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B through the Phosphorylation of I
B
on Tyrosine Residues1
Cancer Biology Research Laboratory, Stanford University School of Medicine, Stanford, California 94305-5468
The response of mammalian cells to stress is controlled by transcriptional regulatory proteins such as nuclear factor
B (NF-
B) 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 I
B
degradation, increased NF-
B DNA binding activity, and transactivation of a reporter gene construct containing two NF-
B DNA binding sites. Pretreatment of cells with protein tyrosine kinase inhibitors and the dominant negative allele of c-Raf-1 (Raf301) inhibited I
B
degradation, NF-
B binding, and transactivation of
B reporter constructs by hypoxia. To demonstrate a direct link between changes in the phosphorylation pattern of I
B
with NF-
B activation, we immunoprecipitated I
B
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 I
B
prevented I
B
degradation and NF-
B binding. In comparison to other activators of NF-
B such as phorbol myristate acetate or tumor necrosis factor, we did not detect changes in the tyrosine phosphorylation status of I
B
following treatment with either of these agents. These results suggest that tyrosine phosphorylation of I
B
during hypoxia is an important proximal step which precedes its dissociation and degradation from NF-
B.
1 This work was supported by Grant CA03353 from the National Cancer Institute to A. J. G. and a NIH Predoctoral Training Grant to A. C. K.
2 To whom requests for reprints should be addressed.
Received 1/14/94. Accepted 2/ 4/94.
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