p14 Arf Promotes Small Ubiquitin-like Modifier Conjugation of Werners Helicase
2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês
10.1074/jbc.m405414200
ISSN1083-351X
AutoresYvonne L. Woods, Dimitris P. Xirodimas, Alan R. Prescott, Alison Sparks, David P. Lane, Mark K. Saville,
Tópico(s)Enzyme Structure and Function
ResumoHere we demonstrate a novel p53-independent interaction between the nucleolar tumor suppressors, p14 Arf and Werners helicase (WRN). Binding of p14 Arf to WRN is multivalent and resembles the binding of p14 Arf to Mdm2. Residues 2–14 and 82–101 of p14 Arf and residues in the central region and C terminus of WRN have particular importance for binding. p14 Arf promotes small ubiquitin-like modifier (SUMO) modification of WRN in a synergistic manner with the SUMO-conjugating enzyme, UBCH9. p14 Arf causes redistribution of WRN within the nucleus, and this effect is reversed by expression of a SUMO-specific protease, thus implicating the SUMO conjugation pathway in WRN re-localization. We establish that the ability to promote SUMO conjugation is a general property of the p14 Arf tumor suppressor. Here we demonstrate a novel p53-independent interaction between the nucleolar tumor suppressors, p14 Arf and Werners helicase (WRN). Binding of p14 Arf to WRN is multivalent and resembles the binding of p14 Arf to Mdm2. Residues 2–14 and 82–101 of p14 Arf and residues in the central region and C terminus of WRN have particular importance for binding. p14 Arf promotes small ubiquitin-like modifier (SUMO) modification of WRN in a synergistic manner with the SUMO-conjugating enzyme, UBCH9. p14 Arf causes redistribution of WRN within the nucleus, and this effect is reversed by expression of a SUMO-specific protease, thus implicating the SUMO conjugation pathway in WRN re-localization. We establish that the ability to promote SUMO conjugation is a general property of the p14 Arf tumor suppressor. The INK4a-ARF locus encodes two different cell cycle inhibitors p16 INK4a and p14 Arf by alternative splicing and the use of alternative reading frames (reviewed in Ref. 1Sherr C.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 731-737Crossref PubMed Scopus (817) Google Scholar). p14 Arf (p19 Arf in mice) is an arginine-rich protein and is localized to the nucleolus. p19 Arf is a tumor suppressor, and p19 Arf null mice are highly tumor prone. p19 Arf acts upstream of p53 to cause growth arrest at the G1 stage of the cell cycle (2Kamijo T. Zindy F. Roussel M.F. Quelle D.E. Downing J.R. Ashmun R.A. Grosveld G. Sherr C.J. Cell. 1997; 91: 649-659Abstract Full Text Full Text PDF PubMed Scopus (1373) Google Scholar).Early observations indicated that Arf mediates a p53-dependent checkpoint that responds to oncogenic, hyperproliferative signals. In mice, p19 Arf expression is induced by oncogenic signals such as Myc, E2F1, and oncogenic Ras, and human p14 Arf expression is also induced by high levels of E2F (3Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (810) Google Scholar). Recent work has argued against a critical role for p19 Arf in regulating Mdm2 function under physiological conditions and has suggested that the situations in which Arf regulates the p53/Mdm2 pathway might be restricted to those in which cellular stresses activate p53 during the tumorigenic process (4O'Leary K.A. Mendrysa S.M. Vaccaro A. Perry M.E. Mol. Cell. Biol. 2004; 24: 186-191Crossref PubMed Scopus (26) Google Scholar).At the molecular level, p14 Arf overcomes the ability of the ubiquitin E3 1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; WRN, Werners helicase; IP, immunoprecipitation; NLS, nuclear localization sequence; NTA, nitrilotriacetic acid; FCS, fetal calf serum; SUMO, small ubiquitin-like modifier; HA, hemagglutinin; IVT, in vitro translation; siRNA, small interfering RNA.1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; WRN, Werners helicase; IP, immunoprecipitation; NLS, nuclear localization sequence; NTA, nitrilotriacetic acid; FCS, fetal calf serum; SUMO, small ubiquitin-like modifier; HA, hemagglutinin; IVT, in vitro translation; siRNA, small interfering RNA. ligase, Mdm2, to