| Popis: |
Sporadic clear cell renal carcinomas (RCC) frequently harbor inactivating mutations in exon 2 of the von Hippel-Lindau (VHL) tumor suppressor gene. In this work, we examine the effect of the loss of exon 2-encoded beta-domain function on VHL biochemical properties. Exon 2-encoded residues are not essential for VHL ability to assemble with elongin BC/Cullin-2 and to display E3-ubiquitin ligase activity in vitro. However, exon 2-encoded beta-domain is required for VHL-mediated NEDD8 conjugation on Cullin-2, proper formation of an extracellular fibronectin matrix, assembly with fibronectin and elongation factor-1alpha (EF-1alpha), a protein that we recently found to be associated with wild-type VHL in vivo. Exon 2-encoded residues are also needed for VHL binding to hypoxia-inducible factor alpha (HIF-alpha) and for its subsequent ubiquitination. Localization studies in HIF-1alpha-null embryonic cells suggest that exon 2-encoded beta-domain mediates transcription-dependent nuclear/cytoplasmic shuttling of VHL independently of assembly with HIF-1alpha and oxygen concentration. Therefore, we suggest that exon 2-encoded sequences of VHL are essential for VHL nuclear/cytoplasmic shuttling and for substrate HIF-alpha recognition and ubiquitination. |
