Role of exon 2-encoded beta -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 (HIFalpha) 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-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. Exon 3-encoded alpha-helical domain is required for VHL complex formation with BC/cullin-2 and E3 ubiquitin ligase activity, for binding to HIFalpha/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 HIFalpha, 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 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.
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Department of Cellular and Molecular Medicine and Kidney Research Center, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
Groulx
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de Paulsen N
Lee
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D000256:Adenoviridae; D002292:Carcinoma, Renal Cell / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D044842:Cullin Proteins; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005091:Exons; D005353:Fibronectins / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D016147:Genes, Tumor Suppressor; D006367:HeLa Cells; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007680:Kidney Neoplasms / Q000235:genetics; D008025:Ligases; D000075687:NEDD8 Protein; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein
C105153:CUL2 protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; D005353:Fibronectins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; C497446:VHL protein, human