Lee, Stephen,Bonicalzi, Marie-Eve.2009-03-232009-03-2320012001Source: Masters Abstracts International, Volume: 40-06, page: 1457.9780612672000http://hdl.handle.net/10393/9314http://dx.doi.org/10.20381/ruor-7749Sporadic 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.99 p.Biology, Molecular.Thesis