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γ and down-regulates the activity of p53
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Biochemical and Biophysical Research Communications 368 (2008) 690–695 www.elsevier.com/locate/ybbrc
Sirt2 interacts with 14-3-3 b/c and down-regulates the activity of p53 Yun-Hye Jin a, Yeon-Jin Kim b, Dae-Won Kim c, Kwang-Hyun Baek d, Bok Yun Kang a, Chang-Yeol Yeo b,*, Kwang-Youl Lee a,* b
a College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju, Republic of Korea Department of Life Science and Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea c Department of Biochemistry, Yonsei University, Seoul, Republic of Korea d Graduate School of Life Science and Biotechnology, Pochon CHA University, CHA General Hospital, Seoul, Republic of Korea
Received 21 January 2008 Available online 4 February 2008
Abstract Sirt2 is a mammalian member of the Sirtuin family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. Although Sir-2.1 (a Caenorhabditis elegans Sirt2 ortholog) has been reported to interact with PAR-5/FTT-2 (a C. elegans 14-3-3 homolog), the molecular significance of the interaction between Sirt2 and 14-3-3 proteins in mammalian cell is not understood. Here, we report that Sirt2 interacts with 14-3-3 b and c among various 14-3-3 isoforms, and that this interaction is strengthened by AKT. Furthermore, Sirt2 deacetylates and down-regulates the transcriptional activity of p53, and 14-3-3 b/c augment deacetylation and down-regulation of the p53 transcriptional activity by Sirt2 in an AKT-dependent manner. Treatment of cells with nicotinamide, an inhibitor of Sirtuins, relieves the inhibition of p53 by Sirt2 and 14-3-3 b/c. Therefore, our results suggest that the interaction between Sirt2 and 14-3-3 b/c is a novel mechanism for the negative regulation of p53 beside the well-characterized Mdm2-mediated repression. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Sirt2; 14-3-3 b/c; p53; AKT
Sirtuins are nicotinamide adenine dinucleotide (NAD+)dependent protein lysine deacetylases and this enzymatic activity is conserved from yeast to mammals. Sirtuins are categorized as class III histone deacetylase as they use NAD+ as a cofactor unlike the classical class I and II histone deacetylases which use zinc as a cofactor [1,2]. There are seven Sirtuin homologs (Sirt1-7) in human [3]. Among them, Sirt1 is most well characterized and it has been shown to regulate cell proliferation, apoptosis, and cell survival [4–6]. Sirt1 can deacetylate and, subsequently, reduce the transcriptional activity of p53 which can be also deacetylated by HDAC1 [7,8]. In addition, Sirt1 plays a significant role in modulating the p53-dependent apoptotic response under DNA damage and oxidative stress [6]. * Corresponding authors. Fax: +82 2 3277 2385 (C.-Y. Yeo), +82 62 530 2949 (K.-Y. Lee). E-mail addresses: [email protected] (C.-Y. Yeo), kwanglee@chonnam. ac.kr (K.-Y. Lee).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.114
p53 is hyper-acetylated in Sirt1-deficient mice and Sirt1deficient mice display defects in the development of retina and heart [9]. In contrast to Sirt1, little is known about the precise function of Sirt2. Sirt2 is predominantly cytoplasmic. Sirt2 can deacetylates lysine-40 on a-tubulin and it co-localizes with microtubules and HDAC6 [10]. Sirt2 is hyper-phosphorylated in M phase where it regulates mitotic progression [11]. In addition, it is reported that Sirt2 is involved in life span extension and epigenetic gene silencing in yeast, Caenorhabditis elegans and Drosophila [12–14]. Recently, in C. elegans, it has been reported that PAR-5/FTT2, a 14-3-3 homolog, can interact with SIR-2.1, a Sirt2 ortholog, and that SIR2.1 can extend life span in a DAF-16-dependent manner which is a homolog of FOXO [15]. 14-3-3 Proteins are highly conserved from yeast to mammals and they are expressed in a wide range of tissues. There are seven 14-3-3 isoforms in mammals and the interactions of each 14-3-3 isoform with numerous binding
Y.-H. Jin et al. / Biochemical and Biophysical Research Communications 368 (2008) 690–695
partners have been studied until now. 14-3-3 Proteins require phosphoserine/phosphothreonine-containing motifs on their binding partners for interaction. 14-3-3 Proteins play critical roles in regulating cellular metabolism, signal transduction, cell cycle, apoptosis, transcription, and stress responses by altering the sub-cellular localization, stability, phosphorylation state, activity, and molecular interaction of their binding partners [16,17]. In this study, we investigated the interaction between Sirt2 and 14-3-3 isoforms. Sirt2 interacts with 14-3-3 b and c and this interaction is regulated by AKT. Furthermore, we found that Sirt2 deacetylates p53 and down-regulates the transcriptional activity of p53. 14-3-3 b/c enhances the inhibitory effect of Sirt2 on p53 in an AKTdependent manner. Therefore, our results suggest a novel inhibitory mechanism for the transcriptional activity of p53 by Sirt2 in addition to the well-characterized Mdm2mediated inhibition of p53.
