letters to nature 30. He, T. C. et al. A simplified system for generating recombinant adenoviruses. Proc. Natl Acad. Sci. USA 95, 2509–2514 (1998). Supplementary Information accompanies the paper on Nature’s website (ç http://www.nature.com/nature). Acknowledgements We thank B. Vogelstein, K. Vousden and T. Jacks for plasmids; J. Chen for 2A-10 antibody; and G. Del Sal for discussion and sharing unpublished data. We also thank E. R. Flores for E1A-retrovirus and advice on ChIP analysis, and Y. Zhang for technical assistance on cell cycle analysis. This work was supported by the NIH (Z.-X.X) and Department of Defense (Z.-X.X). Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to Z.-X.X. (e-mail: [email protected]). .............................................................. The prolyl isomerase Pin1 reveals a mechanism to control p53 functions after genotoxic insults Paola Zacchi*†‡, Monica Gostissa*‡, Takafumi Uchida§, Clio Salvagno*†, Fabio Avolio*, Stefano Voliniak, Ze’ev Ronai{, Giovanni Blandino#, Claudio Schneider*q & Giannino Del Sal*† * Laboratorio Nazionale CIB, AREA Science Park, Padriciano 99, 34012 Trieste, Italy † Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita` degli Studi di Trieste, via L. Giorgeri 1, 34100, Trieste, Italy § Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan k Universita’ di Ferrara, Sezione di Istologia ed Embriologia, Dipartimento di Morfologia ed Embriologia, via Fossato di Mortara 64/b, 44100, Ferrara, Italy { The Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York 10029-6574, USA # Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, via Messi d’oro 156, 00158 Rome, Italy q Dipartimento di Scienze e Tecnologie Biomediche, Universita` degli Studi di Udine, p. le Kolbe 1, 33100 Udine, Italy ‡ These authors contributed equally to this work ............................................................................................................................................................................. The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 post-translational modifi- cations and association with other proteins have been implicated in the regulation of its stability and transcriptional activities1,2. Here we report that, on DNA damage, p53 interacts with Pin1, a peptidyl-prolyl isomerase3, which regulates the function of many proteins involved in cell cycle control and apoptosis4–6.The interaction is strictly dependent on p53 phosphorylation, and requires Ser 33, Thr 81 and Ser 315. On binding, Pin1 generates Figure 1 Pin1 interacts with activated p53 in a phosphorylation-dependent manner. conformational changes in p53, enhancing its transactivation a, Lysates from U2-OS cells treated with different DNA-damaging agents or with activity. Stabilization of p53 is impaired in UV-treated Pin1 2/2 proteasome inhibitor MG132 were subjected to GST or GST–Pin1 pull-down followed by cells owing to its inability to efficiently dissociate from Mdm2. As immunoblotting with DO-1 antibody. Anti-pSer15 antibody was used to detect a consequence, a reduced p53-dependent response was detected DNA-damage-induced phosphorylation on p53. WB, western blot; UV, ultraviolet in Pin1 2/2 cells, and this correlates with a diminished transcrip- radiation; Bleo, bleomycin; Adria, adriamycin. b, U2-OS cells were g-irradiated and tional activation of some p53-regulated genes. Our results processed as in a. c, Lysates from UV-irradiated U2-OS cells were treated with calf suggest that, following stress-induced phosphorylation, p53 intestinal phosphatase (CIP) before incubation with GST and GST–Pin1. d, NIH3T3 cells needs to form a complex with Pin1 and to undergo a confor- infected with retroviruses expressing b-galactosidase (b-Gal), activated Ras (RasV12) or mational change to fulfil its biological roles. E1A were analysed as in a. Murine p53 was detected by anti-p53 polyclonal antibody. The peptidyl-prolyl isomerase Pin1 has recently emerged as an e, Lysates from 293 cells, UV irradiated and untreated, were immunoprecipitated with important regulator of cell proliferation and DNA-replication anti-Pin1 antibody or with normal rabbit serum (NRS). Immunoprecipitates were analysed checkpoint3,7. Pin1 interacts with a number of phosphoproteins by western blotting with DO-1 and anti-Pin1 antibodies. IgG, immunoglobulin-g. Asterisk through recognition of pSer/Thr-Pro motifs by its amino-terminal indicates the light chain of the antibodies used for immunoprecipitation. NATURE | VOL 419 | 24 OCTOBER 2002 | www.nature.com/nature © 2002 Nature Publishing Group 853 letters to nature WW domain8,9. By promoting the