Global and Specific Translational Control by Rapamycin in T Cells ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 25, Issue of June 21, pp. 22175–22184, 2002 Printed in U.S.A.

Global and Specific Translational Control by Rapamycin in T Cells Uncovered by Microarrays and Proteomics* Received for publication, February 28, 2002 Published, JBC Papers in Press, April 9, 2002, DOI 10.1074/jbc.M202014200

Annabelle Grolleau‡, Jessica Bowman‡, Be´renge`re Pradet-Balade§, Eric Puravs¶, Samir Hanash¶, Jose A. Garcia-Sanz§, and Laura Beretta‡储 From the ‡Department of Microbiology and Immunology, and the ¶Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109 and the §Department of Immunology and Oncology, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientı´ficas, Madrid E-28049, Spain

Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysomebound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes. Another was to determine protein synthesis levels using two-dimensional PAGE. Analysis of expression changes at the polysome-bound RNA levels showed that translation of most of the expressed genes was partially reduced following rapamycin treatment. However, translation of 136 genes (6% of the expressed genes) was totally inhibited. This group included genes encoding RNA-binding proteins and several proteasome subunit members. Translation of a set of 159 genes (7%) was largely unaffected by rapamycin treatment. These genes included transcription factors, kinases, phosphatases, and members of the RAS superfamily. Analysis of [35S]methionine-labeled proteins from the same cell populations using two-dimensional PAGE showed that the integrated intensity of 111 of 830 protein spots changed in rapamycin-treated cells by at least 3-fold (70 increased, 41 decreased). We identified 22 affected protein spots representing protein products of 16 genes. The combined microarray and proteomic approach has uncovered novel genes affected by rapamycin that may be involved in its immunosuppressive effect and other genes that are not affected at the level of translation in a context of general inhibition of cap-dependent translation.

Rapamycin is a macrolide antibiotic originally isolated from Streptomyces hygroscopicus (1). It is a potent immunosuppressant with therapeutic applications in the prevention of organ allograft rejection and in the treatment of autoimmune disease (2– 6). The importance of rapamycin as an immunosuppressant drug has focused attention on its mechanism of action. Rapamycin has a similar biochemical structure to cyclosporin A and FK506. However, unlike cyclosporin A and FK506, rapamycin is not a calcineurin inhibitor (7). The primary mode of immu* This work was supported by National Institutes of Health Grant 1RO1-AI50896 (to L. B.) and an EU TMR network grant (Contract ERBFMRXCT980197 (to J. A. G. S.)). The Department of Immunology and Oncology was founded and is supported by the Spanish Research Council (Consejo Superior de Investigaciones Cientı´ficas) and Amersham Biosciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 储 To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109. Tel.: 734-615-5964; Fax: 734-615-6150; E-mail: [email protected]. This paper is available on line at http://www.jbc.org

nosuppressive action of rapamycin is an antiproliferative action reflecting the ability of the drug to disrupt signaling by T cell growth-promoting lymphokines such as IL-21 and IL-4 (8). The growth-inhibitory effects of rapamycin are not limited to T cells, since this drug inhibits the proliferation of many mammalian cell types as well as that of yeast cells (9). Rapamycin blocks progression of the cell cycle at the G1 phase by binding to FKBP12 (FK506-binding protein) (10, 11). The rapamycin-FKBP12 complex inhibits mTOR (mammalian target of rapamycin), also referred to as FRAP (FKBP-rapamycin-associated protein) (9). Targets of mTOR include 4E-BP1 and the 40 S ribosomal protein S6 kinase, p70s6k (12–16). Rapamycin-induced inhibition of p70s6k activity and subsequent dephosphorylation of the ribosomal S6 protein lead to a selective translational repression of mRNA containing a polypyrimidine-rich tract (TOP) motif at their 5⬘ terminus (17). 4E-BP1 is a small heat- and acid-stable protein whose activity is regulated by phosphorylation. Dephosphorylated 4E-BP1 inhibits cap-dependent translation by binding to the mRNA capbinding protein eukaryotic initiation factor 4E (eIF4E) (18, 19). We previously reported that rapamycin blocks the phosphorylation of 4E-BP1 and inhibits specifically cap-dependent initiation of translation (12) and that, in contrast, rapamycin increases internal initiation of translation, a mechanism independent of the cap structure and reported so far for some viral and cellular mRNAs (20). We performed a systematic study to identify global and specific effects of rapamycin on translation. To determine rapamycin-sensitive transcripts, we used a methodology based on the separation of polysomes from mRNPs using sucrose gradient centrifugation followed by oligonucleotide microarray hybridization. This technology has been recently adapted for studies of translational control (21–23) and is based on the assumption that translationally inactive mRNAs are present as free cytoplasmic mRNPs, whereas actively translated mRNAs are contained within polysomes. This enables identification of mRNAs specifically mobilized from free mRNPs onto polysomes and vice versa in T cells in response to rapamycin. A complementary approach used proteomic analysis to systematically analyze gene expression in T cells in response to rapamycin. EXPERIMENTAL PROCEDURES

Cell Culture—The human Jurkat T cell clone E6 –1 (American Type Culture Collection, Manassas, VA) was grown in the presence of 10% heat-inactivated fetal calf serum, using RPMI 1640 medium supplemented with 2 mM L-glutamine, 10 mM Hepes buffer, and gentamycin (20 ␮g/ml). The day prior to performing the polysome profiles, the cells 1 The abbreviations used are: IL, interleukin; eIF4E, eukaryotic initiation factor 4E; IRES, internal ribosome entry site; MAP, mitogenactivated protein.