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Materials and methods Cell culture. All culture media and antibiotics were purchased from Invitrogen. HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics–antimycotics at 37 °C, 5% CO2. Plasmids and antibodies. Myc- or HA-tagged Sirt2, p53, p300-4, 14-3-3 isoforms (e, f, b, and c) and AKT were constructed in a CMV promoterderived mammalian expression vector (pCS4+). Antibodies against Myc (9E10, Santa Cruz), acetyl-lysine (Cell Signaling) and HA (12CA5, Roche) were used. DNA transfection and reporter assay. Transient transfections were performed using the calcium phosphate and Lipofectamin plus methods. For luciferase assays, HEK293 cells were plated on 24-well plates the day before transfection. pCMV b-gal plasmid was co-transfected as an internal control for the efficiency of transfection. For luciferase assays, cells were lysed 36 h after transfection and the luciferase activity was measured using Luciferase Reporter Assay Kit (Promega). Immunoprecipitation and immunoblotting. HEK293 cells were lysed in an ice-cold lysis buffer (25 mM Hepes [pH 7.5], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 250 lM PMSF, 10 lg/ml leupeptin, and 10 lg/ml aprotinin) and cleared by centrifugation. Supernatants were subjected to immunoprecipitation using appropriate antibodies and protein A or protein G-Sepharose beads. Immunoprecipitated proteins were resolved by SDS–PAGE and transferred to PVDF membranes. Proteins were visualized using appropriate primary antibodies, horseradish peroxidasecoupled secondary antibodies and chemiluminescence Western blotting reagent (Amersham Bioscience).
Results Sirt2 interacts with 14-3-3 b/c and their interaction is increased by AKT In C. elegans, Sir-2.1, a Sirt2 ortholog, interacts with PAR-5/FTT-2, a 14-3-3 homolog [15]. We examined whether human Sirt2 can interact with various 14-3-3 isoforms by immunoprecipitation. Sirt2 interacted with 14-33 b and c but not with 14-3-3 f or e (Fig. 1). 14-3-3 Proteins interact with their binding partners only when the binding partners are phosphorylated at specific serine/threonine residues by corresponding kinases. A search of Sirt2 amino
W: Myc (Sirt2) Fig. 1. Sirt2 interacts with 14-3-3 b and c. HEK293 cells were transfected with indicated combinations of Myc-tagged Sirt2 and HA-tagged 14-3-3 isoforms (f, e, b or c). Cell lysates were subjected to immunoprecipitation using anti-Myc antibody and immunoprecipitated proteins were analyzed by Western blotting using anti-HA antibody [IP: Myc (Sirt2); W: HA (143-3)] (top panel). The levels of 14-3-3 isoforms and Sirt2 in total lysates were also examined by Western blotting using anti-HA antibody [W: HA (14-3-3)] or anti-Myc antibody [W: Myc (Sirt2)] (bottom panels).
acid sequence revealed several regions similar to the consensus AKT phosphorylation motif. To determine if AKT affects the interaction between Sirt2 and 14-3-3 b/c, co-immunoprecipitation of 14-3-3 b/c with Sirt2 was examined in the presence of absence of wild type or a kinasedefective form of AKT. The interaction between Sirt2 and 14-3-3 b/c was increased in the presence of wild type AKT while the interaction was dramatically decreased in the presence of the kinase-defective AKT (Fig. 2). The enhancement of interaction between Sirt2 and 14-3-3 b/c by wild type AKT but not by kinase-defective AKT suggests that Sirt2 may be phosphorylated by AKT which subsequently interacts with 14-3-3 b/c. Cooperation of Sirt2 and 14-3-3 b/c can further increase the deacetylation of p53 than by Sirt2 alone It has been reported that Sirt1 deacetylates p53 which is acetylated at lysine-382 [8]. Although Sirt2 is also presumed to deacetylate p53 as p53 deacetylation is inhibited by cambinol, an inhibitor for Sirt1 and Sirt2 [18], direct evidence has not been presented so far. To determine whether Sirt2 deacetylates p53, the levels of p300-induced acetylation of p53 was examined in the presence or absence of
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Sirt2. The level of p53 acetylation was increased by p300 and it was dramatically decreased when Sirt2 was also present (lanes 1–3, Fig. 3). The inhibitory effect of Sirt2 on p53 acetylation was diminished in the presence of nicotinamide (NIA), an inhibitor of Sirtuins (lane 4). As Sirt2 interacts with 14-3-3 b/c, we examined whether 14-3-3 b/c also affects the acetylation of p53. In the presence of 14-3-3 b or c, the deacetylation of p53 by Sirt2 was significantly potentiated (lanes 5–8 vs. lanes 2–4). The inhibitory effects of 14-3-3 b/c on p53 acetylation were also diminished in the presence of nicotinamide (lanes 5–8). However, the inhibitory effects of NIA on 14-3-3 b/c-enhanced p53 deacetylation could not be seen in the absence of Sirt2 (lanes 9–12). These results suggest that acetylated p53 can be deacetylated by Sirt2 and that Sirt2 may deacetylate p53 through the interaction with 14-3-3 b/c.