repress p53 (5Pomerantz 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, 6Zhang Y. Xiong Y. Yarbrough W.G. Cell. 1998; 92: 725-734Abstract Full Text Full Text PDF PubMed Scopus (1391) Google Scholar). It has been proposed that the interaction of p14 Arf with Mdm2 inhibits its E3 ligase activity toward p53, resulting in p53 accumulation and induction of the p53 response (7Honda R. Yasuda H. EMBO J. 1999; 18: 22-27Crossref PubMed Scopus (612) Google Scholar, 8Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar, 9Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar). It has also been suggested that p14 Arf sequesters Mdm2 in the nucleolus causing physical segregation of Mdm2 from p53 (10Weber 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). However, under some circumstances, Arf can stabilize endogenous Mdm2 and p53 without quantitative relocation of either protein (11Llanos S. Clark P.A. Rowe J. Peters G. Nat. Cell Biol. 2001; 3: 445-452Crossref PubMed Scopus (225) Google Scholar). Another model proposes that p53 is targeted for degradation via a nucleolar route of export to the cytoplasm (12Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6937-6941Crossref PubMed Scopus (495) Google Scholar) and that p14 Arf and many other stimuli induce p53 activation by causing nucleolar disruption (13Rubbi C.P. Milner J. EMBO J. 2003; 22: 6068-6077Crossref PubMed Scopus (638) Google Scholar).Additional p53-independent functions of Arf have been reported. Knockout mice lacking p19 Arf, p53, and Mdm2 develop a wider spectrum of tumors, with higher frequency, than those observed in animals lacking both p53 and Mdm2. In addition, p19 Arf halts proliferation of mouse embryo fibroblasts lacking both Mdm2 and p53 at the G1/S-phase boundary of the cell cycle, implying that at least one p19 Arf target, when regulated in an entirely p53- and Mdm2-independent manner, can be rate-limiting for the G1/S transition (14Weber J.D. Jeffers J.R. Rehg J.E. Randle D.H. Lozano G. Roussel M.F. Sherr C.J. Zambetti G.P. Genes Dev. 2000; 14: 2358-2365Crossref PubMed Scopus (332) Google Scholar). Overexpression of p14 Arf reduces the rate of DNA synthesis, resulting in accumulation of an S-phase cell population, independently of p53 (15Yarbrough W.G. Bessho M. Zanation A. Bisi J.E. Xiong Y. Cancer Res. 2002; 62: 1171-1177PubMed Google Scholar). p19 Arf induces expression of anti-proliferative genes that can inhibit growth of cells lacking both p53 and Mdm2 (16Kuo M.L. Duncavage E.J. Mathew R. den Besten W. Pei D. Naeve D. Yamamoto T. Cheng C. Sherr C.J. Roussel M.F. Cancer Res. 2003; 63: 1046-1053PubMed Google Scholar). Strikingly, p14 Arf has also been shown recently to promote conjugation of a small ubiquitin-like modifier (SUMO) to Mdm2 and p53 (17Xirodimas D.P. Chisholm J. Desterro J.M. Lane D.P. Hay R.T. FEBS Lett. 2002; 528: 207-211Crossref PubMed Scopus (84) Google Scholar, 18Chen L. Chen J. Oncogene. 2003; 22: 5348-5357Crossref PubMed Scopus (104) Google Scholar).The human Werners helicase (WRN) gene encodes a 160-kDa protein that is a member of the RecQ family of DNA helicases, also including BLM and RecQ4. WRN is unique within this family, in that it also has an N-terminal 3′–5′-exonuclease activity. In man, germ line mutations in WRN give rise to a rare autosomal recessive disorder, termed Werners syndrome. Werners syndrome is a disease of premature aging that is associated with an elevated risk of cancer (reviewed in Ref. 19Chen L. Oshima J. J. Biomed. Biotechnol. 