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Translational Control by Rapamycin

FIG. 1. Assessment of Jurkat E6 T cell sensitivity to rapamycin. A, cell growth curves of untreated and treated T cells. T cells were seeded at an initial density of 1.5 ⫻ 105 cells/ml without or with 20 ng/ml of rapamycin and were cultured for the indicated times without any change of media. Viable cells were counted after 24, 48, and 72 h of culture. The shown concentrations are the mean of three separate experiments, and the error bars indicate the S.D. B, protein synthesis rates in T cells. T cells (2 ⫻ 105) were preincubated 1 h in methionine-free medium. Rapamycin was added to the cells together with [35S]methionine (100 ␮Ci). Cells were harvested at 4 and 8 h, and radioactivity incorporated into trichloroacetic acid–precipitable material was measured. The effect of rapamycin is expressed as percentage of the control. The experiment was carried out three times, and the error bars indicate S.D. C, effect of rapamycin in 4E-BP1 phosphorylation. After 1- and 4-h exposure to rapamycin, T cells were lysed, and total protein extract was analyzed by Western blotting using polyclonal antibody to 4E-BP1 followed by monoclonal anti-actin. were seeded in fresh medium at a density of 105 cells/ml. When indicated, cells were incubated with 20 ng/ml rapamycin (Calbiochem). For the cell proliferation assay, cells were seeded at an initial density of 1.5 ⫻ 105 cells/ml with or without rapamycin and cultured for 3 days without any change of media. Cell proliferation was monitored every 24 h by determining cell number with a Coulter counter ZM equipped with a Coultronic 256 channelizer (Hialeah, FL). Metabolic Labeling—Jurkat cells were preincubated at 37 °C for 1 h in methionine-free RPMI 1640 medium. Rapamycin and [35S]methionine (100 ␮Ci; PerkinElmer Life Sciences) were added together for the indicated times, and the cells were either lysed in 20 mM Tris-HCl, pH 7.5, buffer containing 5 mM EDTA and 100 mM KCl for the measure of radioactivity incorporation rates after trichloroacetic acid precipitation or were processed for two-dimensional PAGE analysis. Western Blotting Analysis of 4E-BP1—Untreated and rapamycintreated Jurkat cells were rinsed twice with ice-cold phosphate-buffered saline and lysed by successive freeze-thaw cycles, in 20 mM Tris-HCl, pH 7.5, buffer containing 5 mM EDTA and 100 mM KCl. The homogenate was centrifuged at 6000 ⫻ g for 10 min, and the supernatant was collected. Proteins (100 ␮g) were loaded onto a 15% polyacrylamide gel, separated, and transferred onto a 0.22-␮m nitrocellulose membrane (Schleicher and Schuell). Following transfer, membranes were incubated for 2 h in blocking buffer containing 5% milk in 10 mM Tris-HCl, pH 7.5; 2.5 mM EDTA, pH 8; 50 mM NaCl. The membranes were incubated for 2 h with rabbit polyclonal antiserum against 4E-BP1 (TEBU, Le Peray-en-Yvelines, France) and actin (ICN Biomedical, Aurora, OH) at a dilution of 1:1000. The membranes were then incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit antibodies, at a 1:2000 dilution. Immunodetection was realized by ECL (Amersham Biosciences). Two-dimensional PAGE—The procedure followed was as previously described (24). Cells were solubilized in 200 ␮l of lysis buffer containing 9.5 M urea (Bio-Rad), 2% Nonidet P-40, 2% ␤-mercaptoethanol, 2%

carrier ampholytes, pH 4 – 8 (Gallard/Schlessinger, Carle Place, NY), and 10 mM phenylmethanesulfonyl fluoride. Aliquots containing 5 ⫻ 106 cells were applied onto isofocusing gels. Isoelectric focusing was conducted using pH 4 – 8 carrier ampholytes at 700 V for 16 h, followed by 1000 V for an additional 2 h. The first dimension gel was loaded onto the second dimension gel, after equilibration in 125 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 1% dithiothreitol, and bromphenol blue. For the second dimension separation, a gradient of 11–14% of acrylamide (Serva; Crescent Chemicals, Hauppauge, NY) was used. Gels were then either silver-stained or dried and exposed to an x-ray film. The gels were digitized at 1024 ⫻ 1024-pixel resolution using an Eastman Kodak Co. CCD camera. Spots were detected and quantified with Visage software (Genomic Solutions, Ann Arbor MI) as described (25). RNA Isolation and Polysome Fractionation—Total RNA was isolated using Trizol reagent (Invitrogen) and quantitated by absorbance at 260 nm. Cytoplasmic RNA was obtained by lysing cells in 1 ml of polysome buffer containing 10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, and a ribonuclease inhibitor, RNasin (500 units/ml; Promega, Madison, WI). After the removal of nuclei, the cytosolic supernatant was supplemented with 150 ␮g/ml cycloheximide, 665 ␮g/ml heparin, 20 mM dithiothreitol, and 1 mM phenylmethanesulfonyl fluoride. Mitochondria and membrane debris were removed by centrifugation, and postmitochondrial supernatant was applied directly to sucrose gradient for polysome separation as described previously (26). Briefly, 1 ml of postmitochondrial supernatant was overlaid onto a 15– 40% sucrose gradient and spun at 38,000 rpm for 2 h at 4 °C in a SW41Ti rotor (Beckman Instruments, Inc.). Fractions (500 ␮l) were collected from the bottom of each gradient and deproteinated with 100 ␮g of proteinase K in presence of 1% SDS and 10 mM EDTA. After Trizol extraction, the amount of RNA in each fraction was determined photometrically, and RNA integrity was controlled by electrophoresis analysis on denaturing 1.2% formaldehyde-agarose gels and subsequent Northern blot. After RNA transfer to nylon membranes (GeneScreen; PerkinElmer Life Sci-