Sirt2 AKT WT β
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W: Myc (Sirt2) W: HA (AKT) Fig. 2. The interaction between Sirt2 and 14-3-3 b/c is increased by AKT. HEK293 cells were transfected with indicated combinations of Myctagged Sirt2, HA-tagged 14-3-3 b/c, and HA-tagged wild type (AKT WT) or a kinase-defective form (AKT KD) of AKT. Cell lysates were subjected to immunoprecipitation using anti-Myc antibody and immunoprecipitated proteins were analyzed by Western blotting with anti-HA antibody [IP: Myc (Sirt2); W: HA (14-3-3)] (top panel). The levels of 14-3-3 isoforms [W: HA (14-3-3)], Sirt2 [W: Myc (Sirt2)] and AKT [W: HA (AKT)] proteins in total lysates were also examined by Western blotting using anti-Myc and anti-HA antibodies (bottom panels).
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Sirt2 decreases the transcriptional activity of p53 and this inhibition is potentiated by 14-3-3 b/c in an AKT-dependent manner It has been reported that Sirt1 decreases the transcriptional activity of p53 on the promoter of p21WAF1 gene [8]. Therefore, we examined the effect of Sirt2 on the transcriptional activity of p53 using two p53-responsive
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NIA sirt2 P300-4 p53 IP: Acetyl-Lysine W: HA
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W: HA (14-3-3) Fig. 3. Deacetylation of p53 by Sirt2 is potentiated by 14-3-3 b/c and the deacetylation is inhibited by nicotinamide, a Sirtuin inhibitor. HEK293 cells were transfected with indicated combinations of HA-tagged p53, HA-tagged p300-4, Myc-tagged Sirt2, and HA-tagged 14-3-3 b/c. Twelve hours after transfection, cells were treated with 2.5 mM of nicotinamide (lanes 4, 6, 8, 10, and 12) or vehicle alone (lanes 1–3, 5, 7, 9, and 11). p300-4 contains the histone acetyl transferase (HAT) domain of p300. The levels of p53 acetylation were analyzed by immunoprecipitation using anti-acetyl-lysine antibody followed by Western blotting using anti-HA antibody (IP: acetyl-lysine; W: HA) (upper panel). The levels of p53 [W: HA (p53)], Sirt2 [W: Myc (Sirt2)], p300-4 [W: HA (p300-4)] and 14-3-3 b/c [W: HA (14-3-3)] in total lysates were also examined by Western blotting using anti-Myc and anti-HA antibodies (bottom panels).
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slightly enhanced the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c, while the kinase-defective AKT significantly reduced the inhibitory effects of Sirt2 and 14-3-3 b/c on the transcriptional activity of p53 (Fig. 4C). These results suggest that the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c requires the kinase activity of AKT.
luciferase reporters, p53-luc and p21-2300-luc. p53 activated p21 2300-luc and the luciferase activity was decreased by coexpression of 14-3-3 b or c but not by 14-3-3 f or e (Fig. 4A). Sirt2 also decreased the p53-induced luciferase activity from p21 2300-luc and this inhibition was further potentiated by the coexpression of 14-3-3 b or c but not by 14-3-3 f or e (Fig. 4A). Next, we examined the effects of nicotinamide on the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c. p53-induced luciferase activity from p53-luc was reduced by Sirt2 and/or 14-3-3 b/c and the luciferase activity was recovered in the presence of nicotinamide (Fig. 4B). These results suggest that the transcriptional activity of p53 can be inhibited by Sirt2 and this inhibition can be potentiated by 14-3-3 b/c. We have shown that wild type AKT enhances and a kinase-defective form of AKT reduces the interaction between Sirt2 and 14-3-3 b/c. Therefore, we next examined whether AKT has any effect on the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c. Wild type AKT
Discussion Sirtuins, the NAD+-dependent class III HDACs, regulate cell survival, senescence, cell proliferation, and differentiation by deacetylating histones and various non-histone proteins. p53 is first identified non-histone substrate of Sirt1. Deacetylation by Sirt1 reduces the function of p53 as an apoptotic protein, and Sirt1 promotes cell survival under stress [6]. In the current study, we have shown that Sirt2 interacts with 14-3-3 b/c in an AKT-dependent manner, and that Sirt2 and 14-3-3 b/c cooperatively down-reg-
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Fig. 4. Reduction of the transcriptional activity of p53 by Sirt2 and 14-3-3 b/c. The transactivation activities of p53 were determined by luciferase reporter assays. pCMV-b-galactosidase was used as an internal control for transfection efficiency and the luciferase activities were normalized with b-galactosidase activities. Experiments were performed in triplicates, and means and standard deviations of a representative experiment from three independent experiments were shown. (A) Sirt2 and 14-3-3 b/c inhibit the transcriptional activity of p53. HEK293 cells were transfected with p21-2300-luc, pCMV-bgalactosidase, and indicated combinations of cloning vector (V), p53, 14-3-3 isoforms (f, e, b or c) and Sirt2. p21-2300-luc contains p53-responsive elements of p21WAF1 gene. (B) The inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c is relieved by nicotinamide. HEK293 cells were transfected with p53-luc, pCMV-b-galactosidase and indicated combinations of cloning vector (V), p53, 14-3-3 isoforms (f, e, b or c) and Sirt2. Twelve hours after transfection, cell were treated with nicotinamide (+NIA, gray bars) or vehicle alone ( NIA, open bars). (C) The inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c requires AKT. HEK293 cells were transfected with p21-2300-luc, pCMV-b-galactosidase, and indicated combinations of cloning vector (V), p53, 14-3-3 isoforms (b or c), Sirt2 and wild type (AKT) or a kinase-defective form (AKT-KD) of AKT.