2002; 2: 46-54Crossref PubMed Scopus (51) Google Scholar). Fibroblast cells derived from Werners syndrome patients exhibit a prolonged S-phase and display genomic instability (20Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell Res. 1992; 202: 267-273Crossref PubMed Scopus (186) Google Scholar, 21Fukuchi K. Martin G.M. Monnat Jr., R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar). Numerous studies have suggested potential functions of WRN based upon its other binding partners, including roles in DNA replication and restoration of replication at sites of fork blockade, rRNA transcription, homologous recombination, repair of breaks in double-stranded DNA, and telomere maintenance (reviewed in Ref. 22Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Crossref PubMed Scopus (310) Google Scholar).Here we report a novel interaction between p14 Arf and WRN. p14 Arf causes redistribution of WRN within the nucleus and promotes SUMOylation of WRN, in a synergistic manner with the SUMO-conjugating enzyme UBCH9.EXPERIMENTAL PROCEDURESChemicals and Reagents—[35S]Methionine and Amplify reagent were purchased from Amersham Biosciences. Ni2+-NTA-agarose was from Qiagen. Streptavidin-agarose was from Sigma. NuPAGE pre-cast gradient gels were from Invitrogen.Antibodies—WRN protein was detected using a monoclonal antibody to the WRN C terminus (Transduction Laboratories). FLAG-tagged WRN was detected using the monoclonal anti-FLAG antibody clone M2 (Sigma). p14 Arf was detected using a polyclonal rabbit antiserum, a kind gift from Dr. Karen Vousden (Beatson Institute for Cancer Research, Glasgow, Scotland, UK), or by a mouse monoclonal antibody to the p14 Arf protein, Clone 14PO3 (NeoMarkers). Mdm2 was detected using the 4B2 monoclonal antibody (23Chen J. Marechal V. Levine A.J. Mol. Cell. Biol. 1993; 13: 4107-4114Crossref PubMed Scopus (620) Google Scholar). UBCH9 was detected by a sheep polyclonal antibody, generously provided by Professor Ronald T. Hay (University of St. Andrews, Scotland, UK). SUMO-1 (GMP-1) was detected by a mouse monoclonal antibody, clone 21C7 (Zymed Laboratories Inc.), and ubiquitin was detected by a mouse monoclonal antibody, ab7254 (abcam). Immunofluorescence staining was performed with fluorescein isothiocyanate-conjugated anti-mouse IgG (The Jackson Laboratories). Mouse and rabbit immunoglobulins used in control experiments were from Sigma.Expression Constructs—FLAG-WRN expression constructs were kindly provided by Dr. Moshe Oren (Weizmann Institute, Israel) and are described in Ref. 24Blander G. Kipnis J. Leal J.F. Yu C.E. Schellenberg G.D. Oren M. J. Biol. Chem. 1999; 274: 29463-29469Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar. HA-WRN constructs were prepared by removing the sequence encoding the FLAG tag by restriction digestion and replacing it with a sequence encoding the HA tag. WRN point mutants were constructed using the QuickChange XL kit, according to the manufacturer's instructions (Stratagene). UBCH9 and SSP3 expression constructs were generously provided by Professor Ronald T. Hay (University of St. Andrews, Scotland, UK). p14 Arf, His6-SUMO-1, and His6-ubiquitin expression constructs have been described previously (9Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar, 17Xirodimas D.P. Chisholm J. Desterro J.M. Lane D.P. Hay R.T. FEBS Lett. 2002; 528: 207-211Crossref PubMed Scopus (84) Google Scholar). pSUPER vectors for expression of p14 Arf siRNA were kindly provided by Dr. Sonia Laín (Ninewells Hospital, Dundee, Scotland, UK).Cell Culture—H1299 and U2-OS cell lines were obtained from the ATCC. H1299 cells were cultured in RPMI medium and U2-OS cells in Dulbecco's modified Eagle's medium, each supplemented with 10% FCS and containing 50 μg/ml gentamycin. Cells were grown at 37 °C, 5% CO2 in a humidified atmosphere.Transient Transfections—1 × 106 H1299 cells were seeded onto 10-cm tissue culture plates and transfected 24 h later using the calcium phosphate method. Typically, 5 μg of expression plasmids for WRN, p14 Arf, UBCH9, SSP3, and His6-SUMO-1 were transfected per 10-cm plate, unless otherwise stated. DNA amount was normalized with pcDNA3 vector. Two hours prior to transfection, the cells were washed into Dulbecco's modified Eagle's medium supplemented with 10% FCS and 50 μg/ml gentamycin. 18 h post-transfection, the cells were washed twice with phosphate-buffered saline and transferred into RPMI containing10% FCS and 50 μg/ml gentamycin, prior to harvesting at 36 h post-transfection.Immunoprecipitations—For immunoprecipitation (IP), or peptide pull-down, cells were lysed in IP buffer: 50 mm Tris-HCl, pH 7.5, 10% (v/v) glycerol, 0.1% Nonidet P-40, 150 mm NaCl, supplemented with complete protease inhibitor mixture (Roche Applied Science) and passed several times through a narrow gauge needle. Immunoprecipitating antibody was covalently coupled to protein G-Sepharose using the dimethyl pimelimidate method. 