Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha.
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-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha 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-1alpha Pro-564 and HIF-2alpha 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-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.
Program in Cancer Biology, Department of Radiation Oncology, Stanford University, Stanford, California 94305, USA.
Denise A
DA
Chan
Patrick D
PD
Sutphin
Nicholas C
NC
Denko
Amato J
AJ
Giaccia
D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006900:Hydroxylation; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D015151:Immunoblotting; D007501:Iron / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D000600:Amino Acids, Dicarboxylic; D002614:Chelating Agents; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014157:Transcription Factors; D003035:Cobalt; D011392:Proline; D007501:Iron; D008156:Luciferases; C018021:cobaltous chloride; D003676:Deferoxamine; C040947:oxalylglycine