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ulate the transcriptional activity of p53 in an AKT-dependent manner. Our results reveal the existence of a novel negative regulatory pathway for p53 that is controlled by cooperation of Sirt2 and 14-3-3 b/c. Sirt2 controls longevity in various organisms including yeast, C. elegans and Drosophila [12–14]. Sirt1 promotes the survival of mammalian cells by deacetylating a DNA repair factor Ku70 which is induced by calorie restriction [5]. Sirt2 has been shown to regulate cell cycle progression [11], however, the precise molecular mechanism of Sirt2 function in mammalian cells is not known until now. Our study demonstrated that Sirt2 interacts with 14-3-3 b/c among various 14-3-3 isoforms. 14-3-3 Proteins interact only with proteins that are serine/threonine-phosphorylated by specific kinases. We identified that there are several tentative AKT phosphorylation sites on Sirt2, and we have shown that the interaction between Sirt2 and 143-3 b/c and the inhibitory effect on p53 transcriptional activity by Sirt2 and 14-3-3 b/c are enhanced by AKT. In C. elegans, SIR-2.1, a Sirt2 ortholog, was shown to interact with PAR-5/FTT-2, a 14-3-3 homolog, and to regulate the longevity of C. elegans [15]. In this regard, the AKTinduced interaction between Sirt2 and 14-3-3 b/c may control the survival of mammalian cells. Earlier studies indicated that the expression of 14-3-3 proteins is increased in some human cancers, suggesting that these proteins may act as oncogenes. 14-3-3 b has oncogenic effect in the context of cellular proliferation and tumorigenesis, and it is over-expressed in various murine tumor cell lines [19,20]. In addition, some 14-3-3 isoforms (b, c, r, and h) are over-expressed in human lung cancer tissues and, in particular, over-expression of 14-33 c causes polyploidization in H322 lung cancer cells, suggesting that 14-3-3 c may contribute to tumorigenesis by promoting genomic instability [21,22]. Although these studies on 14-3-3 proteins suggest that they can function as oncogenes in some human cancers, precise biological mechanism for this action is not known until now. We have shown that 14-3-3 b/c interacts with Sirt2, a class III HDAC, and this interaction can potentiate the reduction of acetylation and transcriptional activity of p53 which is an important tumor suppressor. A recent study suggests that AROS can act as a positive regulator of Sirt1 [23]. In this regard, our results suggest that 14-3-3 b/c may act as positive regulators of Sirt2. It is well established that p53 is negatively regulated by Mdm2-mediated ubiquitination and degradation. Negative regulators of p53 including p14ARF, L11, cyclin G, and YY1 control the function of p53 by affecting the accessibility of p53 to Mdm2 [24–27]. In this regard, our results suggest that 14-3-3 b/c may promote tumorigenesis by affecting the accessibility of p53 to Sirt2. In conclusion, our results suggest that association of 14-33 b/c and Sirt2 can be a novel negative regulatory mechanism for p53 activity beside the Mdm2-mediated ubiquitination.
Acknowledgments This study was financially supported by Chonnam National University. Y.-J.K. is supported by the second stage of the Brain Korea 21 Project. References [1] G. Blander, L. Guarente, The Sir2 family of protein deacetylases, Annu. Rev. Biochem. 73 (2004) 417–435. [2] M. Hernick, C.A. Fierke, Zinc hydrolases: the mechanisms of zincdependent deacetylases, Arch. Biochem. Biophys. 433 (2005) 71–84. [3] R.A. Frye, Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins, Biochem. Biophys. Res. Commun. 273 (2000) 793– 798. [4] K.R. Chuan, R. Mostoslavsky, D.B. Lombard, W.W. Pang, S. Saito, S. France, D. Kaushal, H.L. Cheng, M.R. Fischer, N. Stokes, M.M. Murphy, E. Appella, F.W. Alt, Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress, Cell Metab. 2 (2005) 67–76. [5] H.Y. Cohen, C. Miller, K.J. Bitterman, N.R. Wall, B. Hekking, B. Kessler, K.T. Howitz, M. Gorospe, R. de Cabo, D.A. Sinclair, Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase, Science 305 (2004) 390–392. [6] J. Luo, A.Y. Nikolaev, S. Imai, D. Chen, F. Su, A. Shiloh, L. Guarente, W. Gu, Negative control of p53 by Sir2a promotes cell survival under stress, Cell 107 (2001) 137–148. [7] J. Luo, F. Su, D. Chen, A. Shiloh, W. Gu, Deacetylation of p53 modulates its effect on cell growth and apoptosis, Nature 408 (2000) 377–381. [8] H. Vaziri, S.K. Dessalin, E.N. Eaton, S. Imai, R.A. Frye, T.K. Pandita, L. Guarente, R.A. Weinberg, hSIR2 (sirt1) functions as an NAD-dependent p53 deacetylase, Cell 107 (2001) 149–159. [9] H. Cheng, R. Mostoslavsky, S. Saito, J.P. Manis, Y. Gu, P. Patel, R. Bronson, E. Appella, F.W. Alt, K.F. Chua, Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice, Proc. Natl. Acad. Sci. USA 100 (2003) 10794–10799. [10] B.J. North, B.L. Marshall, M.T. Borra, J.M. Denu, E. Verdin, The human Sir2 ortholog, SIRT2, is an NAD(+)-dependent tubulin deacetylase, Mol. Cell 11 (2003) 437–444. [11] S.C. Dryden, F.A. Nahhas, J.E. Nowak, A.