The 19 S Proteasomal Subunit POH1 Contributes to the Regulation of c-Jun Ubiquitination, Stability, and Subcellular Localization
2006; Elsevier BV; Volume: 281; Issue: 23 Linguagem: Inglês
10.1074/jbc.m512086200
ISSN1083-351X
AutoresJoseph F. Nabhan, Paula Ribeiro,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoThe AP1 (activator protein 1) transcription factor, c-Jun, is an important regulator of cell proliferation, differentiation, survival, and death. Its activity is regulated both at the level of transcription and post-translationally through phosphorylation, sumoylation, and targeted degradation. The degradation of c-Jun by the ubiquitin proteasome pathway has been well established. Here, we report that POH1, a subunit of the 19 S proteasome lid with a recently described deubiquitinase activity, is a regulator of c-Jun. Ectopic expression of POH1 in HEK293 cells decreased the level of c-Jun ubiquitination, leading to significant accumulation of the protein and a corresponding increase in AP1-mediated gene expression. The stabilization also correlated with a redistribution of c-Jun in the nucleus. These effects were reduced by mutation of a cysteine residue in the Mpr1 pad1 N-terminal plus motif of POH1 (Cys-120) and appeared to be selective for c-Jun, because POH1 had no effect on other proteasomal substrates. Our results identify a novel mechanism of c-Jun regulation in mammalian cells. The AP1 (activator protein 1) transcription factor, c-Jun, is an important regulator of cell proliferation, differentiation, survival, and death. Its activity is regulated both at the level of transcription and post-translationally through phosphorylation, sumoylation, and targeted degradation. The degradation of c-Jun by the ubiquitin proteasome pathway has been well established. Here, we report that POH1, a subunit of the 19 S proteasome lid with a recently described deubiquitinase activity, is a regulator of c-Jun. Ectopic expression of POH1 in HEK293 cells decreased the level of c-Jun ubiquitination, leading to significant accumulation of the protein and a corresponding increase in AP1-mediated gene expression. The stabilization also correlated with a redistribution of c-Jun in the nucleus. These effects were reduced by mutation of a cysteine residue in the Mpr1 pad1 N-terminal plus motif of POH1 (Cys-120) and appeared to be selective for c-Jun, because POH1 had no effect on other proteasomal substrates. Our results identify a novel mechanism of c-Jun regulation in mammalian cells. The 26 S proteasome is the principal site of controlled protein degradation in eukaryotic cells. The 2- to 2.5-MDa proteasome complex consists of a central core (20 S proteasome) and one or two multiprotein regulatory particles (RPs 2The abbreviations used are: RP, regulatory particle; AP1, activator protein 1; CREB, cAMP-response element-binding protein; DUB, deubiquitinase; JAMM, JAB1 MPN Mov34; MPN+, Mpr1 Pad1 N-terminal plus; RRL, rabbit reticulocyte lysate; WCE, whole cell extract; HA, hemagglutinin; CMV, cytomegalovirus; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; Ub, ubiquitin.; 19 S proteasome), which are further organized into base and lid regions (1Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1622) Google Scholar). The 19 S RP mediates the binding, deubiquitination, and unfolding of substrates into the 20 S core, where proteolysis takes place (2Hendil K.B. Hartmann-Petersen R. Curr. Protein Pept. Sci. 2004; 5: 135-151Crossref PubMed Scopus (35) Google Scholar). One of the many cellular proteins targeted for degradation by the proteasome is the AP1 transcription factor, c-Jun (3Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (852) Google Scholar). In addition to being the most potent transcriptional activator of its group, c-Jun is involved in a myriad of cellular activities, including tumorigenesis (4Shaulian E. Karin M. Nat. Cell. Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2296) Google Scholar). Tight control of intracellular concentrations of active c-Jun is therefore required, a process that is achieved through rapid turnover by ubiquitination and degradation. Novel mechanisms for targeted ubiquitination of c-Jun were recently shown to be carried out by c-Jun-specific ubiquitin ligases, such as Itch (5Gao M. Labuda T. Xia Y. Gallagher E. Fang D. Liu Y.C. Karin M. Science. 