Control of Human PIRH2 Protein Stability
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m312712200
ISSN1083-351X
AutoresIan R. Logan, Vasileia Sapountzi, Luke Gaughan, David E. Neal, Craig Robson,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoMurine PIRH2 (mPIRH2) was recently identified as a RING finger-containing ubiquitin-protein isopeptide ligase that interacts with both p53 and the human androgen receptor. mpirh2 is a p53-responsive gene that is up-regulated by UV, and mPIRH2 protein has the capacity to polyubiquitylate p53, perhaps leading to p53 destruction. mpirh2 therefore has properties similar to those of the oncogene mdm2. Here, we have identified human PIRH2 (hPIRH2) as a TIP60-interacting protein. To investigate its regulation, we characterized hPIRH2 in parallel with hPIRH2 variants possessing mutations of conserved RING finger residues. We observed that wild-type hPIRH2 is an unstable protein with a short half-life and is a target for RING domain-dependent proteasomal degradation. Accordingly, we found that hPIRH2 was ubiquitylated in cells. The TIP60-hPIRH2 association appeared to regulate hPIRH2 stability; coexpression of TIP60 enhanced hPIRH2 protein stability and altered hPIRH2 subcellular localization. These results suggest that hPIRH2 activities can be controlled, at the post-translational level, in multiple ways. Murine PIRH2 (mPIRH2) was recently identified as a RING finger-containing ubiquitin-protein isopeptide ligase that interacts with both p53 and the human androgen receptor. mpirh2 is a p53-responsive gene that is up-regulated by UV, and mPIRH2 protein has the capacity to polyubiquitylate p53, perhaps leading to p53 destruction. mpirh2 therefore has properties similar to those of the oncogene mdm2. Here, we have identified human PIRH2 (hPIRH2) as a TIP60-interacting protein. To investigate its regulation, we characterized hPIRH2 in parallel with hPIRH2 variants possessing mutations of conserved RING finger residues. We observed that wild-type hPIRH2 is an unstable protein with a short half-life and is a target for RING domain-dependent proteasomal degradation. Accordingly, we found that hPIRH2 was ubiquitylated in cells. The TIP60-hPIRH2 association appeared to regulate hPIRH2 stability; coexpression of TIP60 enhanced hPIRH2 protein stability and altered hPIRH2 subcellular localization. These results suggest that hPIRH2 activities can be controlled, at the post-translational level, in multiple ways. The tumor suppressor p53 is a transcription factor that, through modulation of gene expression, can induce apoptosis or cell cycle arrest in response to cellular insults, including DNA damage (1Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2694) Google Scholar). Several regulatory mechanisms control p53 activities, including post-translational modifications (2Brooks C.L. Gu W. Curr. Opin. Cell Biol. 2003; 15: 164-171Crossref PubMed Scopus (630) Google Scholar, 3Prives C. Manley J.L. Cell. 2001; 107: 815-818Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) such as ubiquitylation (4Michael D. Oren M. Semin. Cancer Biol. 2003; 13: 49-58Crossref PubMed Scopus (641) Google Scholar). Ubiquitylation can be an acute mechanism controlling the activities of proteins in numerous cellular processes, including transcription (5Muratani M. Tansey W.P. Nat. Rev. Mol. Cell Biol. 2003; 4: 192-201Crossref PubMed Scopus (668) Google Scholar, 6Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (344) Google Scholar), and can occur once (monoubiquitylation) or in chains (polyubiquitylation) on target proteins (7Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Attachment of polyubiquitin chains by ubiquitinprotein isopeptide ligase (E3) 1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; ARF, alternative reading frame; mPIRH2, murine PIRH2; hPIRH2, human PIRH2; RFP, red fluorescent protein; GFP, green fluorescent protein; CMV-Ub, cytomegalovirus-driven ubiquitin. 