Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes
2016; Elsevier BV; Volume: 15; Issue: 6 Linguagem: Inglês
10.1074/mcp.m115.057158
ISSN1535-9484
AutoresNerea Osinalde, Jone Mitxelena, Virginia Sánchez‐Quiles, Vyacheslav Akimov, Kerman Aloria, Jesús M. Arizmendi, Ana M. Zubiaga, Blagoy Blagoev, Irina Kratchmarova,
Tópico(s)Immune Cell Function and Interaction
ResumoAnti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4+ T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies. Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4+ T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies. The underlying principle of cancer immunotherapy is to eliminate malignant cells by tuning the immune system (1.Restifo N.P. Dudley M.E. Rosenberg S.A. Adoptive immunotherapy for cancer: harnessing the T cell response.Nat. Rev. Immunol. 2012; 12: 269-281Crossref PubMed Scopus (1213) Google Scholar, 2.Vanneman M. Dranoff G. Combining immunotherapy and targeted therapies in cancer treatment.Nat. Rev. Cancer. 2012; 12: 237-251Crossref PubMed Scopus (1072) Google Scholar). 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Importantly, over 300 phosphosites were found to be specifically regulated upon IL-2 stimulation, including Ser455 on ATP-citrate lyase (ACLY), a metabolic enzyme responsible for generating acetyl-CoA. By inhibiting or depleting ACLY in T cells we demonstrate the relevance of this enzyme in IL-2-dependent modulation of histone acetylation levels and T-cell proliferation. Moreover, our study provides a collection of candidates for future hypothesis-driven experiments that could clarify the regulatory networks that lead to IL-2-triggered expansion of T cells and may serve as targets for cancer immunotherapy. Human recombinant IL-2 was kindly provided by "AIDS Research and Reference Reagent Program", Division of AIDS (NIH, National Institute of Health, Bethesda, MD). The following antibodies from (Cell Signaling, Danver, MA) were used for Western blotting assays: AKT (#-9272), phospho-AKT (Ser473) (#-4060), ACLY (#-13390), phospho-ACLY (Ser455) (#-4331), ERK1/2 (#-9102), phospho-ERK1/2 pThr202/Tyr204 (#-9101), Histone H3 (#-9715), and phospho-STAT5 (Tyr695) (#-9351). α-tubulin (T6199) was purchased from (Sigma-Aldrich, Copenhaguen, Denmark). Anti-acetyl histone H3 (06–599) and anti-acetyl histone H4 (06–598) used for Western blotting and chromatin immunoprecipitation were purchased from (Millipore, Darmstadt, Munich, Germany). The HPR-conjugated secondary antibodies anti-mouse (NA931) and anti-rabbit (NA934) were purchased from GE Healthcare. AKT inhibitor MK-2206 and HDAC inhibitor SAHA were purchased from (Selleckchem, Munich, Germany) MEK inhibitor U0126 from (Promega, Madison, WI) and ACLY inhibitor SB-204990 from (Tocris, Bristol, UK). Information concerning stable isotope labeled (SIL) peptides purchased for PRM assay and sequences of the qRT-PCR primers as well as primers used for site-directed mutagenesis are provided in supplemental Table S1. Human leukemic Kit225 T lymphocytes were maintained in RPMI 1640 media (Gibco, Hvidovre, Denmark) supplemented with 10% FBS, 1% glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin and 16 U/ml of IL-2 at a density of 1.106 cell/ml at 37 °C and 5% CO2. Cell number was estimated using NucleoCounter (ChemoMetec, Allerod, Denmark). For the large-scale SILAC experiments, Kit225 T cells were grown in media containing either the light (Arg0/Lys0) or heavy (Arg6/Lys4) isotopes of lysine and arginine. Prior to cytokine treatment, cells were IL-2-starved for 48 h to synchronize them at G1 phase of the cell cycle. Stimulation was performed by incubating cells with 200 U/ml of IL-2 for 5 min at 37 °C and rapidly quenched by placing cells on ice and washing with cold PBS. In the SILAC experiments, T cells grown in light media were kept unstimulated and served as control whereas heavily labeled Kit225 T cells were treated with the cytokine. Human U2OS osteosarcoma cell line was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). The