2004; 306: 271-275Crossref PubMed Scopus (338) Google Scholar) and SCF Fbw7 (6Nateri A.S. Riera-Sans L. Da Costa C. Behrens A. Science. 2004; 303: 1374-1378Crossref PubMed Scopus (311) Google Scholar). Whereas much continues to be learned about mechanisms of c-Jun ubiquitination, little is known about the process of deubiquitination and its contribution to the overall regulation of c-Jun. Cellular deubiquitinases (DUBs) play an important role in controlled protein degradation (7Guterman A. Glickman M.H. Curr. Protein Pept. Sci. 2004; 5: 201-211Crossref PubMed Scopus (100) Google Scholar); when bound to the proteasome they mediate the removal of ubiquitin chains as the substrate is being degraded, a step that facilitates substrate translocation into the 20 S chamber and allows for recycling of ubiquitin in the cell. DUBs also provide ubiquitin-editing activity, which can rescue particular substrates from being degraded by removing or trimming the degradation signal. A wide range of DUBs has been identified in eukaryotic cells, including enzymes with specificities for particular ubiquitinated substrates (8Amerik A.Y. Hochstrasser M. Biochim. Biophys. Acta. 2004; 1695: 189-207Crossref PubMed Scopus (779) Google Scholar) but no specific c-Jun deubiquitinase activity has yet been reported. A number of studies have implicated the 19 S proteasomal subunit, POH1 (also known as RPN11/pad1/S13/mpr1) as a possible regulator of AP1. Schizosaccharomyces pombe pad1/POH1 was shown to influence the activity of an AP1-like regulator, pap1. Overexpression of pad1/POH1 in S. pombe led to enhanced AP1-dependent transcription of a downstream target gene without promoting pap1 mRNA expression (9Shimanuki M. Saka Y. Yanagida M. Toda T. J. Cell Sci. 1995; 108: 569-579Crossref PubMed Google Scholar). Reporter assays in mammalian HeLa cells also demonstrated that the overexpression of yeast pad1/POH1 potentiates c-Jun-mediated transcription (10Claret F.X. Hibi M. Dhut S. Toda T. Karin M. Nature. 1996; 383: 453-457Crossref PubMed Scopus (412) Google Scholar). Subsequent studies showed that a flatworm orthologue of POH1, SmPOH, selectively decreased degradation of c-Jun in vitro (11Nabhan J.F. Hamdan F.F. Ribeiro P. Mol. Biochem. Parasitol. 2002; 121: 163-172Crossref PubMed Scopus (14) Google Scholar). More recently, POH1 was identified as an important DUB of the 19 S lid complex of the proteasome (12Maytal-Kivity V. Reis N. Hofmann K. Glickman M.H. BMC Biochem. 2002; 3: 28Crossref PubMed Scopus (186) Google Scholar, 13Verma R. Aravind L. Oania R. McDonald W.H. Yates 3rd, J.R. Koonin E.V. Deshaies R.J. Science. 2002; 298: 611-615Crossref PubMed Scopus (859) Google Scholar, 14Yao T. Cohen R.E. Nature. 2002; 419: 403-407Crossref PubMed Scopus (611) Google Scholar). Its activity within the complex contributes to substrate deubiquitination during proteasomal degradation and may also play a role in the editing of polyubiquitinated substrates as a means to control degradation (15Glickman M.H. Adir N. PLoS Biol. 2004; 2: E13Crossref PubMed Scopus (58) Google Scholar). In addition, POH1 is believed to have other functions outside the proteasome, some of which do not seem to involve substrate deubiquitination (16Penney M. Wilkinson C. Wallace M. Javerzat J.P. Ferrell K. Seeger M. Dubiel W. McKay S. Allshire R. Gordon C. J. Biol. Chem. 1998; 273: 23938-23945Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Rinaldi T. Pick E. Gambadoro A. Zilli S. Maytal-Kivity V. Frontali L. Glickman M.H. Biochem. J. 2004; 381: 275-285Crossref PubMed Scopus (50) Google Scholar, 18Rinaldi T. Ricci C. Porro D. Bolotin-Fukuhara M. Frontali L. Mol. Biol. Cell. 1998; 9: 2917-2931Crossref PubMed Scopus (67) Google Scholar). However, it remains unclear how these various activities relate to the stabilizing effect of POH1 on c-Jun. In this study, we investigate the mechanism of human POH1-induced c-Jun stabilization and explore its implications for the regulation of c-Jun activity in mammalian cells. Constructs and Transfections—Human c-Jun, p27KIP, s5a/RPN10, and POH1 sequences were cloned by reverse transcription-PCR from HEK293 Total RNA and then confirmed by DNA sequencing. cDNA sequences were modified by PCR to introduce mutations and incorporate a FLAG, His, or HA tag, as indicated, and then subcloned into pCI-neo (Promega) or pTracer (Invitrogen). Plasmid pCMV.HA-Ubiquitin was a gift from Dr. Bohmann's laboratory (University of Rochester, Rochester, NY). POH1 Cys-120 mutants and deletion mutants were generated by PCR and subcloned into pCI-neo. All constructs were confirmed by DNA sequencing. For transient transfection of HEK293 cells, we used FuGENE 6 (Roche Applied Science), according to the manufacturer's recommendations. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 20 mm Hepes. Unless otherwise specified, HEK293 cells were seeded in 6-well or 100-mm plates and were transfected with 1 or 4 μg of plasmid DNA, respectively. Total amounts of transfected DNA, in all experiments, were kept constant by addition of empty plasmids. Cell Fractionation and 20 S Proteasome Assays—For glycerol gradient centrifugations, we prepared lysates from 5 × 107 HEK293 cells transfected with 4 μg of pCI-neo or pCI-neo.POH1-FLAG. Cleared cell lysates were layered on a 10–40% continuous glycerol gradient and subjected to a 22-h fractionation centrifugation at 25,000 RPM using an SW-28 rotor as previously reported (19Takeuchi J. Fujimuro M. Yokosawa H. Tanaka K. Toh-e A. Mol. Cell. Biol. 1999; 19: 6575-6584Crossref PubMed Scopus (62) Google Scholar). Samples were collected from the bottom of the centrifugation tubes, subjected to trichloroacetic acid precipitation, and aliquots of precipitated proteins were analyzed by immunoblotting or proteasome activity. 20 S proteasome assays were performed, according to standard procedures, using 0.5 μg of protein per reaction and Suc-LLVY-AMC (Biomol) as a degradation substrate. Reactions were carried out in a black 96-well plate, and fluorescence was recorded with the Fluostar Galaxy Fluorometer (BMG LabTechnologies) equipped with the appropriate excitation (λex = 380 nm) and emission (λem = 440 nm) filters. In Situ Stabilization, in Vitro Degradation, and Reporter Assays—For the in situ stabilization assays, cells seeded in 100-mm plates were transfected with 0.6 μg of pTracer.c-Jun or pTracer alone, and 1.7 μg or 3.4 μg of pCI-neo.POH1-FLAG, one of the Cys-120 mutant constructs or empty vector. For the in situ stabilization assay of p27KIP, cells seeded in 100-mm plates were transfected with 0.6 μg of pTracer.p27KIP-HA and 3.4 μg of pCI-neo.POH1-FLAG. The pTracer construct encodes GFP under the control of a distinct promoter (Invitrogen) and was used as a transfection control. 48 h after transfection, cells were harvested in lysis buffer (20 mm Tris-Cl, 150 mm NaCl, 10 mm EGTA, 2 mm EDTA, 10 mm Na3VO4, 20 mm NaF, 0.5% Nonidet P-40, 1 mm glycerophosphate, and protease inhibitor mixture from Sigma). 5 μg of cell lysate was then analyzed by immunoblotting with the appropriate antibodies. GFP expression was used to monitor transfection efficiency by fluorescence microscopy and protein loading by immunoblotting with anti-GFP (Molecular Probes/Invitrogen). For the in vitro stabilization assays, experiments were carried out as reported previously (11Nabhan J.F. Hamdan F.F. Ribeiro P. Mol. Biochem. Parasitol. 2002; 121: 163-172Crossref PubMed Scopus (14) Google Scholar). Briefly, recombinant proteins were generated using 1 μg of each cDNA template in a coupled rabbit reticulocyte translation system (T7/SP6 TNT Reticulocyte System, Promega) in the presence or absence of [35S]methionine (Amersham Biosciences, 20 μCi/reaction). Approximate protein concentrations were determined by labeling proteins with [35S]methionine and subjecting them to autoradiography followed by densitometry analysis. Reactions were terminated by addition of 50 μg/ml cycloheximide. The 35S-labeled c-Jun, p27KIP, or GFP proteins were incubated with an equimolar amount of in vitro translated unlabeled POH1 or a mutant POH1 protein in degradation buffer (20 mm Tris HCl, 1 mm dithiothreitol, 5 mm MgCl2, 2 mm ATP (Sigma), 15 mm creatine phosphate (Sigma), and 2 units of creatine phosphokinase (Sigma)). 