1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; ARF, alternative reading frame; mPIRH2, murine PIRH2; hPIRH2, human PIRH2; RFP, red fluorescent protein; GFP, green fluorescent protein; CMV-Ub, cytomegalovirus-driven ubiquitin. enzymes results in discriminate irreversible destruction of the p53 protein by the 26 S proteasome (8Michael D. Oren M. Curr. Opin. Genet. Dev. 2002; 12: 53-59Crossref PubMed Scopus (242) Google Scholar). Other key proteins are controlled the same way; polyubiquitylation leads to their destruction, especially upon modification by Lys48-linked polyubiquitin chains (7Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar).The mdm2 (murine double minute-2) oncogene (HDM2 in humans) is a p53 target gene (9Barak Y. Juven T. Haffner R. Oren M. EMBO J. 1993; 12: 461-468Crossref PubMed Scopus (1167) Google Scholar) that is amplified in osteosarcomas (10Oliner J.D. Kinzler K.W. Meltzer P.S. George D.L. Vogelstein B. Nature. 1992; 358: 80-83Crossref PubMed Scopus (1790) Google Scholar, 11Leach F.S. Tokino T. Meltzer P. Burrell M. Oliner J.D. Smith S. Hill D.E. Sidransky D. Kinzler K.W. Vogelstein B. Cancer Res. 1993; 53: 2231-2234PubMed Google Scholar) and exerts negative regulation on p53 (8Michael D. Oren M. Curr. Opin. Genet. Dev. 2002; 12: 53-59Crossref PubMed Scopus (242) Google Scholar). MDM2 is a RING domain-dependent E3 enzyme that catalyzes formation of polyubiquitin chains on p53 (12Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1587) Google Scholar, 13Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar), leading to p53 destruction (14Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3659) Google Scholar). MDM2 can also mediate nucleocytoplasmic shuttling of ubiquitylated p53 so that p53 can be degraded in the cytoplasm (15Boyd S.D. Tsai K.Y. Jacks T. Nat. Cell Biol. 2000; 2: 563-568Crossref PubMed Scopus (285) Google Scholar, 16Geyer R.K. Yu Z.K. Maki C.G. Nat. Cell Biol. 2000; 2: 569-573Crossref PubMed Scopus (296) Google Scholar, 17Gu J. Nie L. Wiederschain D. Yuan Z.M. Mol. Cell. Biol. 2001; 21: 8533-8546Crossref PubMed Scopus (76) Google Scholar). The importance of MDM2-p53 association is demonstrated by the finding that mdm2-/- cells are nonviable due to p53-mediated apoptosis, whereas mdm2-/- p53-/- cells are rescued from apoptosis (18de Rozieres S. Maya R. Oren M. Lozano G. Oncogene. 2000; 19: 1691-1697Crossref PubMed Scopus (115) Google Scholar, 19Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1057) Google Scholar, 20Montes de Oca Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1196) Google Scholar).MDM2 has become the focus of research (8Michael D. Oren M. Curr. Opin. Genet. Dev. 2002; 12: 53-59Crossref PubMed Scopus (242) Google Scholar) establishing regulatory pathways controlling its enzymatic activity, subcellular localization, and abundance, all of which influence MDM2. MDM2 enzymatic activity is inhibited by association with the murine p19ARF (p14 in humans) tumor suppressor (21Pomerantz J. Schreiber-Agus N. Liegeois N.J. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1324) Google Scholar, 22Zhang Y. Xiong Y. Yarbrough W.G. Cell. 1998; 92: 725-734Abstract Full Text Full Text PDF PubMed Scopus (1392) Google Scholar, 23Stott F.J. Bates S. James M.C. McConnell B.B. Starborg M. Brookes S. Palmero I. Ryan K. Hara E. Vousden K.H. Peters G. EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1007) Google Scholar, 24Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (612) Google Scholar, 25Quelle D.E. Zindy F. Ashmun R.A. Sherr C.J. Cell. 1995; 83: 993-1000Abstract Full Text PDF PubMed Scopus (1307) Google Scholar) in response to oncogene activation (26Sherr C.J. Weber J.D. Curr. Opin. Genet. Dev. 2000; 10: 94-99Crossref PubMed Scopus (571) Google Scholar). MDM2 phosphorylation by ATM in response to DNA damage also inhibits MDM2-p53 interaction (27Mayo L.D. Turchi J.J. Berberich S.J. Cancer Res. 1997; 57: 5013-5016PubMed Google Scholar, 28Maya R. Balass M. Kim S.T. Shkedy D. Leal J.F. Shifman O. Moas M. Buschmann T. Ronai Z. Shiloh Y. Kastan M.B. Katzir E. Oren M. Genes Dev. 2001; 15: 1067-1077Crossref PubMed Scopus (527) Google Scholar), and sumoylation can also control MDM2 activity (29Buschmann T. Fuchs S.Y. Lee C.G. Pan Z.Q. Ronai Z. Cell. 2000; 101: 753-762Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Other mechanisms indirectly control MDM2 activities; HAUSP is a ubiquitin protease that can remove ubiquitin groups from p53 upon DNA damage (30Li M. Chen D. Shiloh A. Luo J. Nikolaev A.Y. Qin J. Gu W. Nature. 