The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2.
We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.
Wada
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Division of Molecular Medicine, Department of Internal Medicine, Institute of Molecular Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
Yeh
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ET
Kamitani
T
T
D019943:Amino Acid Substitution; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D044842:Cullin Proteins; D016147:Genes, Tumor Suppressor; D006801:Humans; D008025:Ligases; D016297:Mutagenesis, Site-Directed; D000075687:NEDD8 Protein; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000302:isolation & purification / Q000378:metabolism*; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D006623:von Hippel-Lindau Disease / Q000235:genetics*
D000906:Antibodies; D000911:Antibodies, Monoclonal; C105153:CUL2 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D011506:Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; C497446:VHL protein, human