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GRP78 Regulates Clusterin Stability, Retrotranslocation and Mitochondrial Localization Under ER Stress in Prostate Cancer

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GRP78 Regulates Clusterin Stability, Retrotranslocation and Mitochondrial Localization Under ER Stress in Prostate Cancer Oncogene (2013) 32, 1933–1942 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE GRP78 regulates clusterin stability, retrotranslocation and mitochondrial localization under ER stress in prostate cancer N Li, A Zoubeidi, E Beraldi and ME Gleave Expression of clusterin (CLU) closely correlates with the regulation of apoptosis in cancer. Although endoplasmic reticulum (ER) stress-induced upregulation and retrotranslocation of cytoplasmic CLU (presecretory (psCLU) and secreted (sCLU) forms) has been linked to its anti-apoptotic properties, mechanisms mediating these processes remain undefined. Here, we show using human prostate cancer cells that GRP78 (Bip) associates with CLU under ER stress conditions to facilitate its retrotranslocation and redistribution to the mitochondria. Many ER stress inducers, including thapsigargin, MG132 or paclitaxel, increased expression levels of GRP78 and CLU, as well as post-translationally modified hypoglycosylated CLU forms. ER stress increased association between GRP78 and CLU, which led to increased cytoplasmic CLU levels, while reducing sCLU levels secreted into the culture media. GRP78 stabilized CLU protein and its hypoglycosylated forms, in particular after paclitaxel treatment. Moreover, subcellular fractionation and confocal microscopy with CLUGFP indicated that GRP78 increased stress-induced CLU retrotranslocation from the ER with co- localized redistribution to the mitochondria, thereby reducing stress-induced apoptosis by cooperatively stabilizing mitochondrial membrane integrity. GRP78 silencing reduced CLU protein, but not mRNA levels, and enhanced paclitaxel-induced cell apoptosis. Taken together, these findings reveal novel dynamic interactions between GRP78 and CLU under ER stress conditions that govern CLU trafficking and redistribution to the mitochondria, elucidating how GRP78 and CLU cooperatively promote survival during treatment stress in prostate cancer. Oncogene (2013) 32, 1933–1942; doi:10.1038/onc.2012.212; published online 11 June 2012 Keywords: ER stress; GRP78; clusterin; cancer; retrotranslocation; apoptosis INTRODUCTION further processing for cleavage and assembly of the mature Clusterin (CLU) is a stress-induced, multifunctional secreted and heterodimer, sCLU.8 cytoplasmic molecular chaperone. The human CLU gene codes for Stress within the ER and Golgi network can lead to protein two isoforms (CLU-1, CLU-2) originating from transcriptional start misfolding and retrotranslocation of conformation-altered glyco- sites in exons 1 and 2, respectively (NCBI Reference Sequence: proteins from the ER lumen to the cytosol.9,10 This NM_001831.2, NM_203339.1). Proteolytic removal of the endo- retrotranslocation of misfolded proteins, followed by protein plasmic reticulum (ER)-targeting signal peptide produces a 60-kDa polyubiquitination and proteasomal degradation, termed ER- ER-associated, presecretary form (psCLU) that is extensively associated degradation (ERAD), has an important role in protein glycosylated in the ER. In the Golgi, psCLU is further processed homeostasis.11 However, given that N-glycosylated proteins are and cleaved at Arg227-Ser228 into two 40 kDa a- and b-subunits often observed in the cytosol,10,12,13 it is likely that some that are assembled in an anti-parallel manner into a B80-kDa retrotranslocated glycoproteins are not destined for degradation, mature, heterodimeric secreted form (sCLU).1 CLU expression is but rather retrotranslocate as a prerequisite for specific biological closely linked to treatment resistance and cancer progression due functions. Levels of cytoplasmic CLU, an exclusively N-glycosylated to its stress-induced, anti-apoptotic properties.2–4 Recently, YB-1 protein, increase with retrotranslocation following many ER was shown to regulate stress-induced CLU transcription and stressors, including heat shock, proteasome (for example, expression, with CLU having a dominant downstream role in MG132) or chaperone (for example, Hsp90) inhibition, ER homeo- YB-1-induced cytoprotection and paclitaxel resistance in prostate stasis disruption (for example, tharpsigargin) or chemotherapy cancer cells.5 drugs (for example, taxanes).14,15 Instead of being a target of TheERisanessentialorganelleforthesynthesis,assembly ERAD, retrotranslocated CLU has specific roles in stress responses, and post-translational modification (PTM) of surface and for example, blocking activated BAX and inhibiting mitochondrial secretary proteins. PTMs include: phosphorylation, glycosylation, apoptosis pathway.16,17 Given the role for CLU in anti-cancer ubiquitination, nitrosylation, methylation, acetylation, and