Identification of the von Hippel-Lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex

Mutations of von Hippel-Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in th e vast majority of sporadic clear cell renal carcinomas. The function of th e pVHL protein has not been clarified. pVHL has been shown to form a comple x with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of t he structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, t he ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We sh ow that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified Ubc H5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the V BC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity o f VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We a lso found that pVHL associates with two proteins, p100 and p220, which migr ate 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-elongin C-CUL-2, fail t o associate with p100 and pun and cannot exhibit the E3 ligase activity. Th ese results suggest that pVHL might he 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 ubiquit in conjugation system which leads to the targeted degradation of substrate proteins.