2 μg of antibody was coupled to 20 μl of packed volume protein G-Sepharose per IP reaction. IP reactions typically used 1 mg of cell extract and were carried out for 1 h at 4 °C. IPs were washed four times in 1 ml of IP buffer. Immunoprecipitated proteins were eluted end-over-end for 30 min at ambient temperature in 50 μl of Laemmli sample buffer and boiled with 5% (v/v) β-mercaptoethanol, prior to SDS-PAGE.Peptide Binding Studies—A series of overlapping biotinylated peptides have been described previously (8Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar). Cells were harvested as for the IP experiments. The indicated amount of peptide was coupled to 20 μl of packed volume of streptavidin-agarose in phosphate-buffered saline for 1 h at 4 °C and then washed into IP buffer prior to incubation with cell extract for 1 h at 4 °C. Peptide pull-downs were then washed four times in 1 ml of IP buffer. Interacting proteins were eluted end-over-end for 30 min at ambient temperature in 50 μl of Laemmli sample buffer and boiled with 5% (v/v) β-mercaptoethanol prior to SDS-PAGE.Purification of His6-SUMO-1 Conjugates—H1299 cells were harvested 36 h post-transfection in 1 ml of 6 m guanidinium HCl, 0.1 m Na2HPO4/NaH2PO4, 0.01 m Tris-HCl, pH 8.0, plus 5 mm imidazole and 10 mm β-mercaptoethanol per 10-cm plate. After passing through a narrow gauge needle to reduce viscosity, the lysates were mixed with 50 μl of Ni2+-NTA-agarose beads prewashed with lysis buffer and incubated for 4 h at room temperature. The beads were successively washed with the following: 6 m guanidinium HCl, 0.1 m Na2HPO4/NaH2PO4, 0.01 m Tris-HCl, pH 8.0, plus 10 mm β-mercaptoethanol; 8 m urea, 0.1 m Na2HPO4/NaH2PO4, 0.01 m Tris-HCl, pH 8.0; 10 mm β-mercaptoethanol; 8 m urea, 0.1 m Na2HPO4/NaH2PO4, 0.01 m Tris-HCl, pH 6.3, 10 mm β-mercaptoethanol (buffer A) plus 0.2% Triton X-100; buffer A and then buffer A plus 0.1% Triton X-100. After the last wash with buffer A, the beads were eluted with 200 mm imidazole in 5% SDS, 0.15 m Tris-HCl, pH 6.7, 30% glycerol, 0.72 m β-mercaptoethanol, prior to SDS-PAGE.Immunofluorescence—H1299 cells were seeded onto 4-well NUNC permanox slides and transfected 24 h later by the calcium phosphate method. 36 h post-transfection, the cells were fixed with ice-cold 50:50 (v/v) methanol/acetone. Cell immunofluorescence was visualized by confocal microscopy.In Vitro Translation (IVT)—In vitro translation reactions were carried out using the TnT quick-coupled transcription/translation system, according to the manufacturer's instructions (Promega). 2 μlof[35S]methionine was incubated with 1 μg of the indicated plasmid cDNA in a 40-μl reaction for 3 h at 30 °C.Mass Spectrometry—Mass spectrometry analysis was performed by the Fingerprints proteomics unit, Wellcome Trust Biocenter, Dundee, Scotland, UK. Tryptic peptides were analyzed on a Perspective Biosystems Elite STR matrix-assisted laser desorption time of flight-mass spectrometer (Framingham, MA), with saturated α-cyanocinnamic acid as the matrix. The mass spectrum was acquired in the positive reflector mode and was internally mass calibrated. The tryptic peptide ions obtained were scanned against the Swiss-Prot and Genpep data bases using the MASCOT program.RESULTSp14 Arf Physically Associates with WRN—Residues 1–14 are the most highly conserved in Arf proteins from different mammalian species. This region also contributes strongly to the high affinity interaction between p14 Arf and Mdm2, although other regions of p14 Arf are also involved (8Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar, 25Clark P.A. Llanos S. Peters G. Oncogene. 