2002; 416: 648-653Crossref PubMed Scopus (784) Google Scholar), reversing MDM2 effects. Nucleolar compartmentalization of MDM2 by alternative reading frame (ARF) or the ribosomal protein L11 can separate MDM2 from p53, producing a p53-dependent response to stress (31Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nat. Cell Biol. 1999; 1: 20-26Crossref PubMed Scopus (795) Google Scholar, 32Weber J.D. Kuo M.L. Bothner B. DiGiammarino E.L. Kriwacki R.W. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 2000; 20: 2517-2528Crossref PubMed Scopus (244) Google Scholar, 33Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Cancer Cells. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Evidence also exists that ARF can regulate MDM2 without nucleolar sequestration (34Llanos S. Clark P.A. Rowe J. Peters G. Nat. Cell Biol. 2001; 3: 445-452Crossref PubMed Scopus (225) Google Scholar). MDM2 is an unstable protein capable of autoubiquitylation, which leads to its own destruction (13Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 35Honda R. Yasuda H. Oncogene. 2000; 19: 1473-1476Crossref PubMed Scopus (308) Google Scholar). Association of MDM2 with other proteins can stabilize MDM2. MDM2-TSG101 interaction appears to inhibit MDM2 destruction (36Li L. Liao J. Ruland J. Mak T.W. Cohen S.N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1619-1624Crossref PubMed Scopus (142) Google Scholar), resulting in decreased p53 levels (37Ruland J. Sirard C. Elia A. MacPherson D. Wakeham A. Li L. de la Pompa J.L. Cohen S.N. Mak T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1859-1864Crossref PubMed Scopus (134) Google Scholar). Although nucleolar accumulation inhibits MDM2, it also stabilizes MDM2 (33Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Cancer Cells. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Although this is unlikely to increase MDM2 activity, it demonstrates MDM2 levels can undergo dynamic alterations. Other E3 enzymes such as E6-AP, p300, and PIRH2 (p53-induced RING-H2) can also modify p53 (38Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1953) Google Scholar, 39Grossman S.R. Deato M.E. Brignone C. Chan H.M. Kung A.L. Tagami H. Nakatani Y. Livingston D.M. Science. 2003; 300: 342-344Crossref PubMed Scopus (382) Google Scholar, 40Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakem R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). The murine PIRH2 (mPIRH2) protein was initially cloned from mouse as ARNIP (androgen receptor N-terminal interacting protein) (41Beitel L.K. Elhaji Y.A. Lumbroso R. Wing S.S. Panet-Raymond V. Gottlieb B. Pinsky L. Trifiro M.A. J. Mol. Endocrinol. 2002; 29: 41-60Crossref PubMed Scopus (70) Google Scholar), but was then demonstrated to interact directly with and ubiquitylate p53 (40Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakem R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). mpirh2 is a p53-regulated gene; its transcript and protein levels increase in response to UV irradiation and cisplatin treatment (40Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakem R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). These findings demonstrate that mpirh2 has a similar relationship to p53 as mdm2.Several groups have reported that the histone acetylase TIP60 (Tat-interactive protein of 60 kDa) is involved in transcriptional regulation (42Kamine J. Elangovan B. Subramanian T. Coleman D. Chinnadurai G. Virology. 