p42/p44 Mitogen-activated Protein Kinases Phosphorylate Hypoxia-inducible Factor 1α (HIF-1α) and Enhance the Transcriptional Activity of HIF-1*
- From the Institute of Signaling, Developmental Biology and Cancer Research, UMR CNRS 6543, Centre Antoine Lacassagne, 33 Avenue Valombrose, 06189 Nice, France
Abstract
Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor and erythropoietin in low oxygen conditions. However, the molecular mechanisms that underlie the activation of the limiting subunit, HIF-1α, are still poorly resolved. Results showing that endogenous HIF-1α migrated 12 kDa higher than in vitrotranslated protein led us to evaluate the possible role of phosphorylation on this phenomenon. We report here that HIF-1α is strongly phosphorylated in vivo and that phosphorylation is responsible for the marked differences in the migration pattern of HIF-1α. In vitro, HIF-1α is phosphorylated by p42 and p44 mitogen-activated protein kinases (MAPKs) and not by p38 MAPK or c-Jun N-terminal kinase. Interestingly, p42/p44 MAPK stoichiometrically phosphorylate HIF-1α in vitro, as judged by a complete upper shift of HIF-1α. More importantly, we demonstrate that activation of the p42/p44 MAPK pathway in quiescent cells induced the phosphorylation and shift of HIF-1α, which was abrogated in presence of the MEK inhibitor, PD 98059. Finally, we found that in a vascular endothelial growth factor promoter mutated at sites previously shown to be MAPK-sensitive (SP1/AP2–88-66 site), p42/p44 MAPK activation is sufficient to promote the transcriptional activity of HIF-1. This interaction between HIF-1α and p42/p44 MAPK suggests a cooperation between hypoxic and growth factor signals that ultimately leads to the increase in HIF-1-mediated gene expression.
Footnotes
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↵* This work was supported by grants from CNRS, Le Ministere de la Recherche (ACC-SV9), La Ligue Nationale Contre le Cancer, and l'Association pour la Recherche contre le Cancer, and by European Community Contract B104-CT97-2071.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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↵‡ Recipient of fellowships from the Heart and Stroke Foundation of Canada, le Ministère des Affaires Etrangères Français, and the O'Brien Foundation; presently supported by ELF-Aquitaine. To whom correspondence should be addressed. Tel.: 33-4-92-03-12-28; Fax: 33-4-92-03-12-25; E-mail: drichard@unice.fr.
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↵§ Supported by an EMBO long term fellowship. Recipient of a fellowship from the Human Frontiers Science Program.
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↵¶ Recipient of a scholarship from CNRS and Roussel Uclaf.
- Abbreviations:
- VEGF
-
vascular endothelial growth factor
- HIF
-
hypoxia-inducible factor
- MAPK
-
mitogen-activated protein kinase
- FCS
-
fetal calf serum
- HA
-
hemagglutinin
- PCR
-
polymerase chain reaction
- ER
-
endoplasmic reticulum
- JNK
-
c-Jun N-terminal kinase
- DTT
-
dithiothreitol
- DMEM
-
Dulbecco's modified Eagle's medium
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- Received June 30, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