2002; 21: 4498-4507Crossref PubMed Scopus (35) Google Scholar). A green fluorescent protein fusion peptide encompassing the first 20 residues of p14 Arf can activate p53 when introduced into cells by transfection (8Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar). p19 Arf mutants lacking residues 2–14 are deficient in arresting the proliferation of cultured primary mouse embryo fibroblasts or NIH-3T3 cells (14Weber J.D. Jeffers J.R. Rehg J.E. Randle D.H. Lozano G. Roussel M.F. Sherr C.J. Zambetti G.P. Genes Dev. 2000; 14: 2358-2365Crossref PubMed Scopus (332) Google Scholar), suggesting that this region is critically important in Arf function.We therefore attempted to identify novel p14 Arf-interacting proteins in a peptide binding approach using a biotinylated peptide encompassing the first 20 residues of p14 Arf. As controls, we employed a high affinity, biotinylated, Mdm2-binding peptide (12/1-WT), which was isolated during a screen of phage display libraries for ligands that would interfere with the Mdm2-p53 interaction, and a non-Mdm2-binding mutant peptide in which the key contact residues were mutated to alanine (12/1-Ala) (26Bottger A. Bottger V. Sparks A. Liu W.L. Howard S.F. Lane D.P. Curr. Biol. 1997; 7: 860-869Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). The peptides were coupled to streptavidin-agarose beads and incubated with H1299 (p53 null) cell extract. We observed a high molecular weight protein band, which bound specifically to the p14 Arf-derived peptide and not to 12/1-WT or to 12/1-Ala (data not shown). The band was excised and digested with trypsin, and mass spectrometry analysis revealed peptide masses matching those derived from WRN with 14% protein coverage. The presence of WRN protein in the p14 Arf peptide binding experiment was confirmed by immunoblotting with a monoclonal antibody specific for the WRN C terminus (Fig. 1A). Both p14 Arf-(1–20) and 12/1-WT bound endogenous Mdm2 from the H1299 cell extract, whereas only the p14 Arf-derived peptide captured endogenous WRN (Fig. 1A), thus indicating that p14 Arf can interact with WRN independently from Mdm2.To establish the interaction of endogenous p14 Arf and WRN proteins, reciprocal immunoprecipitation (IP) reactions were carried out in H1299 cells. Endogenous WRN co-immunoprecipitated with anti-p14 Arf antibody but not with control mouse IgG (Fig. 1B). In the reciprocal experiment, endogenous p14 Arf co-immunoprecipitated with anti-WRN antibody but not with control mouse IgG (Fig. 1B). Therefore, the p14 Arf-WRN complex occurs in vivo.Physical Mapping of the p14 Arf/WRN Interaction—In order to define the regions of WRN involved in p14 Arf binding in vivo, FLAG-tagged WRN truncation mutants were expressed with p14 Arf in H1299 cells. The FLAG-tagged WRN truncation mutants have been described previously and encode N-terminal, C-terminal, and central regions of the WRN polypeptide (24Blander G. Kipnis J. Leal J.F. Yu C.E. Schellenberg G.D. Oren M. J. Biol. Chem. 1999; 274: 29463-29469Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The full-length, central region, and C terminus of WRN were co-immunoprecipitated with p14 Arf rabbit serum but not with control rabbit serum (Fig. 2A). In addition, the level of WRN present in the IPs with anti-p14 Arf antibody was increased by co-transfection of p14 Arf (Fig. 2B). Therefore, the co-immunoprecipitation was specific and not because of direct recognition of WRN by the anti-p14 Arf antibody. In each case, the complex of WRN with endogenous p14 Arf was also detected when longer immunoblot exposures were examined (data not shown). Co-transfection of p14 Arf resulted in the increased expression of each of the truncation mutants of WRN but not of the full-length protein (Fig. 2C). p14 Arf also increases levels of full-length Mdm2 (17Xirodimas D.P. Chisholm J. Desterro J.M. Lane D.P. Hay R.T. FEBS Lett. 2002; 528: 207-211Crossref PubMed Scopus (84) Google Scholar). The reason for this striking effect of p14 Arf is currently unknown, and further work to determine its mechanism is in progress.Fig. 2Co-immunoprecipitation of p14 Arf and WRN truncation mutants.A, H1299 cells were transfected with full-length WRN (FL), the central region of WRN (MID), or the N and C termini of WRN (N and C) and p14 Arf. IP reactions were performed with anti-p14 Arf rabbit serum or a rabbit serum IgG control, as described under "Experimental Procedures." Aliquots of IP eluates were