Interaction of Epstein-Barr Virus Latent Membrane Protein 1 with SCFHOS/β-TrCP E3 Ubiquitin Ligase Regulates Extent of NF-κB Activation
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m307962200
ISSN1083-351X
AutoresWeigang Tang, Oleg Pavlish, Vladimir S. Spiegelman, Andrey A. Parkhitko, Serge Y. Fuchs,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe Epstein-Barr virus latent membrane protein 1 (LMP1) is pivotal in the transforming activity of this virus. We found that the common LMP1-95-8 variant interacts with Homologue of Slimb (HOS), a receptor for the SCFHOS/βTrCP ubiquitin-protein isopeptide ligase (E3) via one canonical and one cryptic HOS recognition site. These sites are mutated or deleted in the tumor-derived LMP1-Cao variant, which did not bind to HOS. Mutations within these sites on LMP1-95-8 abrogated HOS binding and increased transforming activity of LMP1. HOS did not regulate stability of LMP1-95-8 unless it was mutated to bear additional lysine residues near the cryptic motif. LMP1 proteins that could not bind to HOS exhibited an increased ability to induce IκB degradation and NF-κB-mediated transcription without further increase in activation of IκB kinases. Expression of LMP1-95-8 reduced the levels of endogenous HOS available to interact with phosphorylated IκBα. Degradation of IκBα and dose dependence of NF-κB activation by LMP1-95-8 were promoted by co-expression of HOS. Our data suggest that LMP1-95-8 is a pseudo-substrate of SCFHOS/βTrCP E3 ubiquitin ligase and that interaction between LMP1 and HOS restricts the extent of LMP1-induced NF-κB signaling. We discuss the potential role of this mechanism in transforming and cytostatic effects of LMP1 variants in cells and Epstein-Barr virus-associated tumors. The Epstein-Barr virus latent membrane protein 1 (LMP1) is pivotal in the transforming activity of this virus. We found that the common LMP1-95-8 variant interacts with Homologue of Slimb (HOS), a receptor for the SCFHOS/βTrCP ubiquitin-protein isopeptide ligase (E3) via one canonical and one cryptic HOS recognition site. These sites are mutated or deleted in the tumor-derived LMP1-Cao variant, which did not bind to HOS. Mutations within these sites on LMP1-95-8 abrogated HOS binding and increased transforming activity of LMP1. HOS did not regulate stability of LMP1-95-8 unless it was mutated to bear additional lysine residues near the cryptic motif. LMP1 proteins that could not bind to HOS exhibited an increased ability to induce IκB degradation and NF-κB-mediated transcription without further increase in activation of IκB kinases. Expression of LMP1-95-8 reduced the levels of endogenous HOS available to interact with phosphorylated IκBα. Degradation of IκBα and dose dependence of NF-κB activation by LMP1-95-8 were promoted by co-expression of HOS. Our data suggest that LMP1-95-8 is a pseudo-substrate of SCFHOS/βTrCP E3 ubiquitin ligase and that interaction between LMP1 and HOS restricts the extent of LMP1-induced NF-κB signaling. We discuss the potential role of this mechanism in transforming and cytostatic effects of LMP1 variants in cells and Epstein-Barr virus-associated tumors. The latent membrane protein 1 (LMP1) 1The abbreviations used are: LMP1latent membrane protein 1EBVEpstein-Barr virusLMP1-95-8B95-8 prototypeLMP1-CaoChinese isolate of LMP1NF-κBnuclear factor κBIκBinhibitor of NF-κBβ-TrCPβ-transducin repeat-containing proteinHOShomologue of SlimbSCFSkp1-Cullin1 1-F-box protein complexIKKIκB kinaseHAhemagglutininE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligase. is a transmembrane signaling protein encoded by the BNLF1 gene of Epstein-Barr virus (EBV). LMP1 plays a pivotal role in immortalization and proliferation of human B-lymphocytes infected with EBV, which is associated with a wide spectrum of human malignancies (reviewed in Ref. 1.Rickinson A. Kieff E. Fields B.N. Knipe D.M. Howley P.M. Virology. Raven Press, Ltd., New York1996: 2397-2446Google Scholar). LMP1 resembles a viral oncogene by virtue of its capability to transform rodent cells in vitro and to induce tumorigenesis in transgenic mice (2.Wang D. Liebowitz D. Kieff E. 