Role of exon 2-encoded β-domain of the von Hippel-Lindau tumor suppressor protein

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 beta -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 alpha (HIF alpha) 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 alpha -null embryonic cells suggest that exon 2-encoded beta -domain mediates transcription-dependent nuclear/cytoplasmic shuttling of VHL independently of assembly with HIF-1 alpha and oxygen concentration. Exon 2-encoded alpha -helical domain is required for VHL complex formation with BC/cullin-2 and E3 ubiquitin ligase activity, for binding to HIF alpha /fibronectin, but this domain is not essential for transcription-dependent nuclear/cytoplasmic trafficking. VHL-/- renal carcinoma cells expressing beta -domain mutants failed to produce an extracellular fibronectin matrix and to degrade HIF alpha which accumulated exclusively in the nucleus of normorric 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 beta -domain mutations inactivate VHL function differently than alpha -domain mutations, potentially providing an explanation for the relationship between different mutations of the VHL gene and clinical outcome.