-S. Goustin, M.A. Tainsky, Role for human SIRT2 NAD-dependent deacetylase activity in control of mitotic exit in the cell cycle, Mol. Cell. Biol. 23 (2003) 3173–3185. [12] S.J. Lin, P.A. Defossez, L. Guarente, Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae, Science 289 (2000) 2126–2128. [13] B. Rogina, S.L. Helfand, Sir2 mediates longevity in the fly through a pathway related to calorie restriction, Proc. Natl. Acad. Sci. USA 101 (2004) 15998–16003. [14] H.A. Tissenbaum, L. Guarente, Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans, Nature 410 (2001) 227– 230. [15] Y. Wang, S.W. Oh, B. Deplancke, J. Luo, A.J.M. Walhout, H.A. Tissenbaum, C. elegans 14-3-3 proteins regulate life span and interact with SIR-2.1 and DAR-16/FOXO, Mech. Ageing Dev. 127 (2006) 741–747. [16] M.K. Dougherty, D.K. Morrison, Unlocking the code of 14-3-3, J. Cell Sci. 117 (2004) 1875–1884. [17] G. Tzivion, J. Avruch, 14-3-3 Proteins: active cofactors in cellular regulation by serine/threonine phosphorylation, J. Biol. Chem. 277 (2002) 3061–3064. [18] B. Heltweg, T. Gatbonton, A.D. Schuler, J. Posakony, H. Li, S. Goehle, R. Kllipara, R.A. DePinho, Y. Gu, J.A. Simon, A. Bedalov, Antitumor activity of a small-molecule inhibitor of human
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Biochemical and Biophysical Research Communications 368 (2008) 690–695 www.elsevier.com/locate/ybbrc
Sirt2 interacts with 14-3-3 b/c and down-regulates the activity of p53 Yun-Hye Jin a, Yeon-Jin Kim b, Dae-Won Kim c, Kwang-Hyun Baek d, Bok Yun Kang a, Chang-Yeol Yeo b,*, Kwang-Youl Lee a,* b
a College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju, Republic of Korea Department of Life Science and Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea c Department of Biochemistry, Yonsei University, Seoul, Republic of Korea d Graduate School of Life Science and Biotechnology, Pochon CHA University, CHA General Hospital, Seoul, Republic of Korea
Received 21 January 2008 Available online 4 February 2008
Abstract Sirt2 is a mammalian member of the Sirtuin family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. Although Sir-2.1 (a Caenorhabditis elegans Sirt2 ortholog) has been reported to interact with PAR-5/FTT-2 (a C. elegans 14-3-3 homolog), the molecular significance of the interaction between Sirt2 and 14-3-3 proteins in mammalian cell is not understood. Here, we report that Sirt2 interacts with 14-3-3 b and c among various 14-3-3 isoforms, and that this interaction is strengthened by AKT. Furthermore, Sirt2 deacetylates and down-regulates the transcriptional activity of p53, and 14-3-3 b/c augment deacetylation and down-regulation of the p53 transcriptional activity by Sirt2 in an AKT-dependent manner. Treatment of cells with nicotinamide, an inhibitor of Sirtuins, relieves the inhibition of p53 by Sirt2 and 14-3-3 b/c. Therefore, our results suggest that the interaction between Sirt2 and 14-3-3 b/c is a novel mechanism for the negative regulation of p53 beside the well-characterized Mdm2-mediated repression. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Sirt2; 14-3-3 b/c; p53; AKT
Sirtuins are nicotinamide adenine dinucleotide (NAD+)dependent protein lysine deacetylases and this enzymatic activity is conserved from yeast to mammals. Sirtuins are categorized as class III histone deacetylase as they use NAD+ as a cofactor unlike the classical class I and II histone deacetylases which use zinc as a cofactor [1,2]. There are seven Sirtuin homologs (Sirt1-7) in human [3]. Among them, Sirt1 is most well characterized and it has been shown to regulate cell proliferation, apoptosis, and cell survival [4–6]. Sirt1 can deacetylate and, subsequently, reduce the transcriptional activity of p53 which can be also deacetylated by HDAC1 [7,8]. In addition, Sirt1 plays a significant role in modulating the p53-dependent apoptotic response under DNA damage and oxidative stress [6]. * Corresponding authors. Fax: +82 2 3277 2385 (C.-Y. Yeo), +82 62 530 2949 (K.-Y. Lee). E-mail addresses: [email protected] (C.-Y. Yeo), kwanglee@chonnam. ac.kr (K.-Y. Lee).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.114
