p21 Ras/Impedes Mitogenic Signal Propagation Regulates Cytokine Production and Migration in CD4 T Cells
2008; Elsevier BV; Volume: 283; Issue: 34 Linguagem: Inglês
10.1074/jbc.m804084200
ISSN1083-351X
AutoresJan Czyzyk, Hui‐Chen Chen, Kim Bottomly, Richard A. Flavell,
Tópico(s)NF-κB Signaling Pathways
ResumoThe propensity of T cells to generate coordinated cytokine responses is critical for the host to develop resistance to pathogens while maintaining the state of immunotolerance to self-antigens. The exact mechanisms responsible for preventing the overproduction of proinflammatory cytokines including interferon (IFN)-γ are not fully understood, however. In this study, we examined the role of a recently described Ras GTPase effector and repressor of the Raf/MEK/ERK cascade called impedes mitogenic signal propagation (Imp) in limiting the induction of T-cell cytokines. We found that stimulation of the T cell receptor complex leads to the rapid development of a physical association between Ras and Imp. Consistent with the hypothesis that Imp inhibits signal transduction, we also found that disengagement of this molecule by the RasV12G37 effector loop mutant or RNA interference markedly enhances the activation of the NFAT transcription factor and IFN-γ secretion. A strong output of IFN-γ is responsible for the distinct lymphocyte traffic pattern observed in vivo because the transgenic or retroviral expression of RasV12G37 caused T cells to accumulate preferentially in the lymph nodes and delayed their escape from the lymphoid tissue, respectively. Together, our results describe a hitherto unrecognized negative regulatory role for Imp in the production of IFN-γ in T cells and point to Ras-Imp binding as an attractive target for therapeutic interventions in conditions involving the production of this inflammatory cytokine. The propensity of T cells to generate coordinated cytokine responses is critical for the host to develop resistance to pathogens while maintaining the state of immunotolerance to self-antigens. The exact mechanisms responsible for preventing the overproduction of proinflammatory cytokines including interferon (IFN)-γ are not fully understood, however. In this study, we examined the role of a recently described Ras GTPase effector and repressor of the Raf/MEK/ERK cascade called impedes mitogenic signal propagation (Imp) in limiting the induction of T-cell cytokines. We found that stimulation of the T cell receptor complex leads to the rapid development of a physical association between Ras and Imp. Consistent with the hypothesis that Imp inhibits signal transduction, we also found that disengagement of this molecule by the RasV12G37 effector loop mutant or RNA interference markedly enhances the activation of the NFAT transcription factor and IFN-γ secretion. A strong output of IFN-γ is responsible for the distinct lymphocyte traffic pattern observed in vivo because the transgenic or retroviral expression of RasV12G37 caused T cells to accumulate preferentially in the lymph nodes and delayed their escape from the lymphoid tissue, respectively. Together, our results describe a hitherto unrecognized negative regulatory role for Imp in the production of IFN-γ in T cells and point to Ras-Imp binding as an attractive target for therapeutic interventions in conditions involving the production of this inflammatory cytokine. The small GTPase Ras is a potent signaling molecule that can bind with numerous downstream effector molecules including the protein kinase Raf (1Stokoe D. MacDonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (847) Google Scholar, 2Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). 