Hypoxia Causes the Activation of Nuclear Factor KB through the Phosphorylation of IKB«on Tyrosine Residues1

The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor KB iNT-Kit i to induce a wide variety of early response genes. In this report, we show that exposure of cells to hypoxia (0.02% (>,) results in IK-HI» degradation, increased N1 -H-IÕ DNA binding activity, and transactivation of a reporter gene con struct containing two NF-KB DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative alÃ-eleof c-Raf-1 (Raf 301) inhibited IKBa degradation, NF-KB binding, and transactivation of KB reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I«Bawith NF-KB activation, we immunoprecipitated IK!!«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 IKÃœIVprevented IK!!«degradation and NF-KB binding. In comparison to other activators of NF-KB such as phorbol myristate ac etate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of IKBa following treatment with either of these agents. These results suggest that tyrosine phosphorylation of Itili« dur ing hypoxia is an important proximal step which precedes its dissociation and degradation from NF-KB.