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In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1\/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B\u2013elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.<\/jats:p>","DOI":"10.1073\/pnas.96.22.12436","type":"journal-article","created":{"date-parts":[[2002,7,26]],"date-time":"2002-07-26T14:41:48Z","timestamp":1027694508000},"page":"12436-12441","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":416,"title":["Identification of the von Hippel\u2013Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex"],"prefix":"10.1073","volume":"96","author":[{"given":"Kazuhiro","family":"Iwai","sequence":"first","affiliation":[{"name":"Department of Molecular and System Biology, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501, Japan; Howard Hughes Medical Institute, Program in Molecular Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; 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