pVHL Mediates Ubiquitination of nCLU Figure 2. pVHL mediates ubiquitination of nCLU. (A) Modification of nCLU by ubiquitin. 293T cells were transfected with indicated plasmids and incubated for 24 h before harvest for immunoprecipitation and western-blot. (B) pVHL enhanced the ubiquitination of nCLU. 293T cells were transfected with HA-Ub, Myc-nCLU and R-VHL plasmids. In 24 h, the cells were harvested and cellular proteins were prepared for immunoprecipitation using HA antibody. (C) HCT116 and 293T cells were transfected with Myc-VHL. In 48 h, the cells were harvested and lysed. Expression of nCLU was determined by western-blot. doi:10.1371/journal.pone.0035848.g002 K48. We found that overexpression of VHL did not enhance polyubiquitination of nCLU in the presence of Ub(K48) (Fig. 3C). Taken together, these results suggest that pVHL mediates K63- linked ubiquitination of nCLU. The a domain of pVHL associates with the E3 ubiquitin ligase complex [2]. We found that the ubiquitination of nCLU was reduced significantly when VHL(1-155) was tested (Fig. 3D, Fig. S2). Because pVHL(1-155) did not have a domain, the results imply that the a domain of pVHL is required for pVHL to mediate ubiquitination of nCLU. Moreover, expression of VHL(D63-155) had little effect on ubiquitination of nCLU (Fig. S2). Next, we determined the effects of Rbx1, a subunit of the pVHL E3 ubiquitin ligase complex, on K63-linked ubiquitination of nCLU. We found that ectopic expression of Rbx1 enhanced K63- linked ubiquitination of nCLU (Fig. 3E). We also determined whether pVHL could mediate K63-linked polyubiquitination of nCLU in vitro. The pVHL complex was isolated by immunopre- cipitation and incubated with purified nCLU in Reaction II Reticulocyte Conjugation Kit as described previously [25]. We found that the isolated pVHL complex mediated K63-linked ubiquitination of nCLU in vitro (Fig. 3F). However, the isolated pVHL(1-155) could not mediate K63-linked ubiqitination of nCLU in vitro. A control experiment was done. The results showed that the ubiquitin chains were attached to GST-nCLU and not to pVHL itself and Ub(K63R) was not linked to nCLU (Fig. S3). Taken together, these results suggest that pVHL E3 ubiquitin ligase complex mediates K63-linked ubiquitination of nCLU. pVHL promotes nucleus localization of nCLU It is believed that nCLU functions in nucleus. We therefore asked whether the K63-linked ubiquitination promoted nuclear translocation of nCLU. To know this, we determined the effect of expression of Ub(K63) on cellular distribution of nCLU. We found that ectopic expression of Ub(K63) enhanced nuclear localization of nCLU (Fig. 4A). However, overexpression of Ub(K48) had little effect on nCLU nuclear translocation. Immuno-staining of the cells also indicated that ectopic expression of Ub(K63) enhanced nuclear localization of nCLU (Fig. 4B). It was noted that overexpression of Ub(K63)+nCLU, but not Ub(K48)+nCLU, led to alteration of cell morphology (Fig. 4B). Taken together, these results suggest that the K63-linked ubiquitination of nCLU promotes nCLU nuclear translocation. Next, we determined the effect of pVHL on nCLU nuclear translocation. The nuclear and cytoplasmic proteins were isolated and western-blot was performed. We found that overexpression of pVHL enhanced protein levels of nCLU in nucleus (Fig. 4C). Immuno-staining of the cells was also performed to indicate nCLU distribution in cells. We found that Myc-nCLU proteins were mainly cytoplasmic when the cells were transfected with Myc- nCLU alone (Fig. 4D). If Myc-nCLU were co-transfected with HA-VHL, the Myc-nCLU proteins in nucleus were increased. These results suggest that pVHL promotes nuclear translocation of nCLU. We noted that expression of exogenous VHL+nCLU altered cell morphology (Fig. 4D). This is similar to the results of Fig. 4B. The altered cell morphology might be due to increased nCLU in nucleus. Discussion It is known that pVHL targets HIFa for proteasomal degradation. As an adaptor protein, pVHL not only binds to HIFa but also binds to many other proteins and has multiple functions [1]. We found in our work that pVHL bound to nCLU and mediated nCLU K63-linked ubiquitination, which may lead to nuclear translocation of nCLU. To our knowledge, this is the first report to demonstrate that pVHL mediates K63-linked ubiquitination of a protein. The addition of ubiquitin to proteins occurs through an enzymatic cascade. Firstly, an activating enzyme, E1, is charged with ubiquitin, then the ubiquitin is transferred to an E2- conjugating enzyme and, finally, it is attached to a substrate by an E3 ligase [29]. There are a few types of polyubiquitin chains [27] and in many conditions the type of polyubiquitin chain formed appears to be directed primarily by the cooperating E2 enzyme. E2 dictates the type of inter-ubiquitin linkages and thereby determines the fate of the substrate [30]. We found that pVHL mediated K63-linked ubiquitination of nCLU (Fig. 3) and promoted nCLU nuclear translocation (Fig. 4). Our results suggest that, for different substrates, pVHL may mediate different types of ubiquitination, probably using different E2 enzymes. Although protein ubiquitination has been best characterized as a mechanism for targeting proteins to the proteasome for degradation, recently it has become clear that this modification can also serve many other functions. For example, K48-linked ubiquitination tags the target protein to be degraded by the PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35848