1996; 216: 357-366Crossref PubMed Scopus (242) Google Scholar, 43Kimura A. Horikoshi M. Genes Cells. 1998; 3: 789-800Crossref PubMed Scopus (167) Google Scholar, 44Yamamoto T. Horikoshi M. J. Biol. Chem. 1997; 272: 30595-30598Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). TIP60 can interact with and enhance human androgen receptor activity (45Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) through human androgen receptor acetylation (46Gaughan L. Logan I.R. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 2002; 277: 25904-25913Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 47Fu M. Rao M. Wang C. Sakamaki T. Wang J. Di Vizio D. Zhang X. Albanese C. Balk S. Chang C. Fan S. Rosen E. Palvimo J.J. Janne O.A. Muratoglu S. Avantaggiati M.L. Pestell R.G. Mol. Cell. Biol. 2003; 23: 8563-8575Crossref PubMed Scopus (219) Google Scholar). TIP60 expression is also altered in prostate cancer (48Halkidou K. Gnanapragasam V.J. Mehta P.B. Logan I.R. Brady M.E. Cook S. Leung H.Y. Neal D.E. Robson C.N. Oncogene. 2003; 22: 2466-2477Crossref PubMed Scopus (179) Google Scholar), which is initially dependent upon androgens for tumorigenesis (49Abate-Shen C. Shen M.M. Genes Dev. 2000; 14: 2410-2434Crossref PubMed Scopus (545) Google Scholar). Other studies have shown TIP60 to associate with alternative transcription factors such as Bcl-3 (50Dechend R. Hirano F. Lehmann K. Heissmeyer V. Ansieau S. Wulczyn F.G. Scheidereit C. Leutz A. Oncogene. 1999; 18: 3316-3323Crossref PubMed Scopus (263) Google Scholar), Myc (51Frank S.R. Parisi T. Taubert S. Fernandez P. Fuchs M. Chan H.M. Livingston D.M. Amati B. EMBO Rep. 2003; 4: 575-580Crossref PubMed Scopus (292) Google Scholar), TEL/ETV (52Nordentoft I. Jorgensen P. Biochem. J. 2003; 374: 165-173Crossref PubMed Scopus (41) Google Scholar), STAT3 (signal transducer and activator of transcription-3) (53Xiao H. Chung J. Kao H.Y. Yang Y.C. J. Biol. Chem. 2003; 278: 11197-11204Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and ING (54Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). There is also an established transcriptional role for TIP60 in amyloid precursor protein, interleukin-1β signaling, and subsequent NF-κB-mediated gene regulation (55Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1045) Google Scholar, 56Baek S.H. Ohgi K.A. Rose D.W. Koo E.H. Glass C.K. Rosenfeld M.G. Cell. 2002; 110: 55-67Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Significantly, TIP60 is part of a large multiprotein complex involved in DNA repair and apoptosis (54Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 57Ikura T. Ogryzko V.V. Grigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar). It is not known if this whole TIP60 complex is involved in its transcriptional activities (56Baek S.H. Ohgi K.A. Rose D.W. Koo E.H. Glass C.K. Rosenfeld M.G. Cell. 2002; 110: 55-67Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar), but TIP60, like its yeast homolog Esa1, seems important in DNA transactions (58Galarneau L. Nourani A. Boudreault A.A. Zhang Y. Heliot L. Allard S. Savard J. Lane W.S. Stillman D.J. Cote J. Mol. Cell. 2000; 5: 927-937Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 59Bird A.W. Yu D.Y. Pray-Grant M.G. Qiu Q. Harmon K.E. Megee P.C. Grant P.A. Smith M.M. Christman M.F. Nature. 2002; 419: 411-415Crossref PubMed Scopus (456) Google Scholar). Additionally, TIP60 was shown to associate with MDM2, resulting in ubiquitin-dependent proteolysis of TIP60 (60Legube G. Linares L.K. Lemercier C. Scheffner M. Khochbin S. Trouche D. EMBO J. 2002; 21: 1704-1712Crossref PubMed Scopus (126) Google Scholar).Here, we demonstrate that human PIRH2 (hPIRH2) is an unstable protein that is ubiquitylated and targeted for RING domain-dependent proteasomal destruction. We also demonstrate that hPIRH2 can interact with TIP60, which bears similarities to the MDM2-ARF/L11 associations; we show that TIP60 can stabilize hPIRH2 protein levels and produce hPIRH2 subcellular relocation and, in some cells, nucleolar compartmentalization. We