10-μl samples were removed at t = 0, 90, and 180 min and analyzed by gel autoradiography. For AP1 reporter assays, cells seeded in 6-well plates were transfected with 1 μg of pAP1-Luc (Clontech/BD Biosciences), 0.25 μg of pTracer.c-Jun, and 0.75 μg of pCI-neo.POH1, a Cys-120 POH1 mutant construct, or empty plasmid. The luciferase assays were later carried out using a luciferase assay kit (Promega) according to the manufacturer's recommendations, and luminescence was recorded using a Fluostar Galaxy Luminometer (BMG LabTechnologies). Reverse Transcription and Quantitative Real-time PCR—Total RNA from HEK293 cells transfected with a POH1 construct or vector (pCI-neo) alone was prepared using the RNeasy Mini kit (Qiagen), according to the manufacturer's instructions. Reverse transcription was performed on 1 μg of total RNA in a reaction volume of 20 μl using Superscript II Reverse Transcriptase (Invitrogen). Bcl1/Cyclin D1, BIM, Bcl3, CREB, and β-tubulin primers were designed using the Oligo Software (Molecular Biology Insights) and specificity was confirmed by BLAST data base analysis (see Table 1 for primer sequences). All oligonucleotides were obtained from Operon (Huntsville, AL) and reconstituted in RNase-free water (Invitrogen). Preliminary validation experiments demonstrated that the amplification efficiencies of the target genes and the internal reference (β-tubulin) were approximately equal. PCR reactions were carried out with the QuantiTect SYBR Green PCR kit (Qiagen) in a final volume of 10 μl using the Rotor-Gene RG3000 Instrument (Corbett Research). Cycling conditions were as follows: 95 °C for 15 min followed by 50 cycles of 95 °C for 15 s, 57 °C for 15 s, and 72 °C for 20 s. The generation of specific PCR products was confirmed by melting curve analysis and agarose gel electrophoresis. Quantitation of relative differences in expression levels were finally calculated using the comparative CT method (20Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (133172) Google Scholar).TABLE 1Regulation of expression of endogenous c-Jun driven genes in cells overexpressing POH1GeneSize of ampliconRelative expression levelsNaN is the number of independent determinations, each done in triplicate.p valuebp2-DΔCT ± S.E.Bcl1/Cyclin D11281.48 ± 0.1960.05BIM1321.34 ± 0.1360.01Bcl31081.44 ± 0.1660.01CREB1001.10 ± 0.2060.65 (NS)bNS, not significant.a N is the number of independent determinations, each done in triplicate.b NS, not significant. Open table in a new tab In Situ Ubiquitination Assays—For the in situ c-Jun ubiquitination assays, cells seeded in 6-well plates were transfected with 0.5 μg of pCI-neo.c-Jun-His, 0.5 μg of pCMV.HA-Ub, and 1 μg of pCI-neo.POH1, POH1C120S, POH1C120A, or empty plasmid and for the in situ p27KIP ubiquitination assays, with 0.5 μg of pCI-neo.p27KIP-His, 0.5 μg of pCMV.HA-Ub, and 1 μg of pCI-neo.POH1 or empty plasmid. 48 h later, cells were lysed directly into denaturing buffer (20 mm Tris Cl, 0.5 m NaCl, 5 mm imidazole, and 6 m urea) and subjected to three freeze-thaw cycles. Ni2+-charged agarose beads (Novagen) were then added to the lysates for 2 h at 4°C and then extensively washed with a 5 mm imidazole buffer followed by a 40 mm imidazole solution. Bound proteins were eluted by addition of reducing sample buffer containing 1 m imidazole and finally analyzed by immunoblotting with anti-HA (Sigma) and anti-c-Jun (Oncogene/EMD Biosciences) or anti-p27KIP (Cell Signaling Technology) antibodies. To assay for the effect of overexpression of POH1 on total protein ubiquitination and proteasome activity, cells seeded in 100-mm plates were transfected with 4 μg of pTracer or pTracer.POH1 and analyzed by FACS using FACS Aria (BD Biosciences) equipped with an argon (λ = 488 nm) laser. 2 × 105 sorted cells were seeded in 6-well plates and left overnight at 37 °C to recover. An equal number of non-transfected or transfected but unsorted cells was seeded as controls. 