pVHL Mediates Ubiquitination of nCLU Figure 3. pVHL mediates K63-linked ubiquitination of nCLU. (A) pVHL enhanced the K63-linked ubiquitination of nCLU. 293T cells were transfected with Myc-nCLU, R-VHL and HA-Ub(K48R) (or Ub(K63R)). In 24 h, the cells were lysed and immunoprecipitation was done using HA antibody. The relative amount of ubiquitinated nCLU was determined by densitometry assay. (B) pVHL enhanced the ubiquitination of nCLU in the presence of Ub(K63). 293T cells were transfected with Myc-nCLU, R-VHL and HA-Ub(K63) plasmids. In 24 h, the cells were harvested and then lysed for immunoprecipitation using HA antibody. (C) pVHL did not enhance nCLU ubiquitination in the presence of Ub(K48). 293T cells were transfected with Myc-nCLU, R-VHL and HA-Ub(K48) plasmids. In 24 h, the cells were harvested and then lysed for immunoprecipitation using HA antibody. (D) pVHL(1- 155) did not mediate ubiquitination of nCLU. 293T cells were transfected with various constructs as indicated. In 24 h, the cell lysates were prepared for immunoprecipitation and western-blot. (E) Overexpression of Rbx1 enhanced K63-linked ubiquitination of nCLU. 293T cells were transfected as indicated. In 24 h, the cells were harvested and cell lysates were prepared for immunoprecipitation. The relative amount of ubiquitinated Myc-nCLU was determined by densitometry assay. (F) pVHL mediated K63-linked ubiquitination of nCLU in vitro. In vitro ubiquitination assay was performed as described under Methods. doi:10.1371/journal.pone.0035848.g003 proteasome, and K29-linked ubiquitination is considered as an indicator of subsequent lysosomal degradation [31]. During the last few years the K63-ubiquitination has emerged as a novel post- translational modification of remarkable functional interest for fine-tuning signal transduction pathways [27,32]. We found that ectopic expression of pVHL promoted nuclear translocation of nCLU (Fig. 4). This might be one function of pVHL that mediates K63-linked ubiquitination of nCLU. There are currently a few examples of ubiquitin serving as a nuclear import signal. For example, TRAF6, an E3 ubiquitin ligase, was shown to mediate K63-linked ubiquitination of p75 cytoplasmic interactor (NRIF) and this modification is necessary for NRIF nuclear translocation [33]. nCLU has been found to trigger cell death when in nucleus in a few systems [22]. However, we did not find that overexpression of nCLU led to cell apoptosis in our work. The difference might be due to different cells used in these works. It should be noted that in our work the increased nucleus nCLU do altered the morphology of these cell (Fig. 4B and D). The physiological function of pVHL that mediates nCLU ubiquitination needs further investigation. For example, it will be interesting to determine whether pVHL- mediated ubiquitination and nucleus translocation of nCLU plays an important role in regulation of cell proliferation and apoptosis. Another important question is whether pVHL is required for nCLU nucleus translocation. In this manuscript, we have disclosed a novel function of pVHL that mediates K63-linked ubiquitination and identified nCLU as a new target of pVHL. These results indicate that pVHL mediates not only K48- but also K63-linked ubiquitination of proteins. It is known that K48-linked ubiquitination usually leads to protein degradation and K63-linked ubiquitination functions as a post- translational modification for protein trafficking and fine-tuning signal transduction pathways. Our results may provide a new sight of pVHL’s function. PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35848