electrophoresed on 4–12% gradient gels and immunoblotted with anti-FLAG antibody. B, H1299 cells were transfected with full-length WRN (FL), the central region of WRN (MID), or the N and C termini of WRN (N and C) with or without p14 Arf. IP reactions were performed with anti-p14 Arf rabbit serum and analyzed, as for A. C, aliquots of IP inputs were electrophoresed and immunoblotted, as for A.View Large Image Figure ViewerDownload (PPT)To delineate the regions of p14 Arf involved in interaction with WRN, we employed a series of 20-mer overlapping biotinylated peptides encompassing the entire polypeptide sequence of p14 Arf (8Midgley C.A. Desterro J.M. Saville M.K. Howard S. Sparks A. Hay R.T. Lane D.P. Oncogene. 2000; 19: 2312-2323Crossref PubMed Scopus (229) Google Scholar). Peptides were coupled to streptavidin-agarose and incubated with H1299 extract. Bound proteins were eluted in sample buffer, resolved by SDS-PAGE, and visualized by immunoblotting. Endogenous WRN was predominantly captured by p14 Arf-derived peptides encompassing regions 1–60 and 81–100 of p14 Arf (Fig. 3, upper panels). This pattern was similar to that observed with endogenous Mdm2 (Fig. 3, lower panels), and the same results were obtained with U2-OS osteosarcoma (p14 Arf null) cells (data not shown).Fig. 3Physical mapping of the p14 Arf/WRN interaction. Peptide binding studies were performed as described under "Experimental Procedures." Aliquots of the inputs and column eluates were electrophoresed on 4–12% gradient gels and immunoblotted with either anti-WRN antibody or the 4B2 antibody.View Large Image Figure ViewerDownload (PPT)To characterize further the multivalent nature of the p14 Arf/WRN interaction, each of the four major WRN-binding peptides from p14 Arf were incubated with IVT 35S-labeled full-length WRN or the N-terminal, central, or C-terminal truncated proteins. Residues 1–20, 11–30, and 1–60 of p14 Arf interacted most strongly in vitro with the central region of WRN and the N terminus (Fig. 4A). These constructs express overlapping regions of WRN (residues 326–365), possibly implicating these WRN residues in the p14 Arf interaction. The central and N-terminal regions of WRN did not interact as strongly with p14 Arf in the IP experiments from cell extracts (Fig. 2). However, these WRN regions completely lack the C-terminal nuclear localization sequence (NLS) (27Matsumoto T. Imamura O. Goto M. Furuichi Y. Int. J. Mol. Med. 1998; 1: 71-76PubMed Google Scholar), and so the observed differences between the in vitro and IP experiments probably reflect a lack of nuclear localization of the N-terminal and central fragments in vivo. Strikingly, residues 81–100 of p14 Arf preferentially bound to the C-terminal WRN (Fig. 4A), suggesting that a distinct p14 Arf-binding site, which interacts with p14 Arf residues 82–101, lies in the WRN C terminus. These data indicate that there are multiple p14 Arf-binding sites in WRN and multiple WRN-binding sites in p14 Arf. This situation resembles the multivalent nature of the p14 Arf/Mdm2 interaction (25Clark P.A. Llanos S. Peters G. Oncogene. 2002; 21: 4498-4507Crossref PubMed Scopus (35) Google Scholar).Fig. 4Multivalent nature of the p14 Arf/WRN interaction.A, 35S-labeled full-length WRN (FL), the central region of WRN (MID), or the N and C termini of WRN (N and C) were prepared by IVT, as described under "Experimental Procedures." The labeled WRN fragments were incubated with p14 Arf peptides encompassing regions 1–20, 11–30, 41–60, and 81–100 coupled to streptavidin-agarose, as described under "Experimental Procedures." Column eluates were electrophoresed on 4–12% gradient gels, incubated in Amplify for 30 min, fixed, dried, and subjected to autoradiography. B, H1299 cells were transfected with WRN and the indicated p14 Arf deletion mutant. The p14 Arf Δ2–14 mutant can form stable oligomers, which survive denaturing electrophoresis (54Menendez S. Khan Z. Coomber D.W. Lane D.P. Higgins M. Koufali M.M. Lain S. J. Biol. Chem. 2003; 278: 18720-18729Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). 