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TABLE I Transcriptionally regulated mRNAs in rapamycin-treated T cells Rapamycin-modulated genes were classified according to their known function and -fold change and represented in clusters containing functionally related genes. ⬃, -fold change calculation for which the smaller value is replaced by an estimate of the minimum value for detectable transcripts. GenBank™ accession no.

Gene description

-Fold change -fold

Up-regulated Signaling/Growth control Z36714 U47414 U41804 U53174 X61123 U68485 U33822 Z15005 Z29630 X77909 Nuclear proteins U15655 U64675 Membrane proteins X83492 M58286 U97502 X57351 M62762 Metabolism/Structure Z14093 L07807 Down-regulated Signaling/Growth control D13639 AB000449 M15353 AB003103 D21090 X83368 AB003698 D11428 M74524 AB003102 AB003177 Nuclear proteins M85085 Y12393 D80003 AB000468 D87448 X79200 U80669 Secreted proteins M60278 Metabolism/Structure D50840 M57423 Z46376 D49489 Not classified D31888 D31887 D13645 D43636 D50923 D87447 D63875 D80004 D80001

Cyclin F Cyclin G2 IL-1 receptor-like 1 ligand RAD9 BTG1 Bridging integrator 1 MAD 1-like 1 CENPE Protein-tyrosine kinase Syk I-␬-B-like 1

⬃11.6 6.1 ⬃4.6 ⬃3.8 ⬃3.4 ⬃2.9 2.7 2.6 2.4 2.1

Ets2 repressor factor BS-63

⬃3.3 2.5

Fas/Apo-1 Tumor necrosis factor receptor Butyrophilin Interferon-induced transmembrane protein 2 Vacuolar H⫹ ATPase proton channel subunit

3.3 ⬃3.2 3.1 2.5 2.1

Branched chain decarboxylase ␣-subunit Dynamin 1

6.8 ⬃5.0

Cyclin D2 Vaccinia related kinase 1 (VRK1) Translation initiation factor 4E (elF4E) 26 S proteasome subunit p55 RAD23B Phosphatidylinositol 3-kinase ␥ Cdc7-related kinase Peripheral myelin protein 22 (PMP22) Ubiquitin-conjugating enzyme E2A 26 S proteasome subunit p44.5 26 S proteasome subunit p27

⬃⫺3.3 ⫺2.5 ⫺2.4 ⫺2.4 ⫺2.3 ⬃⫺2.3 ⫺2.1 ⫺2.1 ⫺2.1 ⫺2 ⫺2

Cleavage stimulation factor CSTF2 Karyopherin ␣-4 RAP250 Ring finger protein 4 Topoisomerase (DNA) II-binding protein Synovial sarcoma, X breakpoint 2 (SSX2) NKX3A

⫺3 ⫺2.9 ⫺2.6 ⫺2.3 ⫺2.3 ⫺2.3 ⫺2.1

Heparin-binding EGF-like growth factor (DTR)

⫺2

Ceramide glucosyltransferase Phosphoribosylpyrophosphate synthetase, III Hexokinase 2 Protein-disulfide isomerase-related protein

⫺5.1 ⫺2.7 ⫺2.6 ⫺2

KIAA0071 KIAA0062 KIAA0020 KIAA0096 KIAA0133 KIAA0258 KIAA0155 KIAA0182 KIAA0179

⫺3.4 ⫺2.7 ⫺2.6 ⫺2.6 ⫺2.5 ⫺2.5 ⫺2.2 ⫺2.2 ⫺2.1

ences) and UV cross-linking, the distribution of 18 and 28 S rRNAs was visualized by methylene blue staining of the membranes (see Fig. 2). Fractions 10 –19 and fractions 1–9 corresponding to polysome-bound and nonpolysome RNA, respectively, were pooled from each sucrose gradient according to the distribution profile. Poly(A⫹) RNA was isolated from total and polysome-bound RNA by using oligo(dT) resin (Oligotex; Qiagen, Chatsworth, CA).

Preparation of cRNA, Gene Chip Hybridization, and Data Analysis— Preparation of cRNA, hybridization, and scanning of the HuGeneFL arrays were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA) and as previously described (27). Briefly, 5 ␮g of poly(A⫹) from both total and polysome-bound RNA were converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Invitrogen). Following second

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FIG. 2. Representative polysome profile of T cells. RNA was extracted from each of the 20 sucrose gradient fractions and subsequently transferred onto a nylon membrane. Staining of the filter with methylene blue indicates the distribution of 28 and 18 S RNA. strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). Aliquots of each sample (10 ␮g of fragmented cRNA in 200 ␮l of hybridization mixture) were hybridized to HuGeneFL arrays at 45 °C for 16 h in an oven set at 60 rpm. Hybridization was revealed with streptavidinphycoerythrin (Molecular Probes, Inc., Eugene, OR), stained with biotinylated anti-streptavidin IgG, followed by a second staining with streptavidin-phycoerythrin. The arrays were scanned using the GeneArray scanner (Affymetrix). Data analysis was performed using GeneChip 4.0 software. The software includes algorithms that determine whether a gene is absent or present and whether the expression level of a gene in an experimental sample is significantly increased or decreased relative to a control sample. The microarrays contained more than one probe for the same transcript in many instances. We verified that the responses were consistent for all probes for a same transcript. The comparison of the data analysis obtained from the two experiments indicated that both experiments were highly reproducible. RESULTS

