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.
Mesh Terms:
Adenoviridae, Carcinoma, Renal Cell, Cell Cycle Proteins, Cell Nucleus, Cullin Proteins, DNA-Binding Proteins, Exons, Fibronectins, Gene Deletion, Genes, Reporter, Genes, Tumor Suppressor, HeLa Cells, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Kidney Neoplasms, Ligases, Nuclear Proteins, Protein Conformation, Protein Structure, Secondary, Protein Transport, Proteins, Sequence Deletion, Transcription Factors, Transfection, Tumor Cells, Cultured, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Ubiquitins, Von Hippel-Lindau Tumor Suppressor Protein
Adenoviridae, Carcinoma, Renal Cell, Cell Cycle Proteins, Cell Nucleus, Cullin Proteins, DNA-Binding Proteins, Exons, Fibronectins, Gene Deletion, Genes, Reporter, Genes, Tumor Suppressor, HeLa Cells, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Kidney Neoplasms, Ligases, Nuclear Proteins, Protein Conformation, Protein Structure, Secondary, Protein Transport, Proteins, Sequence Deletion, Transcription Factors, Transfection, Tumor Cells, Cultured, Tumor Suppressor Proteins, Ubiquitin-Protein Ligases, Ubiquitins, Von Hippel-Lindau Tumor Suppressor Protein
J. Biol. Chem.
Date: Jan. 12, 2001
PubMed ID: 11024059
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