Role of Exon 2-encoded β-Domain of the von Hippel-Lindau Tumor Suppressor Protein

Abstract

Sporadic clear cell renal carcinomas frequently harbor inactivating mutations in exon 2 of the von Hippel-Lindau (VHL) tumor suppressor gene. Here, we examine the effect of the loss of exon 2-encoded ␤-domain function on VHL biochemical properties. Exon 2-encoded residues are required for VHL-mediated NEDD8 conjugation on cullin-2 and assembly with hypoxia-inducible factor ␣ (HIF␣) and fibronectin. These residues are not essential for VHL ability to assemble with elongin BC/cullin-2, to display E3 ubiquitin ligase activity in vitro and to confer energy-dependent nuclear import properties to a reporter protein. Localization studies in HIF-1␣-null embryonic cells suggest that exon 2-encoded ␤-domain mediates transcription-dependent nuclear/cytoplasmic shuttling of VHL independently of assembly with HIF-1␣ and oxygen concentration. Exon 3-encoded ␣؊helical domain is required for VHL complex formation with BC/cullin-2 and E3 ubiquitin ligase activity, for binding to HIF␣/fibronectin, but this domain is not essential for transcription-dependent nuclear/cytoplasmic trafficking. VHL ؊/؊ renal carcinoma cells expressing ␤؊do-main mutants failed to produce an extracellular fibronectin matrix and to degrade HIF␣, which accumulated exclusively in the nucleus of normoxic cells. These results demonstrate that exon 2-encoded residues are involved in two independent functions: substrate protein recognition and transcription-dependent nuclear/cytoplasmic trafficking. They also suggest that ␤-domain mutations inactivate VHL function differently than ␣-domain mutations, potentially providing an explanation for the relationship between different mutations of the VHL gene and clinical outcome.

Silencing of Epidermal Growth Factor Receptor Suppresses Hypoxia-Inducible Factor-2–Driven VHL−/− Renal Cancer

“…Ethanol was aspirated and residual was allowed to air dry at 4jC. Cells were stained for 1 hour at room temperature with anti-fibronectin antibody as described in (29). Staining was visualized with a Ziess Axiovert S100TV microscope.…”

The Positive Regulation of p53 by the Tumor Suppressor VHL

“…28 By performing coimmunoprecipitation experiments, we found that the α-domain of pVHL directly interacts with p53, and this endogenous interaction was strengthened under DNA damaging conditions. The β domain of pVHL is believed to have a role in tumor formation, but its α domain also has an important function in tumorigenesis, 29 as it is required for forming a complete ubiquitin ligase complex, which recruits Elongin C-containing proteins. Therefore, we speculated that p53-binding to the α-domain of pVHL would compete with Elongin C. Interestingly, an increase in Elongin C interrupted the p53-pVHL interaction, implying that this interaction has no influence on the ubiquitin-mediated degradation of p53.…”

Deregulated expression of VHL mRNA variants in papillary thyroid cancer

“…However, the binding with HIFα occurs via the β-domain, which is missing in the pVHL 172 Bonicalzi et al 2001). Such domain is also essential for VHL interaction with the chaperonincontaining t-complex polypeptide 1 (CCT), a cytosolic molecular chaperone that assists in the folding of actin, tubulin and other cytosolic proteins.…”