lipi- treatment resistance, understanding mechanisms that regulate its dation that modulate protein structure and function. N-linked retrotranslocation and cellular redistribution may provide insights glycosylation is among the most common enzyme-directed site- on how to exploit these processes. specific PTM in the ER.6 The addition of specific sugar residues to Considering its complex maturation and secretory pathway, newly synthesized proteins can direct transport of proteins to along with its capacity to retrotranslocate from the ER, CLU likely specific subcellular locations.7 InthecaseofpsCLU,theaddition interacts with ER-resident and cytosolic chaperones. Here, we of highly charged N-glycans modifies its folding to facilitate focus on GRP78 (Bip), an important ER-resident chaperone. Department of Urologic Sciences, The Vancouver Prostate Centre, University of British Columbia, Vancouver, British Columbia, Canada. Correspondence: Dr ME Gleave, Department of Urologic Science, The Vancouver Prostate Centre, University of British Columbia, 2660 Oak Street, Vancouver, British Columbia, Canada V5Z 3J5. E-mail: [email protected] Received 29 November 2011; revised 26 March 2012; accepted 13 April 2012; published online 11 June 2012 GRP78 regulation of CLU activity NLiet al 1934 Although GRP78 is predominantly found within the ER lumen, a pattern of lower molecular weight bands of CLU was observed recent studies have shown that it can redistribute, under ER stress after ER stress, most likely a consequence of lost glycans conditions, to the cytosol, tumor cell surface and mitochon- (Figure 2a, upper panel). We speculated that this CLU hypogly- dria.18–20 GRP78 has also been reported to form a complex with cosylation represents a stress-induced PTM of CLU in the ER. another ER protein, Sigma-1 receptor (Sig-1R), and regulate Although increased levels of CLU and its hypoglycosylated Ca2 þ signaling between ER and mitochondria through inositol forms were observed after TG and MG132 treatment, levels of 1,4,5-triphosphate receptors (IP3Rs).21 Furthermore, subcellular sCLU secreted into cell culture media significantly decreased, as localization of GRP78 correlates with its anti-apoptotic measured by ELISA, normalized to live cell counts (Figure 2a, lower properties.22–24 Although expression levels of both GRP78 and panel). This reduced secretion of sCLU may further contribute to CLU increase in parallel under ER stress, stress-induced retention and accumulation of CLU in the ER–Golgi network. To interactions and coordinated anti-apoptotic functions between further substantiate the link between ER stress and CLU these chaperones have not been reported. hypoglycosylation, we used tunicamycin, which is known to In the present study, we set out to define functional interactions induce ER stress by inhibiting protein glycosylation. Tunicamycin between GRP78 and CLU and discovered that GRP78 and CLU also increased GRP78 and CLU hypoglycosylated protein levels, interact with each other under ER stress. GRP78 stabilizes both while decreasing sCLU secretion, similar to other ER stressors psCLU and sCLU to facilitate their retrotranslocation to the cytosol (Supplementary Figure S2), supporting our hypothesis that the with redistribution to the mitochondria. Our findings indicate that additional CLU bands detected following ER stress result from under ER stress, GRP78 and CLU function cooperatively to mediate PTMs involving CLU hypoglycosylation. anti-apoptotic effects in the mitochondria pathway. Next, to determine whether CLU glycosylation status alters its ER processing and secretion, we mutated six glycosylation sites at aspargines (N) 86, 103, 145, 291, 354 and 374 to glutamine (Q) that RESULTS are equally distributed within the b and a, and at (NP_976084.1). GRP78 associates with CLU under ER stress conditions We found that mutated forms, CLUNbQ and CLUNaQ, had lower To better define the physiological and pathological functions of molecular mass than psCLU and sCLU, similar to the hypoglyco- CLU, we set out to identify CLU-binding partners under ER stress sylated bands observed under ER stress with CLUWT (Figure 2b). conditions. CLU was immunoprecipitated (IP) from human LNCaP Notably, sCLU generated by CLUNaQ had a shift in mobility on prostate cancer cells stably overexpressing CLU-2 (LNCaPCLU-2) after SDS–PAGE and also reduced secretion into media (Figure 2b). treatment with the proteasome inhibitor, MG132 and the Ca2 þ – Given that four of the six glycosylation sites are equally located at ATPase inhibitor, thapsigargin (TG). CLU IP was submitted to two CC domains (N86, 103 at CC1 and N354, 374 at CC2), which trypsin digestion followed by nano-liquid chromatography/tandem were previously reported to interact with various CLU-binding mass spectrometry (LC/MS/MS). Among the high confidence partners,25,26,27 we propose that two
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