpha. We conclude that, at physiological concentrations, TNF-alpha activates endogenous PKB by stimulating PDK2 (increase in Ser(473) phosphorylation) in a PI3-kinase-dependent (wortmannin-sensitive) manner, without causing detectable stimulation of PDK1 (no increase in Thr(308) phosphorylation) or ARNO translocation. Possible explanations of these observations are discussed.
O'toole
A
A
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
Moule
S K
SK
Lockyer
P J
PJ
Halestrap
A P
AP
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D002462:Cell Membrane; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D013912:Threonine / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D007334:Insulin-Like Growth Factor I; D019319:Okadaic Acid; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D012694:Serine; D013912:Threonine; D014409:Tumor Necrosis Factor-alpha; C102474:cytohesin-2; C578562:PDPK1 protein, human