Oncogene (2011) 30, 790–805 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Proteasome inhibitors induce nucleolar aggregation of proteasome target proteins and polyadenylated RNA by altering ubiquitin availability L Latonen1,3, HM Moore1, B Bai2,SJa¨a¨maa1 and M Laiho1,2 1Molecular Cancer Biology Program and Haartman Institute, University of Helsinki, Helsinki, Finland and 2Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA The ubiquitin–proteasome pathway is essential for most Keywords: proteasome inhibitor; proteasome; ubiquitin; cellular processes, including protein quality control, cell polyadenylated RNA; nuclear export; aggresome cycle, transcription, signaling, protein transport, DNA repair and stress responses. Hampered proteasome activity leads to the accumulation of polyubiquitylated proteins, endoplastic reticulum (ER) stress and even cell Introduction death. The ability of chemical proteasome inhibitors (PIs) to induce apoptosis is utilized in cancer therapy. During Ubiquitin is a small conserved protein that is covalently PI treatment, misfolded proteins accrue to cytoplasmic linked to its target proteins by ubiquitylation (Hershko aggresomes. The formation of aggresome-like structures and Ciechanover, 1998; Weissman, 2001; Finley, 2009). in the nucleus has remained obscure. We identify here Ubiquitin is essential for many if not most cellular a nucleolus-associated RNA-protein aggregate (NoA) processes, including protein quality control, cell cycle formed by the inhibition of proteasome activity in and transcriptional control, cellular signaling, protein mammalian cells. The aggregate forms within the transport, DNA repair and stress responses. These nucleolus and is dependent on nucleolar integrity, yet is diverse functions are governed by attachment of mono- a separate structure, lacking nucleolar marker proteins, and polyubiquitin chains on the targets through ribosomal RNA (rRNA) and rRNA synthesis activity. The ubiquitin lysine residues. Polyubiquitylation generally NoAs contain polyadenylated RNA, conjugated ubiquitin leads to proteolytic degradation of the target by the and numerous nucleoplasmic proteasome target proteins. proteasome. Polyubiquitylation occurs predominantly Several of these are key factors in oncogenesis, including through Lys48 residues, although recently ubiquitin transcription factors p53 and retinoblastoma protein (Rb), chains based on most lysine residues have been several cell cycle-regulating cyclins and cyclin-dependent implicated in proteasomal targeting (Ikeda and Dikic, kinases (CDKs), and stress response kinases ataxia- 2008; Saeki et al., 2009; Xu et al., 2009). Monoubiqui- telangiectasia mutated (ATM) and Chk1. The aggregate tylation regulates, for example, membrane transport, formation depends on ubiquitin availability, as shown by nucleocytoplasmic protein localization, protein kinase modulating the levels of ubiquitin and deubiquitinases. activation, DNA repair and chromatin dynamics (Chen Furthermore, inhibition of chromosome region main- and Sun, 2009). Conjugated ubiquitin is released during tenance 1 protein homolog (CRM1) export pathway proteasomal processing and by deubiquitylating aggravates the formation of NoAs. Taken together, we enzymes (Reyes-Turcu et al., 2009), and reused. identify here a novel nuclear stress body, which forms The ubiquitin–proteasome pathway is a major upon proteasome inactivity within the nucleolus and is proteolytic system of all eukaryotic cells (Hershko detectable in mammalian cell lines and in human tissue. and Ciechanover, 1998; Weissman, 2001; Finley, These findings show that the nucleolus controls protein 2009). Inhibition of proteasome activity leads to the and RNA surveillance and export by the ubiquitin accumulation of polyubiquitylated and misfolded pathway in a previously unidentified manner, and provide proteins, ER stress and eventually apoptosis (Kopito, mechanistic insight into the cellular effects of PIs. 2000; Navon and Ciechanover, 2009). Proteasome Oncogene (2011) 30, 790–805; doi:10.1038/onc.2010.469; activity can be inhibited by chemical inhibitors, which published online 18 October 2010 fall into several classes based on their chemical structure, mechanism of inhibition and specificity (Kisselev and Goldberg, 2001). Several in vitro studies Correspondence: Dr M Laiho, Department of Radiation Oncology and Molecular Radiation Sciences, Sidney Kimmel Comprehensive utilize peptide aldehydes (such as MG132, ALLN), Cancer Center, The Johns Hopkins University School of Medicine, which are substrate analogs, or a non-peptide inhibitor 1550 Orleans Street, CRB II, Room 444, Baltimore, MD 21231, USA. lactacystin. The ability