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Tumour necrosis factor-alpha activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308.

ABSTRACT: 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.

SUBMITTER: O'toole A

PROVIDER: S-EPMC1222127 | biostudies-other | 2001 Oct

REPOSITORIES: biostudies-other

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Project description:BackgroundThe humoral system is activated and various cytokines are released due to infections in tissues and traumatic damage. Nuclear factor-kappa B dimers are encoded by nuclear factor-kappa B genes and regulate transcription of several crucial proteins of inflammation such as tumour necrosis factor-alpha.AimsTo investigate the possible effect of polymorphisms on tumour necrosis factor-alpha serum levels with clinical and prognostic parameters of sepsis by determining the nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A) gene polymorphisms and tumour necrosis factor-alpha serum levels.Study designCase-control study.MethodsSeventy-two patients with sepsis and 104 healthy controls were included in the study. In order to determine the polymorphisms of nuclear factor-kappa B-1-94 ins/del ATTG and tumour necrosis factor-alpha (-308 G/A), polymerase chain reaction-restriction fragment length polymorphism analysis was performed and serum tumour necrosis factor-alpha levels were determined using an enzyme-linked immunosorbent assay.ResultsWe observed no significant differences in tumour necrosis factor-alpha serum levels between the study groups. In the patient group, an increase in the tumour necrosis factor-alpha serum levels in patients carrying the tumour necrosis factor-alpha (-308 G/A) A allele compared to those without the A allele was found to be statistically significant. Additionally, an increase in the tumour necrosis factor-alpha serum levels in patients carrying tumour necrosis factor-alpha (-308 G/A) AA genotype compared with patients carrying the AG or GG genotypes was statistically significant. No significant differences were found in these 2 polymorphisms between the patient and control groups (p>0.05).ConclusionOur results showed the AA genotype and the A allele of the tumour necrosis factor-alpha (-308 G/A) polymorphism may be used as a predictor of elevated tumour necrosis factor-alpha levels in patients with sepsis.

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Tumour necrosis factor-alpha activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308.

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