Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein.
It is becoming increasingly evident that the degradation of nuclear proteins requires nuclear-cytoplasmic trafficking of both the substrate proteins, as well as the E3 ubiquitin-ligases. Here, we show that nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein (VHL) is required for oxygen-dependent ubiquitination and degradation of the alpha subunits ... of hypoxia-inducible factor (HIF-alpha). VHL engages in a constitutive transcription-sensitive nuclear-cytoplasmic shuttle unaffected by oxygen tension or levels of nuclear substrate HIF-alpha. Ubiquitinated forms of HIF-alpha, as well as VHL/ubiquitinated HIF-alpha complexes, are found solely in the nuclear compartment of normoxic or reoxygenated VHL-competent cells. HIF-alpha localizes exclusively in the nucleus of hypoxic cells but is exported to the cytoplasm upon reoxygenation. Oxygen-dependent nuclear ubiquitination and nuclear export of HIF-alpha can be prevented by treatment with an HIF-specific prolyl hydroxylase inhibitor. Treatment with inhibitors of RNA polymerase II activity, which interfere with the ability of VHL to engage in nuclear export, also prevents cytoplasmic accumulation of HIF-alpha in reoxygenated cells. This caused a marked increase in the HIF-alpha half-life without affecting its nuclear ubiquitination. We present a model by which VHL-mediated ubiquitination of HIF-alpha and its subsequent degradation are dependent upon dynamic nuclear-cytoplasmic trafficking of both the E3 ubiquitin-ligase and the nuclear substrate protein.
Mesh Terms:
Active Transport, Cell Nucleus, Animals, Blotting, Western, Cell Compartmentation, Cell Hypoxia, Cell Line, Cell Nucleus, Cytoplasm, Enzyme Inhibitors, Fatty Acids, Unsaturated, Fibroblasts, Green Fluorescent Proteins, HeLa Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Ligases, Luminescent Proteins, Mice, Oxygen, Procollagen-Proline Dioxygenase, RNA Polymerase II, Recombinant Fusion Proteins, Transcription Factors, Tumor Suppressor Proteins, Ubiquitin, Ubiquitin-Protein Ligases, Von Hippel-Lindau Tumor Suppressor Protein
Active Transport, Cell Nucleus, Animals, Blotting, Western, Cell Compartmentation, Cell Hypoxia, Cell Line, Cell Nucleus, Cytoplasm, Enzyme Inhibitors, Fatty Acids, Unsaturated, Fibroblasts, Green Fluorescent Proteins, HeLa Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Ligases, Luminescent Proteins, Mice, Oxygen, Procollagen-Proline Dioxygenase, RNA Polymerase II, Recombinant Fusion Proteins, Transcription Factors, Tumor Suppressor Proteins, Ubiquitin, Ubiquitin-Protein Ligases, Von Hippel-Lindau Tumor Suppressor Protein
Mol. Cell. Biol.
Date: Aug. 01, 2002
PubMed ID: 12101228
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