pVHL Mediates K63-Linked Ubiquitination of nCLU Jing Xue1., Dan-dan Lv1., Shi Jiao1., Wenting Zhao1, Xuebing Li1, Heng Sun1, Bing Yan1, Li Fan1, Rong- gui Hu2, Jing Fang 1 * 1 The Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, Shanghai, China, 2 The Institute of Biochemistry and Cell Biology, SIBS, Chinese Academy of Sciences, Shanghai, China Abstract pVHL, product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase that targets proteins for ubiquitination and proteasomal degradation. Hypoxia-inducible factor a (HIFa) is the well-known substrate of pVHL. Besides HIFa, pVHL also binds to many other proteins and has multiple functions. In this manuscript, we report that the nuclear clusterin (nCLU) is a target of pVHL. We found that pVHL had a direct interaction with nCLU. nCLU bound to pVHL at pVHL’s b domain, the site for recognition of substrate, indicating that nCLU might be a substrate of pVHL. Interestingly, pVHL bound to nCLU but did not lead to nCLU destruction. Further studies indicated that pVHL mediated K63-linked ubiquitination of nCLU and promoted nCLU nuclear translocation. In summary, our results disclose a novel function of pVHL that mediates K63-linked ubiquitination and identify nCLU as a new target of pVHL. Citation: Xue J, Lv D-d, Jiao S, Zhao W, Li X, et al. (2012) pVHL Mediates K63-Linked Ubiquitination of nCLU. PLoS ONE 7(4): e35848. doi:10.1371/ journal.pone.0035848 Editor: Marianne Koritzinsky, University Health Network, Canada Received June 30, 2011; Accepted March 23, 2012; Published April 20, 2012 Copyright: 2012 Xue et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by National Natural Science Foundation of China (30970586)(http://www.nsfc.gov.cn); Chinese Academy of Sciences (KSCX2- EW-R-08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jfang@sibs.ac.cn . These authors contributed equally to this work. Introduction pVHL, product of von Hippel-Lindau (VHL) tumor suppressor gene, acts as a multipurpose adaptor protein that controls a diverse array of gene expression programs, as well as extracellular matrix assembly and microtubule-based processes by linking various target proteins to appropriate enzymatic activities [1]. Germline mutations in VHL can be found in patients with VHL disease, a familial tumor syndrome characterized by the development of highly vascularized tumors in multiple organs [2,3]. Major clinical manifestation of VHL disease include hemangioblastomas of the retina and central nervous system, renal cysts and clear cell renal cell carcinoma, pancreatic cysts and tumors, as well as pheochro- mocytomas [2,3]. VHL is also mutated in the majority of sporadic clear cell renal cell carcinoma. pVHL forms a multi-protein complex that contained elongin B, elongin C, Cul2, and Rbx1 and functions as the substrate recognition component of an E3- ubiquitin ligase complex that targets proteins for degradation [4– 6]. The best-understood function of pVHL relates to its role that targets the alpha subunit of hypoxia-inducible factor (HIFa) for destruction [3,7]. pVHL binds to hydroxylated HIFa and mediates HIFa ubiquitination and proteasomal degradation [8,9]. The pVHL-mediated degradation of HIFa is involved in tumor progression as well as oxygen sensing [10]. Besides HIFa, pVHL has been found to bind to a dozen different proteins and mediates other biological processes [1,3,11]. pVHL has a direct interaction with atypical protein kinase C and inhibits its activities [12]. pVHL binds to hydroxylated a-chain of collagen IV [13] and reacts with fibronectin [14]. pVHL is also involved in regulation of NF-kB [15] and Wnt signaling [16]. Clusterin (CLU) is an enigmatic glycoprotein with a nearly ubiquitous tissue distribution, involved in many biological processes [17]. CLU is also believed to be involved in many pathological states including cancer [18]. It may promote cell survival or induce cell apoptosis, depending on its different isoforms. [18,19]. The secreted form of CLU (sCLU) is present in almost all physiological fluids [20] and is considered to be a survival factor [21]. In addition to sCLU, a nuclear form of CLU (nCLU) exists. And nCLU has been found to trigger cell death in a few systems [22]. Previous study demonstrated that expression of sCLU was attenuated in cells lacking wild-type pVHL [23]. However, the effect of pVHL on nCLU is unknown. We found in our work that pVHL bound to nCLU but did not target it for destruction. Interestingly, we found that pVHL mediated K63-linked ubiqui- tination of nCLU and promoted its nuclear translocation. Our results reveal a novel function of pVHL that mediates K63-linked ubiquitination and identify nCLU as a new target of pVHL. Materials and Methods Cell culture and reagents Human embryonic kidney 293T cells and human cervix cancer Hela cells were cultured in DMEM with 10% serum, penicillin (100 U/mL), and streptoMycin (100 mg/mL). Human colon cancer HCT116 cells were cultured in McCoy’s 5A media (M5A) with 10% serum, penicillin (100 U/mL), and streptoMycin (100 mg/mL). pVHL antibody was from BD Biosciences (Bedford, MA). nCLU (B5), HA, Myc and His antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against Ub(K63) (BML-PW0600-0025) was from Enzo Life Sciences (Farmingdale, PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35848