HYPOXIA CAUSES THE ACTIVATION OF NUCLEAR FACTOR KAPPA-B THROUGH THE PHOSPHORYLATION OF I-KAPPA-B-ALPHA ON TYROSINE RESIDUES

The response of mammalian cells to stress is controlled by transcripti onal regulatory proteins such as nuclear factor kappa B (NF-kappa B) t o induce a wide variety of early response genes. In this report, we sh ow that exposure of cells to hypoxia (0.02% O-2) results in I kappa B alpha degradation, increased NF-kappa B DNA binding activity, and tran sactivation of a reporter gene construct containing two NF-kappa B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inh ibitors and the dominant negative allele of c-Raf-1 (Raf 301) inhibite d I kappa B alpha degradation, NF-kappa B binding, and transactivation of kappa B reporter constructs by hypoxia. To demonstrate a direct li nk between changes in the phosphorylation pattern of I kappa B alpha w ith NF-kappa B activation, we immunoprecipitated I kappa B alpha after varying times of hypoxic exposure and found that its tyrosine phospho rylation status increased during hypoxic exposure. Inhibition of the t ransfer of tyrosine phosphoryl groups onto I kappa B alpha prevented I kappa B alpha degradation and NF-kappa B binding. In comparison to ot her activators of NF-kappa B such as phorbol myristate acetate or tumo r necrosis factor, we did not detect changes in the tyrosine phosphory lation status of I kappa B alpha following treatment with either of th ese agents. These results suggest that tyrosine phosphorylation of I k appa B alpha during hypoxia is an important proximal step which preced es its dissociation and degradation from NF-kappa B.