Selection of a Rapamycin-sensitive T Cell Line—In mammalian cells, rapamycin causes partial inhibition of cell proliferation and translation rates ranging from 15 to 70% in different cell lines (3, 4). We investigated the sensitivity of the E6 –1 Jurkat T cell clone to rapamycin by three criteria: (i) inhibition of cell proliferation; (ii) reduction of protein synthesis rates; and (iii) induction of 4E-BP1 dephosphorylation. We first examined the effects of rapamycin on Jurkat T cell proliferation. The cells were cultured without or with 20 ng/ml of rapamycin during 72 h, and viable cells were counted at 24, 48, and 72 h (Fig. 1A). Rapamycin exerted a marked antiproliferative effect in T cells with a 43% inhibition observed at day 3. The translation rate was determined by metabolic labeling of Jurkat cells with [35S]methionine. Protein synthesis was rapidly and strongly inhibited by rapamycin, with a 32 and 44% inhibition observed after 4 and 8 h of rapamycin treatment, respectively (Fig. 1B). Finally, we examined the effects of rapamycin on 4E-BP1 phosphorylation by Western blotting using an anti-4EBP1 antibody. Three isoforms of 4E-BP1 (indicated by arrows, Fig. 1C) were detected following immunoblotting of extracts from Jurkat cells. It has been previously reported that these isoforms reflect different phosphorylation status of this protein (8, 13, 14). Treatment of the cells by rapamycin reduced the amount of the slowly migrating, hyperphosphorylated form of 4E-BP1, with a concomitant increase in the abundance of the faster migrating band corresponding to hypophosphorylated 4E-BP1. Slight dephosphorylation of 4E-BP1 was observed as early as 1 h after rapamycin treatment, whereas maximal dephosphorylation was obtained after 4 h. Therefore, the E6 –1 Jurkat T cells are sensitive to rapamycin treatment and were selected for further studies. Analysis of RNA Expression Levels in Jurkat Cells in Response to a Short Treatment of Rapamycin, Using Oligonucleotide Arrays—Poly(A⫹) mRNA were isolated from Jurkat T cells, with or without rapamycin treatment for 4 h, and poly(A⫹) mRNAs were isolated. Two independent experiments were per-

formed, and RNA transcript levels for different genes were determined using oligonucleotide arrays. Transcripts for ⬃2,800 genes (44%) of the 6,300 unique genes assessed were expressed in Jurkat T cells. We identified a small subset of genes (51) that differed in their expression levels during rapamycin treatment, by 2-fold or greater, in both experiments. The genes identified are presented in Table I, with 19 up-regulated and 32 down-regulated genes. Regulated genes included several growth-related genes that may contribute to the antiproliferative effect of rapamycin. Indeed, negative regulators of cell growth such as cyclin G2, MAD1-like 1, BTG1, bridging integrator 1, Syk, and CENPE were up-regulated, with a concomitant decrease in genes involved in cell cycle progression such as cyclin D2, Cdc7-related kinase, phosphatidylinositol 3-kinase ␥, CSTF2, and eIF4E. Up-regulation of I-␬B-like 1, Fas, and tumor necrosis factor receptor was also observed. Remarkably, expression of three subunits of the 26 S proteasome was decreased. Identification of Translationally Regulated Genes by Rapamycin, Using Oligonucleotide Arrays—To identify genes whose expression is translationally regulated, we combined a sucrose gradient separation of polysomes from mRNPs with microarray analysis. Polysome-bound mRNAs (Fig. 2, fractions 10 –19) were purified from Jurkat cells untreated or treated with rapamycin for 4 h, and poly(A⫹) mRNAs were isolated. Two independent experiments were performed, and polysomebound RNA transcript levels were determined using oligonucleotide arrays. Translation of the large majority of the genes was partially reduced following rapamycin treatment. However, translation of 136 genes was strongly inhibited (by 90% or more) in both experiments (Table II). Genes known to be highly repressed by rapamycin changed their expression accordingly in our analysis. This group included numerous ribosomal proteins and elongation factor proteins. However, for most of the 136 genes uncovered, their high sensitivity to rapamycin was unknown. These novel changes included other RNA-binding proteins such as translation initiation factors 4A and 5A and four genes encoding for nuclear ribonucleoproteins. Remarkably, translation of seven genes encoding proteasome subunits was fully inhibited following rapamycin treatment. Translation of prothymosin ␣, a gene associated with proliferation of T cells, was also strongly repressed by rapamycin. Microarray analysis of the non-polysome gradient fractions (Fig. 2, fractions 1–9) were also performed for both experiments and demonstrated that the 136 strongly repressed transcripts were not lost or degraded during rapamycin treatment or polysome separation. Transcripts levels for 159 genes remained bound to polysome following rapamycin treatment, suggesting that translation of these genes was not affected by rapamycin. Table III lists the genes whose mRNAs were associated with polysomes from both untreated and rapamycin-treated cells. Notably, this list includes mRNAs encoding a large number of kinases and phosphatases as well as DNA-binding proteins. Transcription factors and genes involved in DNA and RNA synthesis included AR1, TFIID, TFIIE, TFIIF, E2F, c-MYB, YY1, CREBP1, HSF1, Rb1, ILF1, LIM domain only 4, RNA polymerase II, DNA polymerase ␣-subunit, and replication factors C1 and C5. Translation of several genes encoding for kinases and phosphatases, such as four members of the mitogen-activated protein kinase family, the PI-3 kinase regulatory subunit, protein kinase C-␫, p72syk, and protein phosphatases 1, 2, and 4, was unaffected by rapamycin. Finally, transcripts for nine members of the Ras superfamily including N-Ras, Rap1a, Rap1b, Rab4, Rab5c, Rac1, and RhoG remained bound to polysomes in rapamycin-treated cells.