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An increased capacity of some natural LMP1 isolates from tumors to induce NF-κB activity is thought to be linked to their higher efficacy in transforming rodent cells in vitro compared with a canonical B95-8 variant of LMP1 (LMP1-95-8) (24.Huen D.S. Henderson S.A. Croom-Carter D. Rowe M. Oncogene. 1995; 10: 549-560PubMed Google Scholar, 25.Hu L.F. Chen F. Zheng X. Ernberg I. Cao S.L. Christensson B. Klein G. Winberg G. Oncogene. 1993; 8: 1575-1583PubMed Google Scholar). These highly transforming LMP1 proteins (including LMP1-Cao, 1510, and C15) bear numerous point mutations and a 30-bp deletion in the C-terminal domain (26.Hu L.F. Zabarovsky E.R. Chen F. Cao S.L. Ernberg I. Klein G. Winberg G. J. Gen. Virol. 1991; 72: 2399-2409Crossref PubMed Scopus (231) Google Scholar, 27.Chen M.L. Tsai C.N. Liang C.L. Shu C.H. Huang C.R. Sulitzeanu D. Liu S.T. Chang Y.S. Oncogene. 1992; 7: 2131-2140PubMed Google Scholar, 28.Miller W.E. Edwards R.H. Walling D.M. Raab-Traub N. J. Gen. Virol. 1994; 75: 2729-2740Crossref PubMed Scopus (164) Google Scholar). Whereas the 30-bp deletion increases transforming activity of LMP1-95-8 (29.Li S.N. Chang Y.S. Liu S.T. Oncogene. 1996; 12: 2129-2135PubMed Google Scholar) and is frequently found in EBV-associated tumors (30.Sandvej K. Peh S.C. Andresen B.S. Pallesen G. Blood. 1994; 84: 4053-4060Crossref PubMed Google Scholar), the mechanisms underlying either increased tumorigenicity or augmented induction of NF-κB by these LMP1 variants remain unknown. Surprisingly, in transfected cells, when LMP1-95-8 is expressed at very high levels, it inhibits NF-κB activity (31.Coffin III, W.F. Geiger T.R. Martin J.M. J. Virol. 2003; 77: 3749-3758Crossref PubMed Scopus (42) Google Scholar, 32.Kaykas A. Worringer K. Sugden B. J. Virol. 2002; 76: 11551-11560Crossref PubMed Scopus (25) Google Scholar) and causes cytostatic and/or cytotoxic effects, indicating a role of the tight regulation of LMP1-95-8 levels in the maintenance of EBV latency (33.Dawson C.W. Eliopoulos A.G. Blake S.M. Barker R. Young L.S. Virology. 2000; 272: 204-217Crossref PubMed Scopus (64) Google Scholar, 34.Floettmann J.E. Ward K. Rickinson A.B. Rowe M. Virology. 1996; 223: 29-40Crossref PubMed Scopus (119) Google Scholar, 35.Kaykas A. Sugden B. Oncogene. 2000; 19: 1400-1410Crossref PubMed Scopus (55) Google Scholar). This inhibition is not observed upon expressing LMP1-Cao (33.Dawson C.W. Eliopoulos A.G. Blake S.M. Barker R. Young L.S. Virology. 2000; 272: 204-217Crossref PubMed Scopus (64) Google Scholar, 36.Johnson R.J. Stack M. Hazlewood S.A. Jones M. Blackmore C.G. Hu L.F. Rowe M. J. Virol. 1998; 72: 4038-4048Crossref PubMed Google Scholar), despite the fact that LMP1-Cao protein is more stable than LMP1-95-8 and accumulates to a greater extent in cells (37.Blake S.M. Eliopoulos A.G. Dawson C.W. Young L.S. Virology. 2001; 282: 278-287Crossref PubMed Scopus (44) Google Scholar). In human cells, LMP1-95-8 protein undergoes rapid degradation, which depends on the ubiquitin-proteasome pathway (38.Aviel S. Winberg G. Massucci M. Ciechanover A. J. Biol. Chem. 2000; 275: 23491-23499Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). However, E3 ubiquitin ligase(s) responsible for ubiquitination of LMP1 have not been identified as yet. We have noted that the C-terminal domain of LMP1-95-8 contains a potential β-TrCP recognition motif (DSGHES) and that this motif is mutated in LMP1-Cao. In this study, we investigated a potential role of SCFHOS/βTrCP E3 ubiquitin ligases in degradation of LMP1. We found that LMP1-95-8, but not LMP1-Cao, interacted