12 h later, cells were washed briefly with phosphate-buffered saline, scraped into 500 μl of phosphate-buffered saline, and lysed mechanically using a Dounce homogenizer. Protein concentrations were determined, and aliquots containing 5 μg of protein were analyzed by immunoblotting with anti-ubiquitin (Chemicon). An equal amount of protein was also assayed for 20 S proteasome activity using Suc-LLVY-AMC substrate, as described above. Immunofluorescence Assays—For the immunofluorescence assays, cells seeded in 6-well plates were typically transfected with a total amount of 1 μg of plasmid/well. 48 h following transfection, cells were cooled on ice for 10 min, washed with phosphate-buffered saline, and later fixed and permeabilized with ice-cold methanol at –20 °C for 10 min. Cells were then washed and incubated with primary antibodies for 1 h at 4 °C. The incubation was followed by multiple washes with phosphate-buffered saline, and cells were supplemented with the appropriate secondary antibodies coupled to rhodamine or fluorescein isothiocyanate and left at 4 °C for 1 h. 5 min before the end of this incubation, 1 μg/ml 4′,6-diamidino-2-phenylindole was added to the secondary antibody solution as a nuclear stain. Finally, cells were washed and mounted on slides for visualization by confocal microscopy. All images were acquired using a Zeiss LSM 510 confocal microscope. Fluorescein isothiocyanate, rhodamine, and 4′,6-diamidino-2-phenylindole signals were obtained by excitation with argon (λ = 488 nm), HeNe (λ = 543 nm), and titanium sapphire (λ = 800 nm, Coherent Inc.) laser lines, respectively. Homology Modeling—A theoretical model of POH1 was obtained based on the available crystal structure of AfJAMM (1R5X) (21Ambroggio X.I. Rees D.C. Deshaies R.J. PLoS Biol. 2004; 2: E2Crossref PubMed Scopus (201) Google Scholar). We first obtained a crude alignment of human POH1 and the AfJAMM template using the Consensus server (22Prasad J.C. Comeau S.R. Vajda S. Camacho C.J. Bioinformatics. 2003; 19: 1682-1691Crossref PubMed Scopus (25) Google Scholar). The alignment was examined to ensure that the positions of seed residues corresponding to JAMM amino acids were conserved. Structures were then generated using the COMPOSER module in Sybyl 6.9 (Tripos). Restraints were imposed on the JAMM residues to prevent exaggerated structural changes in this region during energy minimization. The model energies were minimized using the Powel subroutine with the Kollman_All atom force field at 0.05 kcal/mol Ä until convergence. The final structures were analyzed with the WHATIF server and Ramachandran plot analysis. The structures of AfJAMM and human POH1 were subsequently aligned and superimposed using the Dalilite server (root mean square deviation = 1.4 Ä, www.ebi.ac.uk/DaliLite/), and cartoons were generated using Pymol (23De Lano W.L. The Pymol Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar) and Sybyl 6.9 (Tripos). To assess the effect of the Cys-120 mutations, JAMM-Cys-120 was replaced with Ser or Ala using the Biopolymer module of Sybyl 6.9, and the models were minimized again to convergence. Other Methods—Immunoprecipitations were performed using the Protein-A IP kit from Roche Applied Science. Immunoblotting was carried out according to standard procedures using the indicated antibodies, and dilutions were prepared to the manufacturer's recommendations or determined experimentally as required. We had previously used an in vitro degradation assay to demonstrate that a Schistosoma mansoni orthologue of POH1 stabilizes c-Jun (11Nabhan J.F. Hamdan F.F. Ribeiro P. Mol. Biochem. Parasitol. 2002; 121: 163-172Crossref PubMed Scopus (14) Google Scholar). Here, we repeated this analysis with human POH1 and carried out a deletion mutagenesis study to identify the minimum region responsible for the c-Jun-stabilizing effect. A sequence alignment of POH1 orthologues highlights the positions of previously described functional motifs that were targeted for the mutagenesis (Fig. 1A). The most important of these motifs is a divalent metal ion binding stretch of amino acids named MPN+ (Mpr1 pad1 N-terminal plus) (12Maytal-Kivity V. Reis N. Hofmann K. Glickman M.H. BMC Biochem. 2002; 3: 28Crossref PubMed Scopus (186) Google Scholar) or JAMM (JAB1/MPN/Mov34) (13Verma R. Aravind L. Oania R. McDonald W.H. Yates 3rd, J.R. Koonin E.V. Deshaies R.J. Science. 2002; 298: 611-615Crossref PubMed Scopus (859) Google Scholar). This motif (EXnH(S/T)HX7SXXD) is closely related to the active site of a novel class of Zn2+-associated metalloproteases (21Ambroggio X.I. Rees D.C. Deshaies R.J. PLoS Biol. 2004; 2: E2Crossref PubMed Scopus (201) Google Scholar) and is principally responsible for the DUB activity of the protein. Embedded within the JAMM sequence is a Cys-box-like motif of unknown function. The relevant cysteine (Cys-120) of this motif is not believed to be essential for the DUB activity of POH1 (12Maytal-Kivity V. Reis N. Hofmann K. Glickman M.H. BMC Biochem. 