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TABLE II Translationally repressed mRNAs in rapamycin-treated T cells GenBank™ accession no.

Gene description

GenBank™ accession no.

Signaling/Growth control D00761 Proteasome subunit, ␤ type, 1 D26598 Proteasome subunit, ␤ type, 3

Z26876 Z12962 M14199

D26600 D29012

Proteasome subunit, ␤ type, 4 Proteasome subunit, ␤ type, 6

X56997 D13748

D38048 D00763 X59417 D38047

Proteasome Proteasome Proteasome Proteasome

D23662 M31469 L20688

Ubiquitin-like protein RAN, RAS oncogene family member Rho GDP dissociation inhibitor ␤

U15008 X85372 D13413

L32866 M31303 M22382 X15183

Apoptosis inhibitor 4 (survivin) Oncoprotein 18 (Op18), stathmin Heat shock 60-kDa protein 1 Heat shock 90-kDa protein 1, ␣

D28423 X71428 M60858 M23613

J04988 U48296 X52479 M55268 U77129 M84332 L38490 M17733 X52851 RNA metabolism M17885 X17206 M84711 X55715

subunit, ␤ type, 7 subunit, ␣ type, 4 subunit, ␣ type, 6 26 S subunit, non-ATPase, 8

Heat shock 90-kDa protein 1, ␤ Protein-tyrosine phosphatase, type IVA, Protein kinase C ␣ Casein kinase II ␣ subunit MAP kinase kinase kinase kinase 5 (MAP4K5) ADP-ribosylation factor 1 ADP-ribosylation factor 4-like Thymosin ␤-4 Cyclophilin A Ribosomal Ribosomal Ribosomal Ribosomal

protein, large, P0 protein S2 protein S3A protein S3

M58458 U14970 M77232 Z25749

Ribosomal Ribosomal Ribosomal Ribosomal

protein protein protein protein

X67247

Ribosomal protein S8

U14971 U14972 L01124 M13934 M32405 M60854 M18000 X69150 M81757 L04483 D14530 M31520 X69654 S79522 L19739 U14973 X73460 U14966 M36072 Z28407 U12404 X56932 L25899 L11566 X80822 X63527 U25789 X55954 U14968 L19527 Z49148 L05095 X03342 L38941 U12465 L06499 D23661

Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal Ribosomal

protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein protein

S4, X-linked S5 S6 S7

S9 S10 S13 S14 S15 S16 S17 S18 S19 S21 S23 S24 S26 S27a S27 S29 L3 L5 L7a L8 L10a L13a L15 L18 L18a L19 L21 L23 L27a L27 L29 L30 L32 L34 L35 L37a L37

S72024 J04617 X03689 X12517

Gene description

Ribosomal protein L38 Ribosomal protein L41 Laminin receptor 1 (67 kDa, ribosomal protein SA) (LAMR1) Ubiquitin ribosomal protein fusion product 1 Translation initiation factor 4A, isoform 1 (elF4A1) Translation initiation factor 5A (elF5A) Translation elongation factor 1 ␣ 1 Translation elongation factor TU Small nuclear ribonucleoprotein polypeptide C (snRPC) Small nuclear ribonucleoprotein D2 (snRPD2) Small nuclear ribonucleoprotein polypeptide F Heterogeneous nuclear ribonucleoprotein U (hnRPU) Pre-mRNA splicing factor SRp20 Fus Nucleolin Nucleophosmin (nucleolar phosphoprotein B23, numatrin) RNA binding motif protein 6