with β-TrCP2/HOS. Although SCFHOS ligase did not regulate LMP1 stability, the association between LMP1-95-8 and the SCFHOS complex affected the availability of HOS to interact with phosphorylated IκB as well as IκB degradation and NF-κB activation. These data provide an insight into mechanisms underlying differences between LMP1-95-8 and highly tumorigenic LMP1 variants in their ability to activate NF-κB signaling. Plasmids—pSG5 vector-based constructs for expression of LMP1-95-8 and LMP1-Cao (39.Hahn P. Novikova E. Scherback L. Janik C. Pavlish O. Arkhipov V. Nicholls J. Muller-Lantzsch N. Gurtsevitch V. Grasser F.A. Int. J. Cancer. 2001; 91: 815-821Crossref PubMed Scopus (40) Google Scholar) were kindly provided by F. Grässer (Homburg, Germany). Constructs for expression of Cullin1, Skp1, Roc1, hemagglutinin-tagged HOS and HOSΔF (40.Fuchs S.Y. Chen A. Xiong Y. Pan Z.Q. Ronai Z. 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Site-directed mutagenesis on the pSG5-LMP1-95-8 backbone was carried out using a Quick-Change Site-directed Mutagenesis kit (Stratagene) to create the following substitution mutants: G212S, S350A, S366T, G212S/S350A, G212S/S366T, Triple (G212S/S350A/S366T), 95-8-KK (L339K/M340K) and Triple-KK (G212S/S350A/S366T/L339K/M340K). Cells, Transfection, and Cell Lysate Preparation—293T cells were grown in Dulbecco's modified Eagle's medium in the presence of 10% fetal bovine serum and antibiotics at 37 °C and 5% CO2. Rat1a cells were maintained in Dulbecco's modified Eagle's medium in the presence of 10% calf serum and antibiotics under similar conditions. Transfections were performed according to the calcium phosphate procedure or with the aid of LipofectAMINE Plus (Invitrogen). Cells were harvested and lysed with RIPA buffer (0.5% sodium deoxycholate, 1% Triton X-100, and 0.1% SDS in phosphate-buffered saline) supplemented with inhibitors of phosphatases (20 mm NaF and 1 mm Na3VO4) and a "mixture" of protease inhibitors (Sigma). Lysates were cleared by centrifugation at 16,000 × g for 30 min at 4 °C, and protein concentrations were measured using Bradford reagent (Pierce). pRSV-β-gal expression vector was added to the transfection mixture to normalize the transfection efficiency on the basis of β-galactosidase activity (measured with a Promega kit). Expression of recombinant SCFHOS complex components (HOS-HA, Cullin1, Skp1, and Roc1) in 293T cells and their purification were carried out as described previously (45.Tan P. Fuchs S.Y. Chen A. Wu K. Gomez C. Ronai Z. Pan Z.Q. Mol. Cell. 1999; 3: 527-533Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Immunotechniques—Monoclonal antibodies against HA tag (Roche Applied Science), LMP1 (CS1-4, Dako), IKKγ (Pharmingen), β-catenin (Transduction Laboratories), IKKα and IκBα (Santa Cruz Biotechnology), and α-tubulin (Sigma) as well as Cullin1 polyclonal antibody (NeoMarkers) were purchased. Polyclonal antibody against HOS (HOS-N) was described earlier (46.Spiegelman V.S. Tang W. Katoh M. Slaga T.J. Fuchs S.Y. Oncogene. 2002; 21: 856-860Crossref PubMed Scopus (34) Google Scholar). Pulse-chase analysis was performed as described previously (40.Fuchs S.Y. Chen A. Xiong Y. Pan Z.Q. Ronai Z. Oncogene. 1999; 18: 2039-2046Crossref PubMed Scopus (155) Google Scholar). Briefly, 293T cells grown on 100-mm dishes and transfected with indicated plasmids were starved in medium lacking methionine and cysteine, metabolically labeled with a 35S-labeled methionine/cysteine mixture (PerkinElmer Life Sciences), and chased with medium containing 2 mm unlabeled methionine and cysteine for various time points. Cells were harvested, and LMP1 proteins were immunopurified by CS1-4 antibody, separated by SDS-PAGE, and analyzed by autoradiography. Results were quantified using a Storm 860 Imager (Amersham Biosciences). An IKK immunokinase assay was carried out with IKKγ antibody as described elsewhere (47.Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). Immunoprecipitation and immunoblotting procedures were described earlier. Data were analyzed using Scion Image Software (version Beta 4.0.2). Luciferase Assays—Luciferase assays in the cells plated in 24-well plates and co-transfected with either κB- or jun2-driven luciferase reporter constructs and pRSV-β-gal plasmid were performed using the appropriate kit (Promega) as described earlier (40.Fuchs S.Y. Chen A. Xiong Y. Pan Z.Q. Ronai Z. Oncogene. 1999; 18: 2039-2046Crossref PubMed Scopus (155) Google Scholar). Luciferase activity was normalized with respect to β-galactosidase activity. Foci Formation Assay—Rat1a cells were transfected with LMP1 expressing constructs (0.5 μg) and pBABE-puro (3.5 μg) and grown for 14 days in the presence of puromycin (2 μg/ml, Sigma). The appearance of foci of transformed cells characterized by lack of contact inhibition was observed 14 days after transfection, and the number of foci was scored in a double blind manner. Measurement of Substrate-free HOS Levels—IκBα-derived phosphorylated peptide (KKERLLDDRHDpSGLDpSMKDE, where pS is phosphoserine) and its non-phosphorylated counterpart were synthesized by the Protein Chemistry Core Laboratory of Baylor College of Medicine (Houston, TX). Peptides were covalently coupled with beads using an AminoLink kit (Pierce), and the beads were washed and incubated with 1 mg of lysates from 293T cells transfected with LMP1 constructs for 1 h at 2 °C. After extensive washes with ice-cold phosphate-buffered saline supplemented with Nonidet P-40 (0.1%), the proteins were eluted with Laemmli sample buffer, separated by SDS-PAGE, and analyzed by immunoblotting with HOS-N antibody. LMP1-95-8 but Not LMP1-Cao Interacts with HOS— We sought to investigate whether LMP1 is a target for SCFβ-TrCP/HOS E3 ubiquitin ligases. Both β-TrCP1 and HOS recognize the DpSGXXpS motif (22.Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1083) Google Scholar) and play a redundant role in ubiquitination of IκB and β-catenin (48.Guardavaccaro D. Kudo Y. Boulaire J. Barchi M. Busino L. Donzelli M. Margottin-Goguet F. Jackson P.K. Yamasaki L. Pagano M. Dev. Cell. 2003; 4: 799-812Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Because a similar motif is found within the cytoplasmic C-terminal domain of LMP1-95-9 (Fig. 1C), we chose to focus our studies on interaction of LMP1 with HOS, which, unlike β-TrCP1, is primarily localized in the cytoplasm (49.Davis M. Hatzubai A. Andersen J.S. Ben-Shushan E. Fisher G.Z. Yaron A. Bauskin A. Mercurio F. Mann M. Ben-Neriah Y. Genes Dev. 2002; 16: 439-451Crossref PubMed Scopus (101) Google Scholar, 50.Lassot I. Segeral E. Berlioz-Torrent C. Durand H. Groussin L. Hai T. Benarous R. Margottin-Goguet F. Mol. Cell. Biol. 2001; 21: 2192-2202Crossref PubMed Scopus (206) Google Scholar). LMP1-95-8 protein expressed in 293T cells interacted with immobilized recombinant SCFHOS complex in vitro (Fig. 1A). Co-expression of LMP1-95-8 and HOS-HA in 293T cells followed by immunoprecipitation and immunoblotting analysis revealed that LMP1-95-8, but not LMP1-Cao, was co-immunoprecipitated with HA-tagged HOS (Fig. 1B, lane 4 versus lane 5). As the G212S substitution mutation within the sequence of a putative degradation motif (DSGXXS) in LMP1-Cao was absent from canonical motifs in LMP1-95-8 and other HOS substrates (Fig. 1C), this motif is likely to contribute to the interaction between HOS and LMP1-95-8. Interaction of HOS with LMP1-95-8 Requires Both Canonical and Cryptic HOS Recognition Sites—We next substituted Gly-212 in LMP1-95-8 for serine. Intriguingly, this mutation alone did not affect the ability of the LMP1 protein to interact with HOS (Fig. 2A, lanes 3 and 4), indicating that LMP1-95-8 may contain additional sites for HOS binding that are altered in LMP1-Cao. Although