lentiviral vector pSicoR was obtained from (Addgene, Cambridge, MA) (Addgene Plasmid 11579) in accordance with Material Transfer Agreements (MTAs) and the lentiviral vector pCDF1-MCS2-EF1-Puro was purchased from (System Biosciences Co, Palo Alto, CA). In order to create a DNA construct for ACLY expression, the pSicoR plasmid was modified so that the U6-ShRNA cloning cassette was exchanged with EF1alpha-eYFP construct by PCR methods and conventional cloning. Simiarly, the CMV-eGFP cassette on the pSicoR plasmid was exchanged by EF1-Puro cassette from vector pCDF1-MCS2-EF1-Puro and puromycin resistance gene was substituted by ORF of ACLY cDNA. ACLY cDNA containing each single point mutation of interest was generated by standard site-directed DNA mutagenesis. The sequences of several clones were verified by DNA sequencing and error free clones were chosen for transfections. U2OS cells were transfected using XtremeGENE HD (Roche, Hvidovre, Denmark) following manufacturer′s recommendations. The RNAi sequence potentially targeting human ACLY transcript was generated using available web resources (http://www.dharmacon.com) according to published recommendations for siRNA/shRNA design (34.Birmingham A. Anderson E. Sullivan K. Reynolds A. Boese Q. Leake D. Karpilow J. Khvorova A. A protocol for designing siRNAs with high functionality and specificity.Nat. Protoc. 2007; 2: 2068-2078Crossref PubMed Scopus (155) Google Scholar, 35.Taxman D.J. Livingstone L.R. Zhang J. Conti B.J. Iocca H.A. Williams K.L. Lich J.D. Ting J.P. Reed W. Criteria for effective design, construction, and gene knockdown by shRNA vectors.BMC Biotechnol. 2006; 6: 7Crossref PubMed Scopus (99) Google Scholar). The shRNA sequences were synthesized (DNA technology, Denmark) as two complementary DNA oligonucleotides: T(N19)TTCAAGAGA(rN19)TTTTTTC and TCGAGAAAAAA(N19)TCTCTTGAA(rN19)A where N19 is the 19-nt sense strand of the target sequence and rN19 is the complementary antisense strand. The targeting sequence (N19) we used for silencing ACLY was 5′-GGAGGAAGGGAATGAAACA-3′ whereas the random sequence 5′-GCAATATGACGAGTTAGTA-3′ was used as control. Annealed oligonucleotides were directly cloned into the lentiviral pSicoR-puro vector that allows puromycin resistance-based selection of shRNA-expressing cells. After DNA constructs were verified, lentiviral particles were generated as previously described (36.Chylek L.A. Akimov V. Dengjel J. Rigbolt K.T. Hu B. Hlavacek W.S. Blagoev B. Phosphorylation site dynamics of early T-cell receptor signaling.PLoS ONE. 2014; 9: e104240Crossref PubMed Scopus (45) Google Scholar). Briefly, HEK-293T cells were co-transfected with the lentiviral vector and the virus packaging plasmids using Metafectene and supernatants were collected 48 h and 72 h postinfection. Then viral particles were concentrated by ultracentrifugation and used for infection of Kit225 T cells to generate stable cell lines expressing the described RNAi constructs. Unstimulated (Arg0/Lys0) and IL-2-treated (Arg6/Lys4) Kit225 T cells were subjected to cytosol and nuclear enrichment. Briefly, cells were resuspended in a lysis buffer (25 mm TrisHCl pH 7.5, 100 mm NaCl, 1% Nonidet P-40, complete protease inhibitor mixture tablets (Roche) and phosphoSTOP tablets (Sigma-Aldrich), and centrifuged for 15 min at 13,000 rpm. Cytosolic fraction was recovered and nuclei-containing pellet was disrupted by resuspending in urea buffer (8 m urea, 10 mm TrisHCl pH 7.5) and sonication. After estimating protein abundance by BCA method, light and heavy cell lysates were combined in 1:1 ratio according to their protein concentration and subjected to in-solution digestion using LysC and trypsin as previously described (37.Rigbolt K.T. Prokhorova T.A. Akimov V. Henningsen J. Johansen P.T. Kratchmarova I. Kassem M. Mann M. Olsen J.V. Blagoev B. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation.Sci. Signal. 