Activation of HIF1 alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex

Mutations in the VHL tumor suppressor gene result in constitutive expressio n of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1 alpha, w hich in normal cells is rapidly ubiquitinated and degraded by the proteasom e under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raise d the possibility that VHL may be directly responsible for regulating cellu lar levels of HIF1 alpha by targeting it for ubiquitination and proteolysis , In this report, we test this hypothesis directly. We report development o f methods for production of the purified recombinant VHL complex and presen t direct biochemical evidence that it can function with an E1 ubiquitin-act ivating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1 alpha u biquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for futu re investigations of the mechanisms underlying VHL regulation of oxygen-dep endent gene expression.