2002; 3: 28Crossref PubMed Scopus (186) Google Scholar, 14Yao T. Cohen R.E. Nature. 2002; 419: 403-407Crossref PubMed Scopus (611) Google Scholar) but may be required for deubiquitination of specific subsets of proteins (24Lundgren J. Masson P. Realini C.A. Young P. Mol. Cell. Biol. 2003; 23: 5320-5330Crossref PubMed Scopus (63) Google Scholar). The alignment also identified a short hydrophobic peptide (LALLKML) located near the N terminus that resembles the ubiquitin-binding motif of RPN10 (LALALR(L/V)) (25Fu H. Sadis S. Rubin D.M. Glickman M. van Nocker S. Finley D. Vierstra R.D. J. Biol. Chem. 1998; 273: 1970-1981Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). At the other end of the protein is a newly discovered C-terminal functional domain that has been implicated in some of the non-proteasomal effects of POH1 (17Rinaldi T. Pick E. Gambadoro A. Zilli S. Maytal-Kivity V. Frontali L. Glickman M.H. Biochem. J. 2004; 381: 275-285Crossref PubMed Scopus (50) Google Scholar) and is thought to be unrelated to substrate deubiquitination. We also identified a putative nuclear export signal motif resembling that of signalosome subunit JAB1, which may bind nuclear export protein CRM1 (26Tomoda K. Kubota Y. Arata Y. Mori S. Maeda M. Tanaka T. Yoshida M. Yoneda-Kato N. Kato J.Y. J. Biol. Chem. 2002; 277: 2302-2310Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). We have generated POH1 deletion mutants that lack a large portion of the JAMM motif (POH1ΔJAMM: 105–126) or the predicted Ub-binding peptide (POH1Δ37–44), as well as a series of C-terminal truncations. In addition, to test the role of the Cys-box motif, we produced two single amino acid substitutions of Cys-120, POH1C120S and POH1C120A (Fig. 1B). Wild-type POH1 and mutants were subsequently tested for their effects on c-Jun stability, using in vitro translated proteins in a cell-free degradation assay that contained rabbit reticulocyte lysates as a source of proteasomes and ubiquitination machinery, as well as cycloheximide to prevent new protein synthesis (Fig. 1C). The results show that 35S-labeled c-Jun degraded rapidly in the control sample but was significantly stabilized by addition of wild-type POH1 or a mutant containing an intact JAMM motif. Deletion of up to 143 residues from the C-terminal end did not change the stabilizing effect of POH1 on 35S-c-Jun. Mutant POH1Δ37–44 also decreased degradation of 35S-c-Jun though to a lesser extent than the wild-type or C-terminal deletions. In contrast, samples treated with a ΔJAMM mutant degraded very rapidly, as did the single point substitutions of Cys-120. Surprisingly, the half-life of 35S-c-Jun treated with either the POH1C120S or POH1C120A mutant was essentially indistinguishable from that of the control sample, suggesting this one cysteine was critical for the stabilizing effect of POH1 on c-Jun. To test whether POH1 could stabilize other proteins we repeated the in vitro degradation assays with p27KIP or GFP (Fig. 1D). p27KIP is a cyclin-dependent kinase inhibitor that is ubiquitinated and degraded by the proteasome (27Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1742) Google Scholar), and GFP is a relatively stable protein that is not affected by proteasomal degradation except as a ubiquitin fusion degradation protein (28Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (481) Google Scholar). There was no apparent effect of POH1 on the degradation rate of either protein. Next we expanded the study into a cell-based system and examined the stabilization effect of POH1 on c-Jun in HEK293 cells. Because the mutation of residue Cys-120 of the JAMM motif was sufficient to abrogate the stabilizing effect of POH1 in vitro, we focused on this one residue for further analysis in situ. HEK293 cells were co-transfected with a fixed amount of c-Jun-expressing plasmid and two different amounts of plasmids expressing POH1, POH1C120S, POH1C120A, or vector only. Cells were supplemented with a small