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The observation that a relative lack of Ras/ERK signal is associated with inability of T cells to respond to an antigen further underscores the fundamental role of this signaling pathway in T cell stimulation (15Fields P.E. Gajewski T.F. Fitch F.W. Science. 1996; 271: 1276-1278Crossref PubMed Scopus (357) Google Scholar, 16Li W. Whaley C.D. Mondino A. Mueller D.L. Science. 1996; 271: 1272-1276Crossref PubMed Scopus (408) Google Scholar, 17Zha Y. Marks R. Ho A.W. Peterson A.C. Janardhan S. Brown I. Praveen K. Stang S. Stone J.C. Gajewski T.G. Nat. Immunol. 2006; 7: 1166-1173Crossref PubMed Scopus (228) Google Scholar). It is not surprising to find therefore that T cells and other cell types have developed inhibitors to control the magnitude and duration of the ERK signaling. Among the most prominent endogenous repressors of the Ras/ERK signaling are GTPase-activating proteins (18Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, 19Bos J.L. 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Nature. 2004; 427: 256-260Crossref PubMed Scopus (163) Google Scholar). mitogen-activated protein kinase nuclear factor of activated T cells T cell receptor mitogen-activated protein kinase/extracellular signal-regulated kinase kinase extracellular signal-regulated kinase kinase interleukin impedes mitogenic signal propagation kinase suppressor of Ras monoclonal antibody phycoerythrin interferon fluorescent-activated cell sorter RNA interference epidermal growth factor protein antigen-presenting cell enzyme-linked immunosorbent assay spingosine phosphate. Imp, also known as BRCA1-associated protein (BRAP2), was originally identified as a predominantly cytoplasmic protein that recognizes the nuclear localization signals of BRCA1, SV40 large T antigen, and myosin (29Li S. Ku C.-Y. Farmer A.A. Cong Y.-S. Chen C.-F. Lee W.-H. J. Biol. Chem. 1998; 273: 6183-6189Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). It has been proposed that BRAP2 masks nuclear localization signal motifs causing the mislocalization of specific nuclear proteins and thus serving as a cytoplasmic retention protein (29Li S. Ku C.-Y. Farmer A.A. Cong Y.-S. Chen C.-F. Lee W.-H. J. Biol. Chem. 1998; 273: 6183-6189Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 30Asada M. Ohmi K. Delia D. Enosawa S. Suzuki S. Yuo A. Suzuki H. Mizutani S. Mol. Cell. Biol. 2004; 24: 8236-8243Crossref PubMed Scopus (42) Google Scholar). Interestingly, White and collaborators (28Matheny S.A. Chen C. Kortum R.L. Razidlo G.L. Lewis R.E. White M. Nature. 2004; 427: 256-260Crossref PubMed Scopus (163) Google Scholar) demonstrated that Imp can also disrupt the Ras/ERK pathway possibly by uncoupling Raf kinase from MEK through the inactivation of a scaffolding protein of the Ras pathway called the kinase suppressor of Ras (KSR). The exact mechanism of this inhibition of KSR function is unknown, however. Importantly, Imp is regulated by activated Ras. Specifically, binding of GTP-loaded Ras to a region encompassing the ubiquitin-protease-like zinc finger (UJBP-ZnF) domain of Imp/BRAP targets this molecule for ubiquitination and possible degradation by the proteasome. In summary, activated Ras relays a signal to the Erk cascade in two ways: (i) by recruiting Raf kinase to the plasma membrane and (ii) by relieving Imp-mediated inhibition of KSR-dependent formation of the Raf-MEK complex. It is unknown whether Imp, by providing an additional layer of resistance to the Erk pathway prevents the unnecessary activation of T cells. However, the amplitude and kinetics of ERK activation have been associated with different outcomes for T cells including survival or apoptosis in "young" T cells in the thymus (31Daniels M.A. Teixeiro E. Gill J. Hausmann B. Roubaty D. Holmberg K. Werlen G. Holländer G.A. Gascoigne N.R.J. Palmer E. Nature. 