cis–trans isomerization of this corresponding Ser/Thr to Ala point mutants, and assayed their peptide bond through its carboxy-terminal prolyl isomerase ability to bind GST–Pin1 on overexpression in SaOS-2 cells and UV (PPIase)3 domain, it regulates the function of its substrates. treatment. Under these conditions, wild-type p53 (p53WT) and Analysis of the amino-acid sequence of human p53 revealed p53(S46A) exhibited a similar affinity for Pin1, whereas p53(S33A), several Pin1 consensus motifs, which are phosphorylated by mem- p53(T81A) and p53(S315A) showed a reduced binding. This bers of the MAP kinase10 and the cell-cycle-dependent kinase11 reduction was more pronounced in the case of the double and (Cdk) families on genotoxic insults. Therefore we tested whether triple mutants (Fig. 2a), thus suggesting that these three sites are all Pin1 could associate with p53 by using a glutathione-S-transferase critical for Pin1 binding. (GST)–Pin1 pull-down assay6. Pin1 bound p53 with high affinity Next we verified whether the prolyl-isomerase activity of Pin1 only on treating the cells with several types of DNA-damaging induces conformational changes in p53 by performing a partial agents, including ultraviolet radiation (UV), ionizing radiation and proteolysis assay14 on in vitro-translated p53WT, which is phos- chemotherapeutic drugs (Fig. 1a, b). This binding is not simply due phorylated on Pin1 consensus, as judged by immunoprecipitation to quantitative changes in p53 levels, as no interaction with GST– with the pSer/Thr-Pro specific MPM-2 antibody6, and binds to Pin1 Pin1 was detected when p53 was accumulated by treatment with (not shown). His-tagged p53WT was incubated with GST or proteasome inhibitor (Fig. 1a, lane 15). The interaction relied GST–Pin1 before addition of subtilisin. Pin1 protected p53 from entirely on p53 phosphorylation, as demonstrated by the lack of proteolytic cleavage, whereas lack of protection was observed on p53 binding when p53 was dephosphorylated by phosphatase treatment being stripped of its phosphates by treatment with calf intestinal (Fig. 1c), and required a functional Pin1-WWdomain (Supplemen- phosphatase (CIP; Fig. 2b, left panel). Importantly this effect was tary Fig. S1a). Similar results were obtained with other human and not due to steric hindrance exerted by Pin1 binding, but was murine cell lines (not shown). In addition to DNA damage, over- dependent only on its isomerase activity (Fig. 2b, right panel), expression of oncogenes activating p5312,13 promoted its interaction because GST–Pin1(C109A), a mutant that is catalytically impaired with GST–Pin1 (Fig. 1d). but fully competent to bind its substrates7, was completely The in vivo association between Pin1 and p53 was detected by ineffective. coimmunoprecipitation between either overexpressed (Supplemen- Changes in p53 conformation may impinge on its transactivation tary Fig. S1b) or endogenous proteins (Fig. 1e), and the binding was functions. We therefore performed reporter assays in SaOS-2 cells greatly enhanced on UV treatment. overexpressing p53WT. The ectopic expression of Pin1, but not To identify the Pin1 binding sites on p53, we generated the Pin1-WW mutant, exerted a stimulatory effect toward Bax- and Figure 2 Phosphorylated Ser 33, Thr 81 and Ser 315 are required for the interaction with motifs. WT, wild type. b, Subtilisin cleavage was performed on His-tagged p53 after Pin1, which drives conformational changes in p53. a, Different Ser/Thr-Ala p53 point incubation with GST, GST–Pin1 or GST–Pin1(C109A) (right panel), and on p53 mutants were assayed for interaction with GST–Pin1 as described in Fig. 1 on dephosphorylation (left panel). The reaction products were analysed by SDS–PAGE and overexpression in SaOS-2 cells and UV treatment. A representation of p53 domains is autoradiography. The amount of the various GST-fusions were verified by western blotting presented above (TA, transactivation domain; PP, polyproline region; DBD, DNA binding (lower panels). domain; CT, C-terminal domain), together with the positions of putative Pin1 consensus 854 © 2002 Nature Publishing Group NATURE | VOL 419 | 24 OCTOBER 2002 | www.nature.com/nature letters to nature pG13-luciferase reporters. This effect was not observed when Pin1 performing pull-down assays (Fig. 3d, lanes 1–6) and coimmuno- was co-expressed with p53 mutants S33A/S315A, T81A/S315A or precipitation (Fig. 3d, lanes 7–12) assays on lysates normalized for S33A/T81A/S315A, thus indicating that it is a consequence of a p53 content. This revealed that in UV-treated Pin1 2/2 cells a higher direct interaction