p53 is hyper-acetylated in Sirt1-deficient mice and Sirt1deficient mice display defects in the development of retina and heart [9]. In contrast to Sirt1, little is known about the precise function of Sirt2. Sirt2 is predominantly cytoplasmic. Sirt2 can deacetylates lysine-40 on a-tubulin and it co-localizes with microtubules and HDAC6 [10]. Sirt2 is hyper-phosphorylated in M phase where it regulates mitotic progression [11]. In addition, it is reported that Sirt2 is involved in life span extension and epigenetic gene silencing in yeast, Caenorhabditis elegans and Drosophila [12–14]. Recently, in C. elegans, it has been reported that PAR-5/FTT2, a 14-3-3 homolog, can interact with SIR-2.1, a Sirt2 ortholog, and that SIR2.1 can extend life span in a DAF-16-dependent manner which is a homolog of FOXO [15]. 14-3-3 Proteins are highly conserved from yeast to mammals and they are expressed in a wide range of tissues. There are seven 14-3-3 isoforms in mammals and the interactions of each 14-3-3 isoform with numerous binding
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partners have been studied until now. 14-3-3 Proteins require phosphoserine/phosphothreonine-containing motifs on their binding partners for interaction. 14-3-3 Proteins play critical roles in regulating cellular metabolism, signal transduction, cell cycle, apoptosis, transcription, and stress responses by altering the sub-cellular localization, stability, phosphorylation state, activity, and molecular interaction of their binding partners [16,17]. In this study, we investigated the interaction between Sirt2 and 14-3-3 isoforms. Sirt2 interacts with 14-3-3 b and c and this interaction is regulated by AKT. Furthermore, we found that Sirt2 deacetylates p53 and down-regulates the transcriptional activity of p53. 14-3-3 b/c enhances the inhibitory effect of Sirt2 on p53 in an AKTdependent manner. Therefore, our results suggest a novel inhibitory mechanism for the transcriptional activity of p53 by Sirt2 in addition to the well-characterized Mdm2mediated inhibition of p53.
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Materials and methods Cell culture. All culture media and antibiotics were purchased from Invitrogen. HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics–antimycotics at 37 °C, 5% CO2. Plasmids and antibodies. Myc- or HA-tagged Sirt2, p53, p300-4, 14-3-3 isoforms (e, f, b, and c) and AKT were constructed in a CMV promoterderived mammalian expression vector (pCS4+). Antibodies against Myc (9E10, Santa Cruz), acetyl-lysine (Cell Signaling) and HA (12CA5, Roche) were used. DNA transfection and reporter assay. Transient transfections were performed using the calcium phosphate and Lipofectamin plus methods. For luciferase assays, HEK293 cells were plated on 24-well plates the day before transfection. pCMV b-gal plasmid was co-transfected as an internal control for the efficiency of transfection. For luciferase assays, cells were lysed 36 h after transfection and the luciferase activity was measured using Luciferase Reporter Assay Kit (Promega). Immunoprecipitation and immunoblotting. HEK293 cells were lysed in an ice-cold lysis buffer (25 mM Hepes [pH 7.5], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 250 lM PMSF, 10 lg/ml leupeptin, and 10 lg/ml aprotinin) and cleared by centrifugation. Supernatants were subjected to immunoprecipitation using appropriate antibodies and protein A or protein G-Sepharose beads. Immunoprecipitated proteins were resolved by SDS–PAGE and transferred to PVDF membranes. Proteins were visualized using appropriate primary antibodies, horseradish peroxidasecoupled secondary antibodies and chemiluminescence Western blotting reagent (Amersham Bioscience).
Results Sirt2 interacts with 14-3-3 b/c and their interaction is increased by AKT In C. elegans, Sir-2.1, a Sirt2 ortholog, interacts with PAR-5/FTT-2, a 14-3-3 homolog [15]. We examined whether human Sirt2 can interact with various 14-3-3 isoforms by immunoprecipitation. Sirt2 interacted with 14-33 b and c but not with 14-3-3 f or e (Fig. 1). 14-3-3 Proteins interact with their binding partners only when the binding partners are phosphorylated at specific serine/threonine residues by corresponding kinases. A search of Sirt2 amino
W: Myc (Sirt2) Fig. 1. Sirt2 interacts with 14-3-3 b and c. HEK293 cells were transfected with indicated combinations of Myc-tagged Sirt2 and HA-tagged 14-3-3 isoforms (f, e, b or c). Cell lysates were subjected to immunoprecipitation using anti-Myc antibody and immunoprecipitated proteins were analyzed by Western blotting using anti-HA antibody [IP: Myc (Sirt2); W: HA (143-3)] (top panel). The levels of 14-3-3 isoforms and Sirt2 in total lysates were also examined by Western blotting using anti-HA antibody [W: HA (14-3-3)] or anti-Myc antibody [W: Myc (Sirt2)] (bottom panels).