2006; 444: 724-729Crossref PubMed Scopus (460) Google Scholar) and the generation of helper T cell (Th1 or Th2) cytokines in mature T cells (32Jorritsma P.J. Brogdon J.L. Bottomly K. J. Immunol. 2003; 170: 2427-2434Crossref PubMed Scopus (124) Google Scholar), Thus, it is not unlikely that Imp influences these processes by limiting Erk pathway activity. Consequently, we designed this study to define overall sensitivity of T cells to repressive function of Imp. Molecular Constructs—cDNAs encoding H-Ras and Ras effector loop mutants (RasV12G35, RasV12G37, and RasV12C40) were described previously (33Czyzyk J. Brogdon J.L. Badou A. Henegariu O. Preston Hurlburt P. Flavell R.A. Bottomly K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6003-6008Crossref PubMed Scopus (15) Google Scholar). N17Ras in pcDNA3 was a gift from K. L. Guan (University of Michigan). K-Ras, N-Ras, and wild type Imp were generated by polymerase chain reaction amplification from the mouse cDNA library. ImpC264A was created using oligonucleotides carrying the desired point mutation and PCR amplification with high-fidelity polymerase. A construct encoding a membrane-targeted variant of Imp was generated by introducing the sequence encoding the 19 C-terminal residues of K-Ras4B (MSKDGKKKKKKSKTKCVIM) into the C terminus of full-length Imp (designated Imp-CAAX) (34Clark J.C. Quilliam L.A. Hisaka M.M. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4887-4891Crossref PubMed Scopus (55) Google Scholar). All new constructs were cloned into pMSCV, which contains a cassette consisting of the green fluorescent protein (GFP) and an internal ribosome entry site that permits the translation of two open reading frames from a single messenger RNA. In the pMIGR2 retroviral plasmid, tailless human CD2 replaces GFP. Antibodies—Anti-CD3 mAb OKT3 (eBiosciences, San Diego, CA) and C363.29B followed by goat anti-rat polyclonal antibody (MP Biomedical, Solon, OH) were used to stimulate, respectively, Jurkat human T cells and CD4 T cells, that had been isolated from B10.BR mice. Polyclonal antibodies, anti-Imp (The Biodesign Institute, Arizona State University, Tempe, AZ) and anti-FLAG (Sigma), and mAbs, anti-ERK (clone D-2; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-ERK (clone E-4; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-Ras (clone Ras10; Upstate Biotechnology, Lake Placid, NY) were used in a Western blot analysis. Anti-LFA-1 (M17/4; BD Biosciences) and anti-VLA-4 (R1–2) mAbs were used in adoptive transfer experiments to measure lymphocyte egress from the lymph nodes (35Lo C.G. Xu Y. Proia R.L. Cyster J.G. J. Exp. Med. 2005; 201: 291-301Crossref PubMed Scopus (258) Google Scholar). Phycoerythrin (PE)-conjugated anti-IFN-γ, biotin-conjugated anti-human CD2, antigen-presenting cell (APC), anti-CD4, PE anti-Vβ5 TCR, and biotin anti-Vα2 TCR mAbs were used to stain CD4 T cells and analyze them using fluorescent-activated cell sorter (FACS); all of these antibodies were from BD Biosciences. mAbs, 11B11 (anti-IL4), and XMG1.2 (anti-IFN-γ) were used to make sure that Th1 and Th2 skewing took place during primary stimulation of CD4 T cells. Cell Culture and Activation—Jurkat human T cells and mouse CD4 T cells were maintained in RPMI1640 and Bruff culture medium, respectively. Culture media were supplemented with 10% fetal calf serum, 10 mm HEPES, and antibiotics. Jurkat cells were activated in 96-well plates (EIA/RIA plates, Costar Corporation, Cambridge, MA) precoated overnight with OKT3 mAb at