propose that hPIRH2 stability is likely to be regulated by multiple post-translational mechanisms, some of which are described herein.EXPERIMENTAL PROCEDURESYeast Two-hybrid Screening—Yeast PJ69–4A (a gift from Philip James) was sequentially transformed with pAS2-1 (Clontech) containing full-length TIP60α cDNA and human brain cDNA in pACT-2 (Clontech). Prototrophs growing on synthetic dropout medium lacking adenine, leucine, and tryptophan were assayed for β-galactosidase activity with 2-nitrophenyl-β-d-galactopyranoside substrate (Sigma). Library plasmids from positive clones were isolated and verified by endonuclease digestion and then sequenced with pACT-2 primers (Clontech) and retransformed into PJ69–4A containing pAS2-1 or pAS2-1-TIP60 cDNA. Transformants were assayed for TIP60-specific interactions using 2-nitrophenyl-β-d-galactopyranoside and prototrophic growth on synthetic dropout medium.DNA Manipulation—Sequencing results compared with human expressed sequence tags using BLAST identified one clone containing a partial cDNA encoding hPIRH2. Full-length hPIRH2 cDNA from the IMAGE consortium (clone 4651778) was subsequently characterized in this study. Primers 5′-ggatccatggcggcgacggcccgg-3′ and 5′-gaatccttgctgatccagtgaaat-3′ were used to amplify full-length hPIRH2, and cDNA was cloned into pcDNA4.1 (Invitrogen) using BamHI and EcoRI to produce Myc-tagged hPIRH2 (hPIRH2-Myc).hPIRH2-Myc was subjected to in vitro mutagenesis using QuikChange (Stratagene). Conserved RING domain cysteines were altered to serines using oligonucleotides C145S/C148S (5′-cgacagaattctccaatatctttggaggacattc-3′ and 5′-gaatgtcctccaaagatattggagaattctgtcg-3′) and C164S (5′-gctcatgtcttgccatctggacatc-3′ and 5′-gatgtccagatggcaagacatgagc-3′). Primers 5′-ggatccgggcggcgacggccccg-3′ and 5′-ggggatccctcattgctgatccag-3′ were used for cloning wild-type and C145S/C148S hPIRH2 into pEGFP (Clontech) and pACT-2 using BamHI. 2Details of hPIRH2 deletion constructs are available upon request. Constructs were verified by DNA sequencing. pCMV5-driven expression of human FLAG-TIP60 and TIP60-RFP has been described (46Gaughan L. Logan I.R. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 2002; 277: 25904-25913Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 48Halkidou K. Gnanapragasam V.J. Mehta P.B. Logan I.R. Brady M.E. Cook S. Leung H.Y. Neal D.E. Robson C.N. Oncogene. 2003; 22: 2466-2477Crossref PubMed Scopus (179) Google Scholar). FLAG-TIP60 or FLAG-TIP60D (where "D" represents acetylase-dead) cDNA was subcloned into pcDNA4.1 using EcoRI to produce pcDNA4-FLAG-TIP60 or pcDNA4-FLAG-TIP60D, respectively.Cell Culture—PC3M, 293, LNCaP, and COS-7 cells maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (Sigma) and l-glutamine were transfected with Superfect (QIAGEN Inc.). FLAG-TIP60- or FLAG-TIP60D-expressing doxycycline-induced cells were produced by transfection of pcDNA4-FLAG-TIP60 or pcDNA4-FLAG-TIP60D and pcDNA6-TR into PC3M cells, followed by selection in blasticidin and Zeocin (Invitrogen). Individual clones were expanded and tested for TIP60 induction with 1 μg/ml doxycycline. MG132 and cycloheximide were from Calbiochem. GFP transfectants were grown on glass coverslips before fluorescence microscopy analysis.Immunoprecipitation and Antibodies—The antibodies used were as follows: anti-Myc antibody 9B11 (Cell Signaling), anti-ubiquitin antibody PD41 (Santa Cruz Biotechnology), anti-TIP60 antibody N-17 (Santa Cruz Biotechnology), anti-pentahistidine antibody (QIAGEN Inc.), anti-FLAG antibody M2 (Sigma), anti-GFP antibody (Santa Cruz Biotechnology), anti-nucleolin antibody 4E2 (Research Diagnostics Inc.), and Alexa 350-conjugated goat anti-mouse antibody (Molecular Probes, Inc.). For immunoprecipitation, 5 × 105 293 cells seeded on 90-mm dishes (Corning Inc.) were transfected with the indicated plasmids. 