of proteasome inhibitors (PIs) to E-mail: [email protected] induce cell death is exploited in clinical cancer treatment 3Current address: Institute of Medical Technology, University of Tampere, Tampere, Finland. with bortezomib (Velcade; PS-341; MG-341), which is a Received 13 May 2010; revised 21 August 2010; accepted 7 September Food and Drug Administration-approved PI in clinical 2010; published online 18 October 2010 use in mantle cell lymphoma and multiple myeloma Nucleolar aggregates L Latonen et al 791 (Navon and Ciechanover, 2009). The potential use of nucleoli in PI-treated cells (Klibanov et al., 2001; PIs against solid tumors is under active investigation. Latonen et al., 2003; Karni-Schmidt et al., 2007). To Upon PI treatment, polyubiquitylated proteins accu- study how the nucleoli are affected upon proteasome mulate at sites of proteasomes, forming organized inhibition, we treated WS1 human skin fibroblasts aggregates. Most prominently, this occurs in the and HEL-299 human lung fibroblasts with MG132 pericentrosomal area in the cytoplasm, in structures (10 mM) for 12 h. At this time, cell death is not yet called aggresomes (Wo´jcik et al., 1996; Johnston et al., prevailing (Supplementary Figure S1a). We immuno- 1998). In the nucleus, chemical inhibition of the stained nucleolar substructures using antibodies against proteasome induces transient nuclear stress granules nucleophosmin (NPM; for granular component), fibril- containing stress response proteins such as heat-shock larin (FBL; for dense fibrillar component) and upstream factors (Holmberg et al., 2000), but no aggresome binding factor (UBF; for fibrillar center). Treatment formation in the nucleus has been reported. We and of the cells with MG132 led to drastic changes in others have shown that proteasome inhibitor MG132 the localization of the nucleolar markers (Figure 1a). causes translocation of certain stress response-related NPM formed a ring-shaped structure, which partially nuclear proteins (p53, MDM2, PML, Hsp70) to the overlapped by FBL and UBF (Figure 1a). Under phase- nucleolus (Klibanov et al., 2001; Mattsson et al., 2001; contrast illumination, a dense structure became visible Latonen et al., 2003; Kurki et al., 2004; Karni-Schmidt that appeared to localize in the center of the reorganized et al., 2007). As these proteins are degraded by the nucleolus. Similar relocalization was observed by using proteasome, the question has arisen as to whether the antibodies against nucleolar antigens Ki-67 and ARF nucleolus has a role in the degradation of these proteins. (data not shown). To study this effect in more detail, The nucleolus is membrane-less nuclear organelle, we performed transmission EM (TEM) of mock- and which is responsible for the assembly of ribosomal MG132-treated WS1 cells. Although the nucleolar subunits (Leary and Huang, 2001; Fatica and Tollervey, substructures were visible in the mock-treated cells, 2002). The nucleoli are composed of fibrillar centers, MG132 treatment caused alterations in the nucleolar dense fibrillar component and granular component morphology (Figure 1b). This was apparent by inter- (Olson et al., 2000; Hernandez-Verdun, 2006; Boisvert ruption of the nucleolus by electron-dense material et al., 2007), in which ribosomal RNA (rRNA) (Figure 1b, arrowhead). transcription, maturation of pre-RNA transcripts, Previously, Stavreva et al. (2006) have shown that assembly of pre-ribosomal particles and late RNA a high dose of MG132 (100 mM) inhibits pre-RNA processing occur (Lafontaine and Tollervey, 2001; Leary processing. However, a much smaller, more commonly and Huang, 2001). Proteomic analyses have revealed used dose of MG132 (10 mM), is sufficient to inhibit that besides proteins involved in ribosome synthesis, the proteasome activity. Hence, we analyzed the effect of nucleolus contains a large number of proteins associated MG132 at this lower concentration, used throughout the with cell cycle and mitotic division regulation, DNA study, on nascent rRNA synthesis using fluorouridine repair and control of tumor suppressor proteins and (FUrd) incorporation. As shown in Figure 1c, rRNA oncogenes (Andersen et al., 2005; Boisvert et al., 2007). synthesis was readily detectable in the nucleoli following Here, we investigated the possible role of the nucleolus MG132 treatment for 12 h, and colocalized with FBL, as in the ubiquitin–proteasome pathway. We show that expected. However, the dense aggregate observed within nuclear proteasome targets accumulate to, and are the nucleolar marker was devoid of any rRNA synthetic immobilized in, aggregates forming in the nucleoli. These activity. To further assess the effect of PI treatment