concentrations as indicated in the legend to Fig. 1d. Alternatively, Jurkat cells were incubated with 1.0 μg/ml OKT3 mAb on ice and then treated for 5 min at 37 °C with goat anti-rat secondary antibody. For stimulation with APCs, we isolated CD4 T cells from 6–8-week-old B10.BR and B10.5R mice that were transgenic for AND TCR. This TCR is specific for the moth cytochrome c peptide (VFAGLKKANERADLIAYLKQATK). CD4 T cells were grown in the presence of mitomycin C-treated T cell-depleted splenocytes. The APCs were pulsed with either 5 μg/ml pMCC or 15 μg/ml of a pMCC variant in which arginine had replaced lysine (K99R). For stimulation with phorbol esters, CD4 T cells were incubated with phorbol 12-myristate 13-acetate (100 ng/ml) at 37 °C. Transfections—To obtain Jurkat cells exhibiting stable repression of Imp protein, we electroporated the cells with Imp RNAi expressed from pSilcencer™ 3.1-H1 hygro (Ambion, Foster City, CA) and GFP as a marker for early selection and then subcloned them by limited dilution in the presence of hygromycin as a selection drug. Individual clones were subsequently electroporated with an NFAT-luciferase reporter and a plasmid expressing red fluorescence protein to monitor transfection efficiency. Alternatively, Jurkat T cells were electroporated with the PathDetect trans-reporting system (Elk1-GAL4 transactivator and 5xGAL4-luciferase reporter at a ratio of 1:50) (Stratagene, La Jolla, CA) (36Czyzyk J. Leitenberg D. Taylor T. Bottomly K. Mol. Cell. Biol. 2000; 20: 8740-8747Crossref PubMed Scopus (26) Google Scholar), Imp-CAAX construct, and pRL-CMV plasmid containing the Renilla luciferase under control of the cytomegalovirus immediate-early enhancer-promoter region (Promega Corporation, Madison, WI). Reporters were detected using the dual-luciferase reporter assays system (Promega). To normalize the data, Renilla luciferase activity was measured in unstimulated cells. All electroporations (2 × 107 cells per cuvette) were carried out at 250 V and 960 μF on a Gene Pulser™ (Bio-Rad). Lipofectamine 2000 reagent (Invitrogen) was used to transfect retroviral and lentiviral packaging cell lines, Phoenix Ecotropic cells and 293 T cells. Viral Transductions—Two types of viral infection were performed. For experiments involving the use of a retrovirus, the supernatants were obtained from cultures of Phoenix Ecotropic packaging cells transfected with Ras effector loop mutants or Imp constructs cloned into pMSCV, and applied directly to CD4 T cells. For experiments with lentivirus, 293T cells were cotransfected with pLL3.7 and packaging vectors (38Rubinson D.A. Dillon C.P. Kwiatkowski A.V. Sievers C. Yang L. Kopinja J. Rooney D.L. Zhang M. Ihrig M. McManus M.T. Nat. Genet. 2003; 33: 401-406Crossref PubMed Scopus (1348) Google Scholar), and collected after 60 h. The lentivirus was recovered by ultracentrifugation for 2 h at 23,000 × g and resuspension in culture medium. The viral preparations were delivered to CD4 T cells in the presence of Polybrene (final concentration: 8 μg/ml) by centrifugation for 1.5 h at 2,000 × g (37McCarty N. Paust S. Ikizawa K. Dan I. Li X. Cantor H. Nat. Immunol. 2005; 6: 65-72Crossref PubMed Scopus (52) Google Scholar). RNA Interference—The oligonucleotide sequences used to generate Imp RNAi number 7 were as follows: 5′-GATCCGGGAAGTCAGCCGGGGAGATTCAAGAGATCTCTCCTGGTGACTTGCCTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAAAGGCAAGTCACCAGGAGAGATCTCTTGAATCTCTCCTGGTGACTTGCCG. The sequences targeted by RNAi 8, 9, and 11 were GTCCAACCCAGATGAACTA, GTAAAGATCACAGTAAGGA, and GACAAATAAGATGACCTCC, respectively. To generate Imp that is RNAi-resistant, the target sequence was changed from GGGAAGTCAGCCGGGGAGA to GGTAAATCGGCAGGAGAAA. The sequences of primers used to generate CD8-specific RNAi were exactly the same as those described elsewhere (38Rubinson D.A. Dillon C.P. Kwiatkowski A.V. Sievers C. Yang L. Kopinja J. Rooney D.L. Zhang M. Ihrig M. McManus M.T. Nat. Genet. 