Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein

Mol Cell Biol. 2002 Aug;22(15):5319-36. doi: 10.1128/MCB.22.15.5319-5336.2002.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / drug effects
  • Active Transport, Cell Nucleus / physiology
  • Animals
  • Blotting, Western
  • Cell Compartmentation / physiology
  • Cell Hypoxia / physiology
  • Cell Line
  • Cell Nucleus / metabolism*
  • Cytoplasm / metabolism
  • Enzyme Inhibitors / pharmacology
  • Fatty Acids, Unsaturated / pharmacology
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Ligases / metabolism*
  • Luminescent Proteins / genetics
  • Mice
  • Oxygen / metabolism*
  • Procollagen-Proline Dioxygenase / antagonists & inhibitors
  • RNA Polymerase II / antagonists & inhibitors
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Tumor Suppressor Proteins*
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases*
  • Von Hippel-Lindau Tumor Suppressor Protein

Substances

  • Enzyme Inhibitors
  • Fatty Acids, Unsaturated
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Tumor Suppressor Proteins
  • Ubiquitin
  • Green Fluorescent Proteins
  • Procollagen-Proline Dioxygenase
  • Ubiquitin-Protein Ligases
  • Von Hippel-Lindau Tumor Suppressor Protein
  • RNA Polymerase II
  • Ligases
  • VHL protein, human
  • Oxygen
  • leptomycin B