48 h later, cells were lysed in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride with protease inhibitors, and lysates were clarified by ultra-centrifugation at 15,000 rpm for 20 min and then precleared in 50% protein G-Sepharose (Amersham Biosciences) at 4 °C for 4 h. Samples were mixed with 2 μg of antibody and fresh protein G-Sepharose at 4 °C for 12 h. Immune complexes recovered by centrifugation were washed three times with 100 mm Tris (pH 7.4) and 150 mm NaCl and then resuspended in 40 μl of Laemmli buffer. Samples were resolved by SDS-PAGE and subjected to Western blotting as shown.RESULTSTIP60 Interacts with the RING Finger Protein hPIRH2— Yeast two-hybrid screening with a human brain cDNA library fused to the Gal4 activation domain identified a clone encoding amino acids 31–261 of the RING domain-containing hPIRH2 protein that interacted robustly with TIP60 fused to the Gal4 DNA-binding domain (Fig. 1A). The TIP60-hPIRH2 interaction is specific, as hPIRH2 did not interact with pLAM5′ (Fig. 1A), and TIP60 did not interact with another RING domain-containing protein (BRCA1) in yeast (Fig. 1C).To confirm TIP60-hPIRH2 association, co-immunoprecipitation of proteins produced in vitro and from transiently transfected 293 cells was performed. FLAG-TIP60 and full-length wild-type hPIRH2-Myc proteins were specifically co-immunoprecipitated from 293 cells with anti-TIP60 antibody N-17 as shown in Fig. 1B. Western blotting showed that immunoprecipitation did not recover large amounts of TIP60 protein, in agreement with reports of low TIP60 solubility (60Legube G. Linares L.K. Lemercier C. Scheffner M. Khochbin S. Trouche D. EMBO J. 2002; 21: 1704-1712Crossref PubMed Scopus (126) Google Scholar). Western blotting for the Myc epitope showed specific hPIRH2-Myc coimmunoprecipitation with FLAG-TIP60, demonstrating that the two proteins complex in 293 cells (Fig. 1B).hPIRH2 contains a RING finger motif, identical to that of mPIRH2, between residues 145 and 186. To investigate whether the hPIRH2 RING structure mediates the TIP60 interaction, two conserved key cysteine residues were mutated (Fig. 1D). The mutant hPIRH2 protein carrying mutations of Cys145 and Cys148 (C145S/C148S hPIRH2-Myc) also co-immunoprecipitated with TIP60 (Fig. 1B), suggesting that the hPIRH2 RING domain structure is dispensable for the TIP60-hPIRH2 interaction. 35S-Radiolabeled TIP60 and hPIRH2 proteins, produced in vitro using a coupled transcription/translation system, were also specifically co-immunoprecipitated with anti-His antibody directed against TIP60 (data not shown).TIP60-hPIRH2 interaction was analyzed further in yeast. First, full-length wild-type hPIRH2 was assayed for TIP60 interaction and demonstrated a similar interaction strength as the clone encoding amino acids 31–261 of hPIRH2 (∼60-fold over background levels) (Fig. 1C). Next, to delineate regions of hPIRH2 that interact with TIP60, hPIRH2 deletion constructs were assayed with TIP60 in yeast. A construct encoding amino acids 40–261 of hPIRH2 was capable of TIP60 interaction, although to a much lesser degree than full-length hPIRH2 (Fig. 1C). Additionally, hPIRH2 residues 80–261 interacted with TIP60 to the same degree as residues 40–261, whereas residues 94–261 failed to interact with TIP60 (Fig. 1C). This suggests that hPIRH2 amino acids 31–40 likely mediate the TIP60 interaction, but that amino acids 80–94 may also participate in TIP60 interaction. Overall, it is clear the hPIRH2 N terminus mediates TIP60 interaction(s).TIP60 interacted with full-length C145S/C148S hPIRH2 to the same degree as full-length wild-type hPIRH2 in yeast, but not with the N-terminal deletion construct encoding residues 120–261 containing the hPIRH2 RING domain (Fig. 1C). Together, the deletion construct and C145S/C148S mutation data show that the hPIRH2 RING domain is dispensable for TIP60-hPIRH2 interactions. The mPIRH2 RING domain is also dispensable for p53 interaction (40Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakem R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar), which is mediated by mPIRH2 residues 120–137 (40Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakem R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). TIP60 therefore does not bind to the same residues as p53. Pilot data indicated that the TIP60 atypical zinc finger contributed to the TIP60-hPIRH2 interaction and that histone acetylase-deficient TIP60 could also interact with hPIRH2. 