2003; 33: 401-406Crossref PubMed Scopus (1348) Google Scholar). Quantitative PCR—CD4 T cells were isolated from B10.BR mice and sorted for a naïve population expressing CD62Lhi CD44lo. RNA was prepared using a TRIzol reagent and the reverse transcription was carried out using oligo(dT) as a primer and SuperScript™ II Reverse Transcriptase (Invitrogen) as described in the manufacturer's protocol (Invitrogen). Quantitative PCR assays were performed on a Mx3000P® platform (Stratagene) using 25 μl of a reaction mixture that contained 8 to 10 ng of cDNA, SYBR Green, dNTP, primers, and platinum Taq polymerase (Invitrogen). Cycling conditions were as follows: incubation at 95 °C for 3 min, and 40 cycles at 94 °C for 10 s, 60 °C for 15 s, and 72 °C for 27 s. A melting curve was added between 55 and 95 °C. Amplification of hypoxanthine phosphoribosyltransferase gene mRNA was used as an internal control to normalize the data. For Imp the forward primer was 5′-GTGGAAAGGAAGTGTACCCAG-3′ and the reverse primer was 5′-CTCCGCAGGCAGGTGGCTGATCTG-3′. For S1P1 the forward primer was 5′-GACAACCCAGAGACCATTATG-3′ and the reverse primer was 5′-GATCAAGTCAGAATGCTTCCCTCA-3′. For S1P4 the forward primer was 5′-CGTGTTCAACTCAGCCATTAATCC-3′ and the reverse primer was 5′-CTGAAGCTGAGTGACCGAGAAGTC-3′. For hypoxanthine phosphoribosyltransferase the forward primer was 5′-CCAGCAAGCTTGCAACCTTAACCA-3′ and the reverse primer was 5′-ATGATCAGTCAACGGGGGAC-3′. Mice—AND TCR B10.BR, AND TCR B10.A (5R), and OT2 TCR C57BL/6 mice were used as donors of TCR transgenic CD4 T cells. B10.BR, B10.A (5R), and C57BL/6 mice were used as donors of APCs. In adoptive transfer experiments, C57BL/6 and C57BL/6 IFNγR knock-out mice were also used as recipients of RasV12G37 or GFP control CD4 T cells. All animal experiments in this work were done in accordance with the institutional guidelines of the Yale Animal Resources Center (YARC). Generation of RasV12G37 Transgenic Mice—FLAG-tagged RasV12G37 was subcloned into pBI-EGFP plasmid (Clontech) containing a bidirectional tetracycline-responsive promoter. The transgenic expression cassette was subsequently cut out using AseI and PshAI restriction enzymes, then purified, and injected into the embryos of F2(C57BL/6JxSJL/J) mice at Yale Animal Genomic Services. The transgene-positive weanling mice were identified by PCR analysis of the tail biopsies. The following primers were used to amplify a 225-bp fragment containing the 5′ sequence of FLAG-Ras: 5′-CCAGCCTCCGCGGCCCCGAATTCG-3′ and 5′-GATGACCACCTGCTTCCGGTAGG-3′, and a 296-bp fragment containing the 5′ sequence of the EGFP gene: 5′-GCTCGTTTAGTGAACCGTCAGATC-3′ and 5′-GCTTGCCGGTGGTGCAGATGAAC-3′. To identify the founders, the animals that belonged to the F1 generation of a breeding cross between 296-bp positive/225-bp positive and M2 mice (these mice express reverse tetracycline-controlled transactivator), were fed a grain-based doxocycline diet (2.3 g/kg) (BioServ, Laurel, MD) for 2 weeks and then screened by FACS for the presence of EGFP-positive CD4 T cells in the peripheral blood and lymph nodes. Adoptive Cell Transfer Experiments—C57BL/6 or C57BL/6 IFNγR knock-out mice were injected with ∼5 × 106 CD4 T cells transduced with either RasV12G37 or an empty vector control. After 48 h, the mice were sacrificed and lymphocytes were isolated from their inguinal lymph nodes for staining with a mixture of anti-CD4 and anti-TCR mAbs (see below) and