Activation of HIF1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex

Abstract

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1␣, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1͞Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1␣ by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1␣ ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.

Significance of HIF-1-active cells in angiogenesis and radioresistance

“…In normoxia, HIF-1a is hydroxylated at its proline residues of the oxygen-dependent degradation (ODD) domain by prolyl hydroxylases (Bruick and McKnight, 2001;Epstein et al, 2001). The modification accelerates the interaction of HIF-1a with the von Hippel-Lindau (VHL) tumor suppressor protein, resulting in the rapid ubiquitination and subsequent degradation of HIF-1a by the 26S proteasome (Cockman et al, 2000;Kamura et al, 2000;Ohh et al, 2000;Tanimoto et al, 2000).…”

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1α resulting in new vessel formation

“…Whereas HIF-1b, the previously described aryl hydrocarbon receptor nuclear translocator, is quite stable under normoxic conditions, HIF-1a is extremely unstable and is quickly degraded by the ubiquitin-proteasome system (Salceda and Caro, 1997;Huang et al, 1998;Kallio et al, 1999). The tumorsuppressor von Hippel-Lindau (VHL) protein interacts with hydroxylated HIF-1a at Pro564 of HIF-1a in the presence of oxygen, leading to the proteolysis of HIF-1a (Kamura et al, 2000;Tanimoto et al, 2000). Specific HIF prolyl hydroxylases (PHDs) catalyse the hydroxylation of this proline residue in the oxygen-dependent degradation (ODD) domain of HIF-1a (Berra et al, 2003;Cioffi et al, 2003;Erez et al, 2003).…”

The VHL tumor suppressor and HIF: insights from genetic studies in mice

“…pVHL associates with the elongins B and C, cullin2 and Rbx [8][9][10][11][12] and functions as the substrate recognition component of an E3-ubiquitin ligase that ubiquitylates HIF-a. [13][14][15][16][17][18][19] All three HIF a-subunits, HIF-1a, HIF-2a and HIF-3a interact with pVHL. 7,20 This pVHL-HIF-a interaction is highly conserved between species, requires iron-and oxygen-dependent hydroxylation of specific proline residues (Pro402 and Pro564 in human HIF-1a; Pro405 and Pro531 in human HIF-2a) within the oxygen-dependent degradation domain of HIF-a and is necessary for the execution of HIF proteolysis under normoxia.…”