amount of c-Jun to facilitate detection of the protein because the endogenous level was found to be low. The c-Jun construct also encoded GFP, which was used as a loading control, and the total amount of plasmid was kept constant by addition of empty vector, as needed. Lysates of each test sample were subsequently analyzed by Western blotting using antibodies against c-Jun, recombinant POH1 (anti-FLAG) and GFP (Fig. 2A). The results show that overexpression of wild-type POH1 caused a strong, dose-dependent increase in the cellular levels of c-Jun, as predicted from the in vitro degradation assays. The POH1 cysteine mutants expressed at about the same level as the wild-type, based on densitometry analysis of anti-FLAG immunoreactivity. However, the mutants produced a much weaker or virtually no stabilization of c-Jun, depending on the dosage. At lower plasmid concentrations, the mutants had a minimal effect on c-Jun levels compared with ∼6-fold stabilization produced by the wild type. Doubling the amount of plasmid revealed some stabilization but the effect was small, representing ∼24% of that produced by the wild type in the case of C120S and 38% in the case of C120A. Experiments were repeated using a p27KIP expression plasmid in place of c-Jun to assess the specificity of the response. There was no apparent stabilization of p27KIP in cells overexpressing POH1 (Fig. 2B), even at the high dosage of POH1 expressing plasmid (3.4 μg). To rule out possible artifacts due to co-transfection with c-Jun or p27KIP expression plasmids, we also monitored the effect of POH1 overexpression on the endogenous levels of these proteins. Western blot analyses of HEK293 cells transfected with wild-type POH1-FLAG showed ∼6-fold increase in the level of endogenous c-Jun, whereas the levels of endogenous p27KIP and another proteasomal substrate (CREB) were unchanged (data not shown), thus confirming that c-Jun was selectively stabilized in these cells. Subsequent studies showed that the POH1-induced accumulation of c-Jun correlated with an increase in AP1-transcriptional activity (Fig. 2C). Transfection with wild-type POH1 stimulated AP1-mediated expression of a reporter gene (luciferase) ∼2.5-fold, whereas the POH1C120A and POH1C120S mutants had no significant effect compared with the mock-transfected control. To test if this increased AP1 activity can influence expression of endogenous AP1-driven genes, we monitored the effect of POH1 overexpression on mRNA levels of three known c-Jun driven genes, Bcl1/Cyclin D1, BIM, and Bcl3 (29Eferl R. Wagner E.F. Nat. Rev. Cancer. 2003; 3: 859-868Crossref PubMed Scopus (1704) Google Scholar) (Table 1) by quantitative Real-time reverse transcription-PCR. As a control, we determined the expression levels of CREB, which is not known to be influenced by c-Jun. The results showed that transfection with POH1 caused a small but statistically significant increase of ∼40% in the expression level of each test gene, Bcl1, BIM, and Bcl3 compared with pCI-neo-transfected controls, whereas CREB expression levels were not significantly changed. An important question arising from these findings is whether the recombinant POH1 expressed in HEK293 cells was incorporated into the cellular proteasome and how that might influence the integrity and function of the complex. Transfection with a POH1 or POH1-FLAG plasmid under standard conditions caused an approximate 2-fold increase in total POH1 protein, as determined from densitometry analyses of anti-POH1 immunoreactivity (Fig. 3A). The recombinant POH1 was found to co-immunoprecipitate with the proteasome, and the same was true for the two POH1 cysteine mutants, which co-immunoprecipitated with the proteasome to about the same extent as the wild-type (Fig. 3B). Subsequent glycerol density gradient centrifugation analysis (19Takeuchi J. Fujimuro M. Yokosawa H. Tanaka K. Toh-e A. Mol. Cell. Biol. 1999; 19: 6575-6584Crossref PubMed Scopus (62) Google Scholar) determined that ∼90% of all detectable POH1-FLAG sedimented with the proteasome in the higher density fractions. Moreover, the distribution of the recombinant POH1 in these fractions was virtually identical to that of the native protein, sug
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