Abstract
Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1alpha and HIF-1beta subunits. O2-dependent degradation of the HIF-1alpha subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1alpha is regulated by the competition of RACK1 and HSP90 for binding to HIF-1alpha. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1alpha. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1alpha. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1alpha. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1alpha degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1alpha each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1alpha. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1alpha degradation. These results indicate that intracellular calcium levels can regulate HIF-1alpha expression by modulating calcineurin activity and RACK1 dimerization.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Aryl Hydrocarbon Receptor Nuclear Translocator / metabolism
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Calcineurin / metabolism*
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Calcium / metabolism*
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Calmodulin / metabolism*
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Cell Line
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Cyclosporine / pharmacology
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Dimerization
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Elongin
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Enzyme Activation / drug effects
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Enzyme Activation / physiology
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Enzyme Inhibitors / pharmacology
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GTP-Binding Proteins / metabolism*
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Gene Expression Regulation / drug effects
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Gene Expression Regulation / physiology*
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HSP90 Heat-Shock Proteins / metabolism
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Humans
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Hypoxia-Inducible Factor 1, alpha Subunit / biosynthesis*
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Neoplasm Proteins / metabolism*
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Oxygen / metabolism
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Oxygen Consumption / drug effects
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Oxygen Consumption / physiology
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Phosphorylation / drug effects
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Proteasome Endopeptidase Complex / metabolism
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Protein Binding / drug effects
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Protein Binding / physiology
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Protein Structure, Tertiary / physiology
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Receptors for Activated C Kinase
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Receptors, Cell Surface / metabolism*
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Transcription Factors / metabolism
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Ubiquitin / metabolism
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitination / drug effects
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Ubiquitination / physiology
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Von Hippel-Lindau Tumor Suppressor Protein / metabolism
Substances
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ARNT protein, human
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Calmodulin
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ELOC protein, human
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Elongin
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Enzyme Inhibitors
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HIF1A protein, human
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HSP90 Heat-Shock Proteins
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Hypoxia-Inducible Factor 1, alpha Subunit
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Neoplasm Proteins
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RACK1 protein, human
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Receptors for Activated C Kinase
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Receptors, Cell Surface
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Transcription Factors
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Ubiquitin
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Aryl Hydrocarbon Receptor Nuclear Translocator
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Cyclosporine
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Ubiquitin-Protein Ligases
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Von Hippel-Lindau Tumor Suppressor Protein
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Calcineurin
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Proteasome Endopeptidase Complex
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GTP-Binding Proteins
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VHL protein, human
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Oxygen
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Calcium