U50839 Nuclear protein U09477 p53-binding protein M14483 Prothymosin ␣ member 1 D17268 Wilm tumor-related protein J05614 Proliferating cell nuclear antigen (PCNA) D16581 8-Oxo-dGTPase U96915 Sin3-associated polypeptide p18 (SAP18) D63874 High-mobility group, protein 1 (HMG1) D21205 Zinc finger protein 147 U86602 Nucleolar protein p40 U18271 Thymopoietin Cell surface proteins M24194 Guanine nucleotide-binding protein (G protein), ␤ polypeptide 2-like 1 D15057 Defender against cell death 1 (DAD1) S71824 Neural cell adhesion molecule 1 (NCAM1) D29963 CD151 M31525 Major histocompatibility complex, class II, DN ␣ (HLA-DNA) D49824 Major histocompatibility complex, class I, B (HLAB) L11370 Protocadherin 1 (cadherin-like 1) Secreted proteins ␤2-Microglobulin S82297 D14838 Fibroblast growth factor 9 (FGF9) M37435 Colony-stimulating factor 1 (CSF1) Metabolism/Structure M13792 Adenosine deaminase X02152 Lactate dehydrogenase A X13794 Lactate dehydrogenase B U94586 NADH:ubiquinone oxidoreductase M91432 Medium-chain acyl-CaA dehyddrogenase D00596 Thymidylate synthetase ␣-enolase M14328 M24485 Glutathione S-transferase p. Y10807 Arginine methyltransferase D-Dpachrome tatuomerase U49785 J04603 Triose-phosphate isomerase X15341 Cytochrome c oxidase subunit VIa polypeptide 1 AC002115 Cytochrome c oxidase subunit VIb X13238 Cytochrome c oxidase subunit VIc M32879 Cytochrome P450, subfamily XIB Z71460 Vacuolar type H⫹-ATPase 115-kDa subunit D13118 ATP synthase subunit c D14710 ATP synthase ␣-subunit D16562 ATP synthase ␥-subunit J02683 ADP/ATP carrier protein ␤-tubulin V00599 ␤-actin X00351 D14678 Kinesin-like 2 M31212 Myosin, light chain Y00282 Ribophorin II U93205 Chloride intracellular channel 1 Not classified D21261 KIAA0120 D31885 KIAA0069 D23673 KIAA0864 D23673 KIAA0864

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Translational Control by Rapamycin TABLE III Translationally unaffected mRNAs in rapamycin-treated T cells


Gene description

Signaling/Growth control Z11695 MAP kinase 1 L05624 MAP kinase kinase 1 L20321 MAP kinase kinase 2 X75346 MAP kinase-activated protein kinase 2 M37712 p58/Cdc2-like 1 L11285 Serine/threonine kinase 2 U48736 Serine/threonine-protein kinase PRP4 S80267 Tyrosine kinase p72syk M34181 cAMP-dependent protein kinase catalytic subunit (C-␤ isoform) L33881 Protein kinase C, ␫ U00957 A-kinase anchor protein 10 X57206 Inositol 1,4,5-trisphosphate 3-kinase B M61906 Phosphoinositide 3-kinase, regulatory subunit L33801 Glycogen synthase kinase-3 ␤ M80359 MAP/microtubule affinity-regulating kinase 3 Y10275 Phosphoserine phosphatase M25393 Protein-tyrosine phosphatase, non-receptor type 2 M83738 Protein-tyrosine phosphatase, non-receptor type 9 X80910 Protein phosphatase 1, catalytic subunit, ␤ isoform L76703 Protein phosphatase 2, regulatory subunit B U79267 Protein phosphatase 4, regulatory subunit 1 X02751 N-Ras D78132 Ras-related GTP-binding protein M22995 Rap1a X08004 Rap1b X79353 Rab GDI-alpha M28211 Rab4 U11293 Rab5C M29870 Rac1 X61587 Rho G M19645 Bip/GRP78 D88378 Proteasome inhibitor subunit 1 X78140 Ubiquitin-conjugating enzyme E2D 1 U58522 Ubiquitin-conjugating enzyme E2 M74091 Cyclin C Z36714 Cyclin F X77794 Cyclin G1 M83822 Cdc-4 like X82554 S-phase response (cyclin-related) Y08915 D90070 U18242 D26069 U16811 S78085 U65410 X17576 U40038 U23435

Immunoglobulin binding protein 1 PMA-induced protein 1 Noxa Calcium-modulating cyclophilin ligand Centaurin ␤ 2 BCL2-antagonist killer 1 (BAK1) Programmed cell death 2 Mad2 NCK-␣ GTP-binding protein ␣ q Abl-binding protein 3

M38591 S100 calcium-binding protein A10 RNA metabolism X64707 Ribosomal protein L13 U23946 RNA binding motif protein 5 U26032 Translation initiation factor elF-2␣ L19161 Translation initiation factor elF-2␥ U94855 Translation initiation factor 3 subunit 5 M75715 Translation termination factor 1 X95384 Translational inhibitor protein p14.5 X85237 Splicing factor 3a, subunit 1 (SAP114) M90104 Splicing factor Sc35 U77664 Ribonuclease P M67468 Fragile X mental retardation 1 (FMR1) Y11651 RNA 3⬘-terminal phosphate cyclase Nuclear proteins Y08765 Zinc finger protein 162 U09825 Zinc finger protein 173 U37251 Zinc finger protein 177 X95808 Zinc finger protein 261 X59739 Zinc finger protein, X-linked M37197 CCAAT-box-binding transcription factor U19345 Transcription factor 20 (AR1) X83928 Transcription factor TFIID X63469 Transcription factor TFIIE ␤ U15641 Transcription factor E2F4


X16901 M64673 M77698 M98833 U24576 M13666 X15875 M19701 X64229 U15655

Genesescription

General transcription factor IIF, polypeptide 2 Heat shock transcription factor 1 (HSF1) Transcription factor YY1 Transcription factor FLI-1 Transcription factor LIM domain only 4 c-Myb ATF2/CREBP1 Retinoblastoma 1 DEK oncogene Ets2 repressor factor