for FACS analysis. In some experiments, we blocked further entry of transferred cells by injecting the mice intraperitoneally with 100 μg of anti-LFA1 and VLA-4 mAbs (33Czyzyk J. Brogdon J.L. Badou A. Henegariu O. Preston Hurlburt P. Flavell R.A. Bottomly K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6003-6008Crossref PubMed Scopus (15) Google Scholar), and performing analysis after 15 h, as described above. Migration Assay—Transwell chemotaxis assays were performed using 24-well plates and 3-μm pore width filters (BD Biosciences). The lower chamber of the transwell chamber contained 0.6 ml of SP diluted in chemotaxis medium (RPMI1640, 0.5% bovine serum albumin, 10 mm Hepes). The cells were diluted in chemotaxis medium, and 0.1 ml (3 × 105 cells) was added to the top chamber. After 8 h at 37 °C, the cells that had migrated into the lower chamber were collected and counted in four high-power fields using a hemocytometer. The assays were performed in duplicate for each SP concentration. Intracellular Cytokine Staining and FACS Analysis—GFP-sorted CD4 T cells expressing both siRNA from the pLL3.7 lentiviral vector and Ras effector loop mutants from a pMIGR2 retroviral plasmid were stimulated with the antigen for 12 h and cultured for an additional 4 h in the presence of GolgiStop reagent (BD Biosciences) to trap cytokines in the endoplasmic reticulum. Cells were then processed with biotin-conjugated anti-human CD2 mAb followed by streptavidin-Alexa 647 (Invitrogen) for extracellular staining, and PE-labeled anti-IFN-γ mAb for intracellular staining. In adoptive transfer experiments, GFP-positive CD4 T cells were stained with APC anti-CD4, PE anti-Vβ5 TCR, and biotin anti-Vα2 TCR followed by Per-CP streptavidin. Western Blots and Immunoprecipitation—T cells were lysed in a buffer containing 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris (pH 7.4), 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride and protease inhibitor (Roche Diagnostics). Protein samples from precleared cell lysates were fractioned under reducing conditions on a sodium dodecyl sulfate-9% polyacrylamide gel. To verify expression of Imp constructs, proteins samples were fractioned on a SDS 6–18% gradient polyacrylamide gel. After electrophoresis, proteins were electroblotted onto nitrocellulose membranes (Bio-Rad), blocked with either 5% nonfat dry milk or 3% bovine serum albumin in phosphate-buffered saline (staining with p-ERK mAb), probed with the first antibody, and incubated with the electrochemiluminescent (ECL) anti-mouse IgG-horseradish peroxidase-linked antibody (Amersham Biosciences). The immunoblots were developed using an ECL detection system (Amersham Biosciences). For immunoprecipitation, immune complexes were recovered with protein A/G-conjugated-Sepharose beads (Amersham Biosciences) and further analyzed by the immunoblot procedure exactly as described above. Yeast Two-hybrid Assay—We evaluated the relationship between Imp and Ras using the yeast two-hybrid assay, H-RasV12 or H-RasV12 effector loop mutants were cloned inframe with the GAL4 DNA-binding domain within pGBKT7, and a region of Imp encompassing residues 273 to 377 was cloned in-frame with a 768–881-amino acid fragment of the GAL4 activation domain within the pGAD vector. Next, AH109- and Y187-competent yeast cells transformed with pGAD and pGBKT7 plasmids, respectively, were mated and transferred onto plates with SD minimal media containing a –leucine (Leu)/-tryptophan (Trp) dropout supplement. Positive clones were transferred onto new plates with a quadruple dropout supplement (-adenine (Ade)/-histidine(His)/-Leu/-Trp) and incubated at 30 °C for 5 days until colonies