Energy-Dependent Nucleolar Localization of P53 in Vitro Requires Two Discrete Regions Within the P53 Carboxyl Terminus

Oncogene (2007) 26, 3878–3891 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus O Karni-Schmidt1, A Friedler2,3, A Zupnick1, K McKinney1, M Mattia1, R Beckerman1, P Bouvet4, M Sheetz1, A Fersht2 and C Prives1 1Department of Biological Sciences, Columbia University, New York, USA; 2Department of MRC-CPE, Center for Protein Engineering, Medical Research Council, Cambridge, UK; 3Department of Organic Chemistry, The Hebrew University of Jerusalem, Givat Ram, Jerusalem, Israel; 4Laboratoire de Biologie Molećulaire de la Cellule/UMR 5161, Ecole Normale Supe´rieure de Lyon 46, Alleé d’Italie 69364 Lyon Cedex, France The p53 tumor suppressor is a nucleocytoplasmic shuttling concentration, is presumably inactive as a transcription protein that is found predominantly in the nucleus of cells. factor and is diffusely distributed throughout the cell. In In addition to mutation, abnormal p53 cellular localiza- response to DNA damage or other forms of cellular tion is one of the mechanisms that inactivate p53 function. stress, p53 protein accumulates, frequently becomes To further understand features of p53 that contribute to extensively post-translationally modified and concen- the regulation of its trafficking within the cell, we analysed trates within the nucleus (Giaccia and Kastan, 1998; the subnuclear localization of wild-type and mutant p53 in Appella and Anderson, 2001; Liang and Clarke, 2001; human cells that were either permeabilized with detergent Prives and Manley, 2001). or treated with the proteasome inhibitor MG132. We, One property of the nucleus, analogous to cytoplas- here, showthat either endogenously expressed or exoge- mic organelles, is the existence of subnuclear domains. nously added p53 protein localizes to the nucleolus in These nuclear compartments concentrate specific sets detergent-permeabilized cells in a concentration- and ATP of functionally related proteins. Within the nucleus, hydrolysis-dependent manner. Two discrete regions within the nucleolus is the site of ribosomal RNA (rRNA) the carboxyl terminus of p53 are essential for nucleolar transcription, modification, maturation and ribosomal localization in permeabilized cells. Similarly, localization assembly (Carmo-Fonseca et al., 2000; Olson et al., of p53 to the nucleolus after proteasome inhibition in 2000; Leung et al., 2003). Although easily visualized unpermeabilized cells requires sequences within the as a discrete subnuclear entity, the nucleolus is not carboxyl terminus of p53. Interestingly, genotoxic stress separated from the surrounding nucleoplasm by markedly decreases the association of p53 with the membranes, and thus proteins localize to nucleoli by nucleolus, and phosphorylation of p53 at S392, a site processes that do not involve active membrane trans- that is modified by such stress, partially impairs its port. In recent years, multiple proteins involved in nucleolar localization. The possible significance of these cell-cycle progression were identified, whose regulation findings is discussed. involves sequestration within the nucleolus. In response Oncogene (2007) 26, 3878–3891. doi:10.1038/sj.onc.1210162; to oncogenic stress, the p14ARF protein may inactivate published online 22 January 2007 the mouse double minute (MDM)2 oncoprotein (an inhibitor of p53) by inducing nucleolar sequestration Keywords: p53; nucleolus; DNA damage; NoLS of MDM2 (Zhang et al., 1998; Tao and Levine, 1999; Weber et al., 1999; Ashcroft et al., 2000). The requirement of MDM2 localization to nucleoli for its inactivation by p14ARF, however, is not obliga- tory (Llanos et al., 2001). Among other cell cycle Introduction regulators and checkpoint factors that associate with the nucleolus are the Werner’s Syndrome Helicase (Marci- The human p53 protein contains 393 amino acids and is niak et al., 1998), the telomere maintenance enzyme comprised of several functional domains (Prives and (hTERT) (Wong et al., 2002) and Cdc14, a protein Hall, 1999). Appropriate subcellular localization is phosphatase in yeast crucial for promoting exit from crucial for regulating p53 function. Under unstressed mitosis Visintin et al., 1998). These and other findings conditions, the p53 protein is generally present at a low imply a novel role for the nucleolus’s participation in the regulation of cell cycle proteins (as reviewed in Visintin and Amon, 2000; Lamond and Sleeman, 2003; Leung Correspondence: Dr C Prives, Department of Biological Sciences, and Lamond, 2003; Mayer and Grummt, 2005; Olson Columbia University, 816 Fairchild, NY 10027 USA. E-mail: [email protected] and Dundr, 2005). Received 18 July 2006; revised 22 September 2006; accepted 23 October