the DpSGXXpS motif is known to mediate HOS binding in IκB and β-catenin, it has been shown that motifs with more than three amino acids between the phosphoserines may also be recognized by β-TrCP proteins in precursors of NF-κB transcription factors (51.Orian A. Gonen H. Bercovich B. Fajerman I. Eytan E. Israel A. Mercurio F. Iwai K. Schwartz A.L. Ciechanover A. EMBO J. 2000; 19: 2580-2591Crossref PubMed Google Scholar) and ATF4 transcription factors (50.Lassot I. Segeral E. Berlioz-Torrent C. Durand H. Groussin L. Hai T. Benarous R. Margottin-Goguet F. Mol. Cell. Biol. 2001; 21: 2192-2202Crossref PubMed Scopus (206) Google Scholar). Another DSG sequence containing Ser-350 residue within LMP1-95-8 is absent in LMP1-Cao as a part of 30-bp deletion frequently found in LMP1 isolates from tumors (30.Sandvej K. Peh S.C. Andresen B.S. Pallesen G. Blood. 1994; 84: 4053-4060Crossref PubMed Google Scholar). Moreover, distally located serine 366 is substituted for threonine in LMP1-Cao protein (Fig. 2B), and mutation of Ser-366 is one of the most frequently observed among natural LMP1 isolates (see "Discussion"). We further assessed a potential role of Ser-350 and Ser-366 in HOS binding. Neither S350A nor S366T individual mutants lacked the ability to interact with HOS (Fig. 2A, lanes 5 and 6). However, double mutations of LMP1-95-8 such as G212S/S350A or G212S/S366T as well as the triple amino acid substitution (Triple, G212S/S350A/S366T) dramatically inhibited the ability of LMP1 to interact with HOS (Fig. 2A, lanes 7–9). These results suggest the existence of a cryptic HOS recognition site that requires the integrity of serines 350 (within the DSG sequence) and 366 in LMP1-95-8, which are deleted or mutated in LMP1-Cao. In addition, these data indicate that both canonical and cryptic HOS recognition sites are required for binding of HOS to LMP1-95-8 and provide a plausible explanation for the inability of LMP1-Cao, in which both sites are altered, to interact with HOS. HOS Is Not Required for LMP1-95-8 Degradation—As LMP1-Cao protein, which cannot bind HOS (Fig. 1B), is more stable than LMP1-95-8 (37.Blake S.M. Eliopoulos A.G. Dawson C.W. Young L.S. Virology. 2001; 282: 278-287Crossref PubMed Scopus (44) Google Scholar), we were prompted to investigate the role of HOS in the regulation of LMP1 protein stability. To this end, we compared the rate of proteolysis of LMP1-95-8 with its Triple mutant, which exhibited no binding to HOS (Fig. 2A). Contrary to our expectations, pulse-chase analysis of LMP1 proteins showed that the half-life of the Triple mutant was no longer than that of LMP1-95-8 (Fig. 3A). This result indicates that abrogation of HOS binding is not sufficient for stabilization of LMP1 proteins. Furthermore, LMP1-95-8 protein was not stabilized by co-expression of the dominant negative mutant HOSΔF (Fig. 3A). This mutant, which was shown to abrogate degradation of known HOS/β-TrCP substrates (40.Fuchs S.Y. Chen A. Xiong Y. Pan Z.Q. Ronai Z. Oncogene. 1999; 18: 2039-2046Crossref PubMed Scopus (155) Google Scholar, 41.Spiegelman V.S. Tang W. Chan A.M. Igarashi M. Aaronson S.A. Sassoon D.A. Katoh M. Slaga T.J. Fuchs S.Y. J. Biol. Chem. 2002; 277: 36624-36630Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), was evidently expressed under the conditions of our experiments, resulting in accumulation of β-catenin (Fig. 3B). In some experiments we observed a modest stabilization of Triple-LMP1 protein in cells overexpressing HOS; the significance of these observations remains unclear. In addition, although SCFHOS could be successfully bound to LMP1-95-8 (Fig. 1A), we failed to ubiquitinate this protein in vitro under conditions that enabled efficient ubiquitination of IκBα. 2W. Tang, V. Spiegelman, and S. Fuchs, unpublished data. Taken together, these results suggest that despite the ability of LMP1-95-8 to interact with SCFHOS E3 ubiquitin ligase, LMP1-95-8 is not a genuine substrate for this E3 ligase. These findings are in line with conclusions of another group that the C-terminal domain of LMP1-Cao does not contribute to its increased stability (37.Blake S.M. Eliopoulos A.G. Dawson C.W. Young L.S. Virology. 