appeared. Cytokine Assays—IFN-γ and IL-4 levels from cell supernatants were determined by enzyme-linked immunosorbent assay (ELISA, Endogen, Cambridge, MA). The lower limit of sensitivity for the ELISA for IFN-γ and IL-4 was 0.6 ng/ml and 10 pg/ml, respectively. Statistical Analysis—Mann-Whitney analysis was performed to assess influence of RasV12G37 on T cell retention in the lymph nodes. The level of significance was accepted at p < 0.05. Imp Is a Potent Down-regulator of T Cells Responses—In our first attempt to examine the potential role of Imp in T cell activation, we used human Jurkat T cells that had been transfected with anti-Imp-specific RNAi. We used RNAi 7 because it had the strongest inhibitory effect on Imp (Fig. 1A). Two independent Jurkat clones (17.11 and 17.12) that had been stably transfected with this RNAi demonstrated up-regulation of the phosphorylated form of ERK following stimulation with anti-CD3 mAb, compared with control clones (8.1 and 8.3) carrying CD8a-specific RNAi (Fig. 1B). In addition, in 17.11 and 17.12 Jurkat clones the NFAT reporter demonstrated a markedly increased transcriptional response, compared with control clones (Fig. 1C). Of note, unstimulated Jurkat T cells expressing distinct levels of Imp demonstrated similar levels of p-ERK (Fig. 1B) and NFAT reporter activity (Fig. 1C, inset). We then explored the possibility that overexpression of Imp inhibits T cells from inducing a cytokine response. We found that eptopically expressed wild type Imp has a relatively weak suppressive effect in wild type Jurkat T cells (data not shown). Rapid degradation or lack of Imp in close proximity to Ksr in the overexpression system may be responsible for this insufficiency. Therefore, we generated a membrane-targeted form of full-length Imp in which a consensus C-terminal CAAX (C-cysteine, A = aliphatic amino acid, and X = any amino acid) and the sequence encoding the 19 C-terminal residue of K-Ras4B were introduced (Imp-CAAX) (34Clark J.C. Quilliam L.A. Hisaka M.M. Der C.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4887-4891Crossref PubMed Scopus (55) Google Scholar) (Fig. 2A). In addition, we generated another Imp mutant in which the first cysteine in RING-H2 was changed to alanine (C264A) (Fig. 2A). This Imp variant is thought to be resistant to degradation (28Matheny S.A. Chen C. Kortum R.L. Razidlo G.L. Lewis R.E. White M. Nature. 2004; 427: 256-260Crossref PubMed Scopus (163) Google Scholar). In contrast to wild type Imp, the Imp-CAAX variant appeared to be more effective in repressing the IL-2, IL-4, and IFN-γ response in normal CD4 T cells (Fig. 2C). Similar effects were seen with overexpression of Imp-CAAX, which partially blocked NFAT reporter activity in Jurkat T cells. Interestingly, Jurkat cells (e.g. 17.11), which expressed lowered levels of Imp, were more sensitive to the repressing activity of Imp-CAAX (Fig. 2D, right panel) compared with Jurkat cells (e.g. 8.1) expressing normal levels of this molecule (Fig. 2D, left panel). Additionally, a biochemical analysis of the phosphorylated form of ERK revealed Imp-dependent down-regulation of this kinase in antigen-stimulated CD4 T cells (Fig. 2E). Thus, our observations suggest that Imp is a negative regulator of T cell activation. p21 Ras-mediated Repression of the Inhibitory Effect of Imp Is Responsible for Enhanced Cytokine Output by CD4 T Cells—According to a recent study (28Matheny S.A. Chen C. Kortum R.L. Razidlo G.L. Lewis R.E. White M. Nature. 2004; 427: 256-260Crossref PubMed Scopus (163) Google Scholar) Imp is a novel effector of Ras. Therefore, we decided to explore the possibil
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