Role of Prolyl Hydroxylation in Oncogenically Stabilized Hypoxia-inducible Factor-1α*
- From the Program in Cancer Biology, Department of Radiation Oncology, Stanford University, Stanford, California 94305
Abstract
Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1α and proline 531 of HIF-2α are hydroxylated under normoxia. Importantly, HIF-1α Pro-564 and HIF-2α Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1α Pro-564 and HIF-2α Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1α and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1α is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1α under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1α stabilization. Thus, these results indicate distinct pathways for HIF-1α stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1α expression through prolyl hydroxylation.
Footnotes
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↵* This investigation was supported by NCI, National Institutes of Health Grants CA67166 (to A. J. G. and N. C. D.) and CA09302 (D. A. C. and P. D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Center for Clinical Sciences Research, Dept. of Radiation Oncology, Stanford University, 269 Campus Dr., Stanford, CA 94305. Tel.: 650-723-7366; Fax: 650-723-7382; E-mail: giaccia@stanford.edu.
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Published, JBC Papers in Press, August 16, 2002, DOI 10.1074/jbc.M206922200
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↵2 D. A. Chan, unpublished results.
- Abbreviations:
- HRE
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hypoxia-responsive element
- HIF-1α
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hypoxia-inducible factor-1α
- VHL
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von Hippel-Lindau
- DFO
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desferrioxamine
- DMOG
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dimethyloxalyglycine
- GAPDH
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glyceraldehyde-3-phosphate dehydrogenase
- HKO
-
HIF-1α knockout
- MEF
-
mouse embryo fibroblast
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- Received July 11, 2002.
- The American Society for Biochemistry and Molecular Biology, Inc.