Within the nucleus, p53 has been reported to be 2006; published online 22 January 2007 associated with at least three subnuclear compartments ATP-dependent p53 nucleolar localization O Karni-Schmidt et al 3879 including the nucleolus, promyelocytic leukemia bodies loss of p53 upon permeabilization may reflect the release and cajal bodies (Mogaki et al., 1993; Benninghoff et al., of a subpopulation of p53 that is not bound to nuclear 1999; Rubbi and Milner, 2000; Horky et al., 2001; structures. Klibanov et al., 2001; Wesierska-Gadek et al., 2002; Parallel cultures to those in Figure 1a were either Young et al., 2002; Zimber et al., 2004). Approximately directly fixed with 4% paraformaldehyde (unpermeabi- half of nuclear p53 is soluble, whereas the remainder lized) or first permeabilized with PBS/1% Triton X-100 associates with nuclear structures (Zerrahn et al., 1992). for 2 min before fixation. Cells were then incubated p53 can interact with a number of nucleolar components with anti-p53 antibodies followed by incubation with such as L5 ribosomal protein (Marechal et al., 1994), secondary antibodies and analysis by laser scanning topoisomerase I (Gobert et al., 1996), nucleolin (Daniely microscopy and differential interference contrast (DIC). et al., 2002), nucleophosmin (Colombo et al., 2002) and As expected, without permeabilization wild-type and nucleostemin (Tsai and McKay, 2002). Intriguingly as mutant p53 proteins were detected in the nucleoplasm of well, the first cellular DNA-binding site of p53 to be unpermeabilized cells and appeared to be excluded from identified (RGC) is located within the ribosomal gene nucleoli (Figure 1b). Interestingly, after permeabiliza- cluster (Kern et al., 1991). Recently, it was reported that tion, nucleoplasmic staining was generally weaker in mutant p53 localizes to nucleoli in cells treated with a wild-type p53 cells, whereas in approximately 10–15% compound (PRIMA-1) that was reported to reactivate of cells expressing p53Q22/S23 and p53H175 almost exclusive mutant forms of p53 (Bykov et al., 2002a, b; Rokaeus nucleolar staining was observed (Figure 1c). By con- et al., 2006). trast, wild-type p53 was never observed in the nucleoli. Under non-stressed conditions, p53 was reported to Co-staining cells with anti-nucleolin antibody confirmed localize to the nucleolus in permeabilized Hep G2 cells the nucleolar localization of mutant p53. Our observa- (Rubbi and Milner, 2000). The nuclear and nucleolar tions suggest that some of the p53 in the nucleus is not association of p53 in such cells is RNA-dependent as tightly attached to nuclear components and can freely RNase A treatment of permeabilized cells abrogates diffuse outside the nucleus, whereas a subpopulation of detection of most of the anchored p53. In addition, p53 in some cells can be anchored by attaching to treatment of human fibroblasts with MG132, a protea- nuclear and nucleolar components as was reported some inhibitor, causes p53 to accumulate in the previously (Rubbi and Milner, 2000; Klibanov et al., nucleolus and nucleoplasm both in directly fixed cells 2001). and in permeabilized cells (Klibanov et al., 2001; Both mutant p53Q22/S23- and p53H175-expressing Pokrovskaja et al., 2001; Latonen et al., 2003). Although cells contain at least two-fold more p53 than those its functional significance is unknown, the nucleolar expressing wild-type p53 when normalized to cellular sequestration of p53 was reported to be one of the actin levels (Figure 1 and 2). We therefore tested mechanisms contributing to the reactivation of p53 in whether the level of p53 protein can determine HeLa cells after treatment with cisplatin (Horky et al., sequestration of the mutant p53 into the nucleolus 2001; Wesierska-Gadek et al., 2002). by varying the amount of tetracycline in the culture In the present study, we have established conditions medium of mutant p53-expressing cells (Chen for characterizing the association of endogenously et al., 1996). Indeed, when the level of mutant p53 was expressed or exogenously added p53 with nucleoli and lowered to that of fully expressed wild-type p53 neither have identified some of the requirements for this p53Q22/S23 nor p53H175 relocalized to the nucleolus process. (Figure 2). To determine whether endogenously expressed p53 in other cell lines could relocalize to nucleoli after permeabilization, we examined HT-29 cells RESULTS and T98G cells that each express high levels of mutant forms of p53. Note that the levels of endogenous The level of p53 within permeabilized H1299 cells mutant p53 in those cells are similar to those of p53H175 determines nucleolar sequestration that are expressed inducibly in H1299 cell line To investigate the localization

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