2011; 4: rs3Crossref PubMed Scopus (363) Google Scholar). Proteolytic digestion products were desalted on a Sep-Pak C18 cartridge (Waters, Milford), lyophilized for 2 days and enriched in phosphopeptides by consecutive incubations with TiO2 beads (38.Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jorgensen T.J. 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Shotgun proteomic analyses were carried out using a reverse phase liquid chromatography system (EASY-nLC 1000 ultrahigh pressure, Thermo Fisher Scientific) interfaced with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA) via a nanoelectrospray source (Thermo Fisher Scientific). Acidified peptides were loaded on an analytical in-house packed column (20 cm × 75 μm, ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH)) in solvent A (0.5% acetic acid) and eluted by a nonlinear 120 min solvent B gradient (0.5% acetic acid, 80% acetonitrile) at a flow rate of 250 nl/min. Q Exactive was operated in a top 10 data dependent mode. Survey scans were acquired at a resolution of 70,000 (m/z 400) and fragmentation spectra at 35,000 (m/z 400). Precursors were fragmented by higher energy C-trap dissociation (HCD) with normalized collision energy of 25 eV. The maximum injection time was 120 ms for survey and 124 ms for MS/MS scan whereas the AGC target values of 1e6 and 1e4 were used for survey scans and for MS/MS scans, respectively. Repeated sequencing of peptide was minimized by excluding the selected peptide candidates for 45 s. All raw data files acquired were searched against the UniProt human database version 2014.01 (with 88,479 sequence entries) with MaxQuant proteomics computational platform version 1.3.0.5 and using Andromeda search engine (41.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3450) Google Scholar). SILAC doublets were selected for the analysis where light and heavy labels were set as Arg0/Lys0 and Arg6/Lys4. Precursor and fragment mass tolerances were set to 7 and 20 ppm, respectively. Enzyme specificity was set to trypsin, allowing for cleavage N-terminal to proline and between aspartic acid and proline (with a maximum of 2 missed cleavages). Carbamidomethylation of C was set as fixed modification whereas oxidation of M, protein N-terminal acetylation, NQ deamidation and STY phosphorylation were selected as variable modifications for database searching. For the analysis of phosphopeptides, 1% FDR, a minimum localization probability of 0.75 and a score difference of at least 5 was used (42.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2810) Google Scholar). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via PRIDE partner repository (43.Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. 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Phosphopeptides were air dried in a Speedvac and resuspended with the SIL phosphopeptide mixture. Samples were loaded onto an Acclaim PepMap100 precolumn (75 μm × 2 cm, ThermoFisher Scientific) connected to an Acclaim PepMap RSLC (50 μm × 15 cm, Thermo Scientific) analytical column. Peptides were eluted with a 90 min linear gradient from 3% to 30% of acetonitrile in 0.1% of formic acid at a flow rate of 300 nl/min directly onto the nanoES Emitter (ThermoFisher Scientific). The Q Exactive was operated in Targeted-MS2 mode and method optimization was achieved by analysis of SIL phosphopeptide by Full MS, Full MS/dd-MS2 (Top10) and Targeted-MS2. Then, selected m/z values were incorporated in an inclusion list and specific retention time windows were applied based on method optimization results. Spectra were acquired at a resolution of 17,500 (m/z 200). Peptide selection was done with an isolation window of 2.0 Th and normalized collision energy of 28 was applied for peptide fragmentation. The maximum injection time was 500 ms and an AGC target value of 5e5 was used. For peptide relative quantification Skyline v2.6.0.6851 software was used (45.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2964) Google Scholar). All integrated peaks were manually inspected to ensur
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