M25269 ELK1, member of Ets oncogene family X60787 Interleukin enhancer binding factor 1 X85786 DNA-binding regulatory factor X5 M63256 DNA binding protein CDR2 X06745 DNA polymerase ␣-subunit X74874 RNA polymerase II largest subunit L14922 Replication factor C1 L07540 Replication factor C5 X59543 Ribonucleotide reductase M1 polypeptide X59618 Ribonucleotide reductase M2 polypeptide U20979 Chromatin assembly factor 1, subunit A M63483 Matrin 3 Y08612 Nucleoporin NUP88 U66615 SMARCC1 L22343 Interferon-induced protein 75 D79984 SUPT6H X66397 TPR (to activated MET oncogene) U05237 Fetal Alzheimer antigen FAL2 U14680 BRCA1 U17989 Nuclear autoantigen GS2NA X86098 Adenovirus 5 E1A-binding protein U90547 Ro/SSA ribonucleoprotein homolog X85133 Retinoblastoma-binding protein 6 D80000 SMC1-like 1 Membrane proteins Z17227 Interleukin-10 receptor, ␤ M58286 Tumor necrosis factor receptor X63717 Fas/APO-1 Y00285 Insulin-like growth factor II receptor U28811 Cysteine-rich fibroblast growth factor receptor (CFR-1) L77886 Protein-tyrosine phosphatase, receptor type, K X69878 vEGFR3/Fms-related tyrosine kinase 4 M11507 Transferrin receptor (CD71) X64647 T cell receptor ␣ U51587 Golgi autoantigen U51240 Lysosomal associated multitransmembrane protein U01691 Annexin V X65362 Sodium channel 1 U18009 Vesicle amine transport protein 1 S79873 Lysosomal associated membrane protein 2 (LAMP2) X69819 Intercellular adhesion molecule 3 (ICAM-3) X92396 Synaptobrevin-like 1 X68194 Synaptophysin-like protein X92098 RNP24 Secreted proteins M32304 Metalloproteinase inhibitor 2 U41745 Platelet-derived growth factor-associated protein 1 Metabolism/Structure M98045 Folylpolyglutamate synthase M34338 Spermidine synthase M95623 Hydroxymethylbilane synthase D11370 D-Amino acid oxidase X80695 Cytochrome c oxidase assembly 1-like U79270 Cytochrome c oxidase assembly protein U80034 Mitochondrial intermediate peptidase L35546 Glutamate-cysteine ligase D84307 Phosphoethanolamine cytidylyltransferase U09646 Carnitine palmitoyltransferase II L25441 Protein geranylgeranyltransferase type I ␤-subunit M34192 Isovaleryl-coA dehydrogenase (IVD) M11058 3-Hydroxy-3-methylglutaryl-coenzyme A reductase X05276 Tropomyosin 4 X53416 Actin-binding protein (filamin) (ABP-280) L35035 Ribose 5-phosphate isomerase (RPI) J05016 Protein-disulfide isomerase related protein (ERp72)

Translational Control by Rapamycin TABLE III—continued GenBank™ accession no.

U11313 M27891 M14219 M20469 D87120 X79537 U77718 L19783 Z22551 U85946 U83115 X99050 D87459 Not classified M92439 X99961

Gene description

Sterol carrier protein 2 Cystatin C Decorin Clathrin, light polypeptide Predicted osteoblast protein Glycogenin Desmosome-associated protein pinin Phosphatidylinositol glycan, class H Kinesin receptor Secretory protein Sec10-like 1 ␤␥-crystallin like protein UV radiation resistance-associated gene WASP family, member 1 Leucine-rich protein Novel protein (HSNOV1)

Of the 19 mRNAs whose intracellular levels increased in rapamycin-treated cells, five (cyclin F, Ets2 repressor factor, Apo-1/Fas, tumor necrosis factor receptor, and Syk) were found to be greatly enriched in the polysomal fractions from rapamycin-treated cells. Proteomic Profiling of Rapamycin-treated T Cells—Protein changes during rapamycin treatment of the Jurkat T cells were investigated by proteomics. Metabolic labeling was performed in untreated and rapamycin-treated Jurkat cells, and equal amounts of total [35S]methionine-labeled proteins were separated by two-dimensional gel electrophoresis. Following exposure to films, the autoradiograms were digitized, and twodimensional protein patterns were matched by computer analysis. In this study, 830 protein spots were matched and quantitated. Whereas the overall two-dimensional patterns of untreated and rapamycin-treated cells were largely similar, some protein changes were reproducibly detected. We selected protein spots whose intensities changed in all experiments by 3-fold or greater in response to rapamycin. A set of 111 protein spots was identified, with 70 up-regulated and 41 down-regulated protein spots (Fig. 3). We used analogy with a two-dimensional protein map data base developed in the laboratory (28)2 to identify these spots. Of the 111 spots, we identified 22 spots corresponding to 16 genes, including 11 genes listed in Table II or III. Table IV indicates the assignment of these 22 protein spots. Computer analysis determined the radioactivity incorporated in each spot from control and rapamycin-treated cells. Intensities of lactate dehydrogenase B, ␣-enolase, ␤-tubulin, ␤-actin, Op18, ADP-ribosylation factor 1, LAMR1, and eIF4A1 isoforms decreased, and intensities of annexin V and Ro/SSA antigen increased following rapamycin treatment, in good agreement with the microarray data. Discordant microarray and proteomic data were obtained for Hsp60. The other proteins identified (aldehyde dehydrogenase, tropomyosin 5, 143-3 ␴, 14-3-3 ␨/␦, and calmodulin) were not represented in the microarray. DISCUSSION