2001; 282: 278-287Crossref PubMed Scopus (44) Google Scholar). LMP1-95-8 Is a Pseudo-substrate of SCFHOS Because of the Absence of Ubiquitin-Acceptor Lysines—The recently solved SCFβ-TrCP1 structure predicts that efficiency of ubiquitination by this ligase depends on the location of the ubiquitin-accepting lysine(s) relative to the destruction motif (52.Wu G. Xu G. Schulman B.A. Jeffrey P.D. Harper J.W. Pavletich N.P. Mol. Cell. 2003; 11: 1445-1456Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). LMP1-95-8 contains a single lysine residue (Lys-330) that is not essential for its ubiquitination (38.Aviel S. Winberg G. Massucci M. Ciechanover A. J. Biol. Chem. 2000; 275: 23491-23499Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Lack of convenient ubiquitin conjugation site(s) in proximity to HOS-binding sequences similar to Lys-21/22 within IκBα (23.Rodriguez M.S. Wright J. Thompson J. Thomas D. Baleux F. Virelizier J.L. Hay R.T. Arenzana-Seisdedos F. Oncogene. 1996; 12: 2425-2435PubMed Google Scholar) or Lys-19 within β-catenin (52.Wu G. Xu G. Schulman B.A. Jeffrey P.D. Harper J.W. Pavletich N.P. Mol. Cell. 2003; 11: 1445-1456Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar) may explain why LMP1-95-8 is not a bona fide substrate for SCFHOS. To investigate this possibility, we substituted two amino acids positioned at a similar spacing from the cryptic HOS recognition site within LMP-95-8 with two lysine residues and compared the rates of degradation of LMP-95-8 and of this 95-8-KK mutant (L339K/M340K). Pulse-chase analysis showed that introduction of the lysines noticeably decreased the half-life of the mutant LMP1 protein (Fig. 4, A and D). Furthermore, inhibition of HOS activities by co-expressing HOSΔF protein stabilized 95-8-KK (Fig. 4, B and D). This result indicates that introduction of lysines in proximity to the HOS cryptic recognition site creates a protein whose degradation is regulated by SCFHOS. To confirm the role of HOS in controlling stability of 95-8-KK, we introduced the L339K/M340K substitution mutation into the background of the Triple mutant, which cannot bind HOS (Fig. 2A). The resulting Triple-KK mutant protein exhibited a half-life similar to that of LMP1-95-8, and the stability of Triple-KK was not increased by co-expression of HOSΔF (Fig. 4, B and D). To corroborate our hypothesis that an increase in the rate of ubiquitination of 95-8-KK results in destabilization of this protein, we carried out in vivo ubiquitination experiments. Co-transfection of LMP1-95-8 with hexahistidine-tagged ubiquitin in 293T cells followed by purification of ubiquitinated proteins under stringent denaturing conditions (44.Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar) revealed that LMP1 is covalently conjugated with ubiquitin in vivo under these conditions (Fig. 4C). Compared with the LMP1-95-8 protein, the 95-8-KK mutant exhibited a noticeably higher extent of ubiquitination (Fig. 4C, lane 5 versus lane 3), and this increase was inhibited by co-expression of HOSΔN (lane 6 versus lane 5). Mutation of HOS recognition sites in the Triple-KK protein abrogated the effect of introducing lysines (Fig. 4C, lane 7). In addition, the dominant negative HOSΔF mutant did not inhibit ubiquitination of either LMP1-95-8 or Triple-KK proteins. These results indicate that introduction of lysines into an LMP1 protein capable of binding to HOS converts the protein into a substrate for HOS-dependent ubiquitination. In all, our evidence suggests that LMP1-95-8 is a pseudo-substrate o
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