acid sequence revealed several regions similar to the consensus AKT phosphorylation motif. To determine if AKT affects the interaction between Sirt2 and 14-3-3 b/c, co-immunoprecipitation of 14-3-3 b/c with Sirt2 was examined in the presence of absence of wild type or a kinasedefective form of AKT. The interaction between Sirt2 and 14-3-3 b/c was increased in the presence of wild type AKT while the interaction was dramatically decreased in the presence of the kinase-defective AKT (Fig. 2). The enhancement of interaction between Sirt2 and 14-3-3 b/c by wild type AKT but not by kinase-defective AKT suggests that Sirt2 may be phosphorylated by AKT which subsequently interacts with 14-3-3 b/c. Cooperation of Sirt2 and 14-3-3 b/c can further increase the deacetylation of p53 than by Sirt2 alone It has been reported that Sirt1 deacetylates p53 which is acetylated at lysine-382 [8]. Although Sirt2 is also presumed to deacetylate p53 as p53 deacetylation is inhibited by cambinol, an inhibitor for Sirt1 and Sirt2 [18], direct evidence has not been presented so far. To determine whether Sirt2 deacetylates p53, the levels of p300-induced acetylation of p53 was examined in the presence or absence of
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Sirt2. The level of p53 acetylation was increased by p300 and it was dramatically decreased when Sirt2 was also present (lanes 1–3, Fig. 3). The inhibitory effect of Sirt2 on p53 acetylation was diminished in the presence of nicotinamide (NIA), an inhibitor of Sirtuins (lane 4). As Sirt2 interacts with 14-3-3 b/c, we examined whether 14-3-3 b/c also affects the acetylation of p53. In the presence of 14-3-3 b or c, the deacetylation of p53 by Sirt2 was significantly potentiated (lanes 5–8 vs. lanes 2–4). The inhibitory effects of 14-3-3 b/c on p53 acetylation were also diminished in the presence of nicotinamide (lanes 5–8). However, the inhibitory effects of NIA on 14-3-3 b/c-enhanced p53 deacetylation could not be seen in the absence of Sirt2 (lanes 9–12). These results suggest that acetylated p53 can be deacetylated by Sirt2 and that Sirt2 may deacetylate p53 through the interaction with 14-3-3 b/c.
Sirt2 AKT WT β
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W: Myc (Sirt2) W: HA (AKT) Fig. 2. The interaction between Sirt2 and 14-3-3 b/c is increased by AKT. HEK293 cells were transfected with indicated combinations of Myctagged Sirt2, HA-tagged 14-3-3 b/c, and HA-tagged wild type (AKT WT) or a kinase-defective form (AKT KD) of AKT. Cell lysates were subjected to immunoprecipitation using anti-Myc antibody and immunoprecipitated proteins were analyzed by Western blotting with anti-HA antibody [IP: Myc (Sirt2); W: HA (14-3-3)] (top panel). The levels of 14-3-3 isoforms [W: HA (14-3-3)], Sirt2 [W: Myc (Sirt2)] and AKT [W: HA (AKT)] proteins in total lysates were also examined by Western blotting using anti-Myc and anti-HA antibodies (bottom panels).
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Sirt2 decreases the transcriptional activity of p53 and this inhibition is potentiated by 14-3-3 b/c in an AKT-dependent manner It has been reported that Sirt1 decreases the transcriptional activity of p53 on the promoter of p21WAF1 gene [8]. Therefore, we examined the effect of Sirt2 on the transcriptional activity of p53 using two p53-responsive
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W: HA (14-3-3) Fig. 3. Deacetylation of p53 by Sirt2 is potentiated by 14-3-3 b/c and the deacetylation is inhibited by nicotinamide, a Sirtuin inhibitor. HEK293 cells were transfected with indicated combinations of HA-tagged p53, HA-tagged p300-4, Myc-tagged Sirt2, and HA-tagged 14-3-3 b/c. Twelve hours after transfection, cells were treated with 2.5 mM of nicotinamide (lanes 4, 6, 8, 10, and 12) or vehicle alone (lanes 1–3, 5, 7, 9, and 11). p300-4 contains the histone acetyl transferase (HAT) domain of p300. The levels of p53 acetylation were analyzed by immunoprecipitation using anti-acetyl-lysine antibody followed by Western blotting using anti-HA antibody (IP: acetyl-lysine; W: HA) (upper panel). The levels of p53 [W: HA (p53)], Sirt2 [W: Myc (Sirt2)], p300-4 [W: HA (p300-4)] and 14-3-3 b/c [W: HA (14-3-3)] in total lysates were also examined by Western blotting using anti-Myc and anti-HA antibodies (bottom panels).
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slightly enhanced the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c, while the kinase-defective AKT significantly reduced the inhibitory effects of Sirt2 and 14-3-3 b/c on the transcriptional activity of p53 (Fig. 4C). These results suggest that the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c requires the kinase activity of AKT.