3K. Halkidou, I. R. Logan, S. Cook, D. E. Neal, and C. N. Robson, unpublished data. hPIRH2 Has a Short Half-life and Is a Target for Proteasomal Destruction—The presence of a RING domain in several proteins has identified their role as E3 enzymes that covalently attach ubiquitin peptides to substrates (61Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). mPIRH2 is a bona fide RING domain-dependent E3 enzyme that can mediates p53 ubiquitylation (40Leng R.P. Lin Y. Ma W. Wu H. Lemmers B. Chung S. Parant J.M. Lozano G. Hakem R. Benchimol S. Cell. 2003; 112: 779-791Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). Several E3 enzymes can also autoubiquitylate in cells, targeting themselves for proteasomal destruction (13Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 61Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar). One such RING domain-containing E3 enzyme (MDM2) not only modifies itself, but also p53 and other proteins such as TIP60 by attachment of polyubiquitin chains (13Fang S. Jensen J.P. Ludwig R.L. Vousden K.H. Weissman A.M. J. Biol. Chem. 2000; 275: 8945-8951Abstract Full Text Full Text PDF PubMed Scopus (862) Google Scholar, 35Honda R. Yasuda H. Oncogene. 2000; 19: 1473-1476Crossref PubMed Scopus (308) Google Scholar, 60Legube G. Linares L.K. Lemercier C. Scheffner M. Khochbin S. Trouche D. EMBO J. 2002; 21: 1704-1712Crossref PubMed Scopus (126) Google Scholar). Proteins ubiquitylated (either in cis or in trans) may be expected to exhibit a relatively short half-life due to rapid turnover by the proteasome (7Pickart C.M. Trends Biochem. Sci. 2000; 25: 544-548Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) and accumulation in high molecular mass polyubiquitylated species upon proteasomal inhibition. Experiments to examine the possibility that hPIRH2 may regulate its own expression were undertaken. First, the half-life of hPIRH2 was examined. hPIRH2 expression was examined in COS-7 cells transiently transfected with either wild-type or C145S/C148S hPIRH2-Myc and then treated with cycloheximide, a potent inhibitor of protein translation in eukaryotes (62Lin H.K. Wang L. Hu Y.C. Altuwaijri S. Chang C. EMBO J. 2002; 21: 4037-4048Crossref PubMed Scopus (357) Google Scholar). Western analysis showed wild-type hPIRH2 exhibited a relatively short half-life, as hPIRH2 protein levels decreased within 3 h and more dramatically within 5 h (Fig. 2A, left panels). Densitometry analysis indicated that the hPIRH2 half-life (normalized to α-tubulin levels) was ∼3.5 h. The C145S/C148S hPIRH2 RING domain double mutant showed no reduction in protein levels with cycloheximide treatment (Fig. 2A, right panels), illustrating that it is a stable protein and suggesting that destruction of hPIRH2 is dependent upon an intact hPIRH2 RING domain. Interestingly, high molecular mass C145S/C148S hPIRH2 RING domain mutant species were also detected that were not present in wild-type hPIRH2-transfected cells. Similar results were obtained in other cell lines tested. 3K. Halkidou, I. R. Logan, S. Cook, D. E. Neal, and C. N. Robson, unpublished data.Fig. 2hPIRH2 is unstable and targeted for proteasomal degradation. A, COS-7 cells transfected with wild-type (wt) hPIRH2-Myc (left panels) or C145S/C148S (C145/8S) hPIRH2-Myc (right panels). After 48 h, cells were treated for the indicated times with 40 μg/ml cycloheximide. Cell lysates were Western-blotted as shown. The arrow indicates unmodified hPIRH2. B, transfection of wild-type hPIRH2-Myc as described for A. Cells were treated with 5 μm MG132 for the indicated times. Western blotting was performed as described for A. C, transfection of wild-type, C145S/C148S, or C164S hPI
Referência(s)