36 h post-transfection the cells were lysed, and IP reactions were carried out as described under "Experimental Procedures," employing anti-WRN monoclonal antibody. Aliquots of the inputs and IP reactions were electrophoresed on 4–12% gradient gels and immunoblotted with anti-p14 Arf rabbit serum, or anti-WRN.View Large Image Figure ViewerDownload (PPT)To confirm the regions of p14 Arf required to interact with WRN in vivo, previously described p14 Arf truncation mutants were employed (9Xirodimas D. Saville M.K. Edling C. Lane D.P. Lain S. Oncogene. 2001; 20: 4972-4983Crossref PubMed Scopus (155) Google Scholar). The p14 Arf Δ2–14, Δ82–101, and Δ2–14/ Δ82–101 truncation mutants were expressed in H1299 cells together with WRN. Full-length p14 Arf and the Δ2–14 and Δ82–101 mutants co-immunoprecipitated with WRN; however, the Δ2–14/82–101 mutant was greatly impaired in its ability to co-immunoprecipitate with WRN (Fig. 4B), indicating that removal of both the 2–14 and 82–101 regions of p14 Arf are required to ablate interaction with WRN in vivo.p14 Arf Promotes WRN Conjugation to SUMO-1—Recently, it was demonstrated that p14 Arf could promote modification of Mdm2 and p53 with the ubiquitin-like protein SUMO-1 (17Xirodimas D.P. Chisholm J. Desterro J.M. Lane D.P. Hay R.T. FEBS Lett. 2002; 528: 207-211Crossref PubMed Scopus (84) Google Scholar, 18Chen L. Chen J. Oncogene. 2003; 22: 5348-5357Crossref PubMed Scopus (104) Google Scholar). Murine WRN has been reported to interact with the SUMO-conjugating enzyme UBCH9 by yeast two-hybrid analysis (28Kawabe Y. Seki M. Seki T. Wang W.S. Imamura O. Furuichi Y. Saitoh H. Enomoto T. J. Biol. Chem. 2000; 275: 20963-20966Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We therefore compared the abilities of p14 Arf and UBCH9 to promote SUMO-1 conjugation of WRN. H1299 cells were transfected with expression constructs for FLAG-WRN, p14 Arf or UBCH9, and His6-tagged SUMO-1. Cells were harvested in a urea buffer, and a fraction was used to monitor the expression of WRN by immunoblotting. The remaining lysate was incubated with Ni2+-NTA-agarose beads to purify His6-SUMO-1-conjugated proteins, as described under "Experimental Procedures." Bound proteins were eluted from the Ni2+-NTA-agarose beads and analyzed by immunoblotting with an anti-WRN C-terminal antibody. Some unmodified, full-length WRN itself binds to Ni2+-NTA-agarose (Fig. 5A, indicated with *).Fig. 5p14 Arf promotes WRN conjugation to SUMO-1.A, H1299 cells were transfected with the indicated combination of plasmids expressing full-length WRN (FL), His6-SUMO-1 (S), p14 Arf, or UBCH9. Cells were lysed and SUMO assays performed as described under "Experimental Procedures." Aliquots of the inputs and Ni2+-NTA column eluates were electrophoresed on 3–8% or 4–12% gradient gels and immunoblotted with anti-WRN antibody, or anti-UBCH9 antibody (lower panel), respectively. HA-tagged UBCH9 migrates above the endogenous protein upon SDS-PAGE, due to the additional mass contributed by the tag. B, H1299 cells were transfected with the indicated combination of plasmids expressing the C terminus of WRN (C), p14 Arf, His6-SUMO-1 (S), or UBCH9. Lysates were prepared and analyzed as for A. Ni2+-Ag, Ni2+-agarose.View Large Image Figure ViewerDownload (PPT)Higher molecular weight species of WRN were enriched in the Ni2+-NTA-agarose purified material compared with the input, and this enrichment was entirely dependent upon cotransfection with His6-SUMO-1 (Fig. 5A). These high molecular weight species therefore represent SUMO-modified WRN. It is possible that the major band observed contains multiple modified species of WRN that are difficult to fully resolve due to the large molecular weight of SUMO-modified forms of WRN (∼180 kDa). Following co-transfection of p14 Arf and His6-SUMO-1, the single major band of modified WRN was significantly enhanced, and a ladder of multiple higher molecular weight bands was also observed (Fig. 5A). SUMO-1 cannot multimerize; therefore, the laddering pattern might represent SUMO modification of WRN at multiple single sites. Overexpression of HA-tagged UBCH9 also promoted His6
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