luciferase reporters, p53-luc and p21-2300-luc. p53 activated p21 2300-luc and the luciferase activity was decreased by coexpression of 14-3-3 b or c but not by 14-3-3 f or e (Fig. 4A). Sirt2 also decreased the p53-induced luciferase activity from p21 2300-luc and this inhibition was further potentiated by the coexpression of 14-3-3 b or c but not by 14-3-3 f or e (Fig. 4A). Next, we examined the effects of nicotinamide on the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c. p53-induced luciferase activity from p53-luc was reduced by Sirt2 and/or 14-3-3 b/c and the luciferase activity was recovered in the presence of nicotinamide (Fig. 4B). These results suggest that the transcriptional activity of p53 can be inhibited by Sirt2 and this inhibition can be potentiated by 14-3-3 b/c. We have shown that wild type AKT enhances and a kinase-defective form of AKT reduces the interaction between Sirt2 and 14-3-3 b/c. Therefore, we next examined whether AKT has any effect on the inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c. Wild type AKT
Discussion Sirtuins, the NAD+-dependent class III HDACs, regulate cell survival, senescence, cell proliferation, and differentiation by deacetylating histones and various non-histone proteins. p53 is first identified non-histone substrate of Sirt1. Deacetylation by Sirt1 reduces the function of p53 as an apoptotic protein, and Sirt1 promotes cell survival under stress [6]. In the current study, we have shown that Sirt2 interacts with 14-3-3 b/c in an AKT-dependent manner, and that Sirt2 and 14-3-3 b/c cooperatively down-reg-
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Fig. 4. Reduction of the transcriptional activity of p53 by Sirt2 and 14-3-3 b/c. The transactivation activities of p53 were determined by luciferase reporter assays. pCMV-b-galactosidase was used as an internal control for transfection efficiency and the luciferase activities were normalized with b-galactosidase activities. Experiments were performed in triplicates, and means and standard deviations of a representative experiment from three independent experiments were shown. (A) Sirt2 and 14-3-3 b/c inhibit the transcriptional activity of p53. HEK293 cells were transfected with p21-2300-luc, pCMV-bgalactosidase, and indicated combinations of cloning vector (V), p53, 14-3-3 isoforms (f, e, b or c) and Sirt2. p21-2300-luc contains p53-responsive elements of p21WAF1 gene. (B) The inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c is relieved by nicotinamide. HEK293 cells were transfected with p53-luc, pCMV-b-galactosidase and indicated combinations of cloning vector (V), p53, 14-3-3 isoforms (f, e, b or c) and Sirt2. Twelve hours after transfection, cell were treated with nicotinamide (+NIA, gray bars) or vehicle alone ( NIA, open bars). (C) The inhibition of p53 transcriptional activity by Sirt2 and 14-3-3 b/c requires AKT. HEK293 cells were transfected with p21-2300-luc, pCMV-b-galactosidase, and indicated combinations of cloning vector (V), p53, 14-3-3 isoforms (b or c), Sirt2 and wild type (AKT) or a kinase-defective form (AKT-KD) of AKT.
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ulate the transcriptional activity of p53 in an AKT-dependent manner. Our results reveal the existence of a novel negative regulatory pathway for p53 that is controlled by cooperation of Sirt2 and 14-3-3 b/c. Sirt2 controls longevity in various organisms including yeast, C. elegans and Drosophila [12–14]. Sirt1 promotes the survival of mammalian cells by deacetylating a DNA repair factor Ku70 which is induced by calorie restriction [5]. Sirt2 has been shown to regulate cell cycle progression [11], however, the precise molecular mechanism of Sirt2 function in mammalian cells is not known until now. Our study demonstrated that Sirt2 interacts with 14-3-3 b/c among various 14-3-3 isoforms. 14-3-3 Proteins interact only with proteins that are serine/threonine-phosphorylated by specific kinases. We identified that there are several tentative AKT phosphorylation sites on Sirt2, and we have shown that the interaction between Sirt2 and 143-3 b/c and the inhibitory effect on p53 transcriptional activity by Sirt2 and 14-3-3 b/c are enhanced by AKT. In C. elegans, SIR-2.1, a Sirt2 ortholog, was shown to interact with PAR-5/FTT-2, a 14-3-3 homolog, and to regulate the longevity of C. elegans [15]. In this regard, the AKTinduced interaction between Sirt2 and 14-3-3 b/c may control the survival of mammalian cells. Earlier studies indicated that the expression of 14-3-3 proteins is increased in some human cancers, suggesting that these proteins may act as oncogenes. 14-3-3 b has oncogenic effect in the context of cellular proliferation and tumorigenesis, and it is over-expressed in various murine tumor cell lines [19,20]. In addition, some 14-3-3 isoforms (b, c, r, and h) are over-expressed in human lung cancer tissues and, in particular, over-expression of 14-33 c causes polyploidization in H322 lung cancer cells, suggesting that 14-3-3 c may contribute to tumorigenesis by promoting genomic instability [21,22]. Although these studies on 14-3-3 proteins suggest that they can function as oncogenes in some human cancers, precise biological mechanism for this action is not known until now. We have shown that 14-3-3 b/c interacts with Sirt2, a class III HDAC, and this interaction can potentiate the reduction of acetylation and transcriptional activity of p53 which is an important tumor suppressor. A recent study suggests that AROS can act as a positive regulator of Sirt1 [23]. In this regard, our results suggest that 14-3-3 b/c may act as positive regulators of Sirt2. It is well established that p53 is negatively regulated by Mdm2-mediated ubiquitination and degradation. Negative regulators of p53 including p14ARF, L11, cyclin G, and YY1 control the function of p53 by affecting the accessibility of p53 to Mdm2 [24–27]. In this regard, our results suggest that 14-3-3 b/c may promote tumorigenesis by affecting the accessibility of p53 to Sirt2. In conclusion, our results suggest that association of 14-33 b/c and Sirt2 can be a novel negative regulatory mechanism for p53 activity beside the Mdm2-mediated ubiquitination.
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