pVHL Mediates Ubiquitination of nCLU Figure 1. pVHL binds to nCLU. (A) 293T cells were transfected as indicated. In 24 h, the cells were harvested and cellular proteins were prepared. Immunoprecipitation was performed using HA antibody. (B) 293T cells were transfected with Myc-VHL and HA-nCLU. In 24 h, the cells were harvested and immunoprecipitation was performed using HA antibody. (C) Direct interaction between nCLU and pVHL. Equal amount of bacterial lysates containing His-pVHL were incubated with the glutathione-sepharose beads that already captured GST or GST-nCLU. The beads were washed and His- pVHL retained on beads was determined by immunoblotting. CBB, Coomassie Blue Staining. (D) nCLU bound to pVHL at b domain. Equal amount of bacterial lysates containing His-pVHL, His-pVHL(1-155) or His-pVHL(D63-155) were incubated with the glutathione-sepharose beads that already captured GST or GST-nCLU. The beads were washed and His-pVHL or the truncated His-pVHL retained on beads was determined by immunoblotting. (E) A various constructs encoding different nCLUs were designed. 293T cells were transfected with Myc-VHL and nCLU construct as indicated. 24 h post-transfection, the cells were harvested for immunoprecipitation using HA antibody. doi:10.1371/journal.pone.0035848.g001 results suggest that nCLU binds to the b domain of pVHL and nCLU might be a substrate of pVHL. We next determined the site of nCLU that pVHL bound to. Our results showed pVHL had no interaction with nCLU if the N- terminal coil-coil domain of nCLU was deleted (Fig. 1E), suggesting that this domain is required for nCLU-pVHL interaction. sCLU also has the coil-coil domain, however, it does not bind to pVHL (Fig. 1A). sCLU was translated from the first ATG and nCLU was translated from the second ATG (Fig. 1E). So, the N-terminal of sCLU is different from that of nCLU. This might be the reason why nCLU binds to pVHL but sCLU does not. This needs further investigation. pVHL mediates ubiquitination of nCLU pVHL functions as the substrate recognition component of an E3-ubiquitin ligase that targets proteins for degradation. pVHL interacted with nCLU (Fig. 1). Previous study demonstrated that nCLU undergoes proteasomal degradation [26]. We therefore tested whether pVHL mediated ubiquitination of nCLU. We found that nCLU had polyubiquitination (Fig. 2A) and overex- pression of VHL increased polyubiquitination of nCLU (Fig. 2B). The identity of the band below IgG is unknown (Fig. 2A and B). We determined whether pVHL promoted degradation of nCLU. Unexpectedly, ectopic expression of VHL didn’t inhibit expression of nCLU (Fig. 2C). pVHL mediates K63-linked polyubiquitination of nCLU It is known that, the K48-linked polyubiquitination leads to protein proteasomal degradation and the K63-linked polyubiqui- tination plays a role in protein trafficking, kinase and phosphatase activation [27]. One of the examples is the non-degradative K63- polyubiquitination of NEMO that leads to NF-kB activation [28]. This drove us to determine the kind of ubiquitination of nCLU that pVHL mediated. We employed in our work two ubiquitin mutants Ub(K48R) and Ub(K63R) where K48 or K63 was mutated to arginine (R). 293T cells were transfected with nCLU, VHL and Ub(K48R) or Ub(K63R) constructs. Overexpression of VHL enhanced polyubiquitination of nCLU in the presence of Ub(K48R) (Fig. 3A). However, overexpression of VHL had little effect on the polyubiquitination of nCLU in the presence of Ub(K63R) (Fig. 3A), implying that pVHL mediates K63-linked ubiquitination of nCLU. Next, we employed a construct encoding Ub(K63) in our experiments. Ub(K63) is the ubiquitin that the lysine residues are mutated to R except K63. We found that overexpression of VHL enhanced polyubiquitination of nCLU in the presence of Ub(K63) (Fig. 3B). We also used a construct encoding Ub(K48). All lysine residues of it are mutated to R except PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35848