Hypoxia Causes the Activation of Nuclear Factor κB through the Phosphorylation of IκBα on Tyrosine Residues

Abstract The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor κB (NF-κB) to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% O2) results in IκBα degradation, increased NF-κB DNA binding activity, and transactivation of a reporter gene construct containing two NF-κB DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf301) inhibited IκBα degradation, NF-κB binding, and transactivation of κB reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of IκBα with NF-κB activation, we immunoprecipitated IκBα after varying times of hypoxic exposure and found that its tyrosine phosphorylation status increased during hypoxic exposure. Inhibition of the transfer of tyrosine phosphoryl groups onto IκBα prevented IκBα degradation and NF-κB binding. In comparison to other activators of NF-κB such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of IκBα following treatment with either of these agents. These results suggest that tyrosine phosphorylation of IκBα during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-κB.

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