To develop a better understanding of rapamycin’s molecular mechanism in T cells, we utilized two complementary approaches to identify specific genes regulated by rapamycin in T cells. One relies on the quantitative analysis of translated mRNAs by DNA microarrays. The other relies on quantitative analysis and identification of proteins by proteomics. In addition, we quantitated polysome-bound mRNAs as a measure of their translation efficiency (29). Ribosomal proteins and elongation factors contain a polypyrimidine tract at the 5⬘-end of 2

E. Puravs and S. Hanash, unpublished data.

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their mRNAs and have been described as translationally repressed by rapamycin (17). Indeed, translation of a large number of ribosomal proteins and elongation factors was found to be strongly repressed by rapamycin in our study. We have uncovered a large number of additional genes. Part of the regulated genes have functions related to RNA processing and translation. Translation initiation factors 4A and 5A were strongly repressed. Translation of prothymosin ␣ was also strongly repressed by rapamycin. Interestingly, prothymosin ␣ has been reported to enhance cell-mediated immunity as well as proliferative and cytotoxic responses of T cells (30 –33). In vivo, prothymosin ␣ has been shown to exert a potentiating effect on human CD4⫹ T cell proliferation in response to antigens, which was associated with a prothymosin-induced increase in IL-2 production. It was also demonstrated that prothymosin ␣, in combination with IL-2, can render cell to cell interactions more effective, resulting in increased killing of autologous tumors (34). Remarkably, translation of seven genes encoding proteasome subunit members was abolished, which would explain in molecular terms the reported inhibition of proteasome activator expression and proteasome activity by rapamycin (35). The proteasome-mediated degradation pathway regulates a wide variety of cellular activities, including cell growth and immune and inflammatory responses. Within the immune system, the proteasome is essential for production of peptides for major histocompatibility complex class I antigen presentation. More recent studies have suggested a possible role for the proteasome in regulating the levels of cell surface receptors. In particular, a functional proteasome is required for optimal endocytosis of the IL-2 receptor-ligand complex and is essential for the subsequent lysosomal degradation of IL-2, possibly by regulating trafficking to the lysosome (36). In addition, several studies have implicated the proteasome in the regulation of Jak-STAT signal transduction, including IL-2-induced activation of STAT5 (37, 38). Adhesion molecules are essential in interaction between T cells and antigen-presenting cells, between T help cells and T effector cells, and between T cells and endothelial cells. It has been recently demonstrated that proteasome inhibitors repress T lymphocyte aggregation and then potentially cell-cell interactions in the immune system (39). Finally, a role of proteasomes in T cell activation, proliferation, and apoptosis has been reported (40, 41) including a requirement of the proteasome activity for T cells to progress from the G0 to S phase. Most interestingly, inhibition of proteasome activity is a common feature of immunosuppressant drugs such as cyclosporin A and FK506 (42). This raised the intriguing possibility that the proteasome is one of the common downstream targets of these drugs. In addition, our data elucidated the mechanisms by which rapamycin is inhibiting the expression of some proteasome proteins. Therefore, we identified important downstream targets of rapamycin such as prothymosin ␣ and proteasome subunits that may modulate the immune response following rapamycin treatment and mediate the immunosuppressive effects of this drug. Translation of the majority of eukaryotic mRNAs is initiated through a cap. Some mRNAs, however, are translated by a cap-independent mechanism, mediated by ribosome binding to internal ribosome entry site (IRES) elements located in the 5⬘-untranslated region. So far, only a handful of cellular IRES have been described (43). We previously demonstrated that rapamycin inhibits specifically cap-dependent translation, whereas cap-independent translation is unaffected or slightly increased (12, 20). We identified 159 genes that are still translated in the presence of rapamycin. These genes are candidates for IRES-driven mRNAs. Remarkably, these genes included

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FIG. 3. Two-dimensional profiles of T cells. A, up-regulated (white arrows) and down-regulated (black arrows) protein spots are reported on a representative silver-stained two-dimensional gel corresponding to the protein expression profile in rapamycin-treated T cells. These results are representative of three independent experiments. B, close-up sections of [35S]methionine protein labeling two-dimensional gels from untreated (left panel) and rapamycin-treated (right panel) T cells, corresponding to boxed sections in A, are shown for comparison.

Translational Control by Rapamycin TABLE IV Identified regulated proteins in rapamycin-treated T cells Spot number

Description

-Fold change -fold

143 180 183 207 247 327 341 344 345 368 449 475 498 524 548 627 662 667 679 680 681 688

Ro/SSA antigen Aldehyde dehydrogenase Aldehyde dehydrogenase Hsp60 ␤-tubulin ␣ enolase ␤-actin ␤-actin ␤-actin LAMR1 Lactate dehydrogenase B Annexin V Tropomyosin 5 14–3-3 ␴ 14–3-3 ␨/␦ elF4A1 ADP-ribosylation factor 1 Op18 Op18 Op18 Op18 Calmodulin

3.4 3.3 3.3 3.2 ⫺3.4 ⫺15.2 ⫺4.5 ⫺4.2 ⫺4 ⫺29 ⫺3.8 3.8 ⫺4.2 ⫺4.4 8.6 ⫺4.3 ⫺4.3 ⫺4.7 ⫺5 ⫺6.1 ⫺8.7 ⫺4.7

three genes reported to harbor an IRES, the human immunoglobulin heavy chain-binding protein Bip/GRP78 (44), the cyclindependent kinase p58 (PITSLRE) (45), and the transcription activator TFIID (46). Additional genes unaffected by rapamycin included specific families such as kinases and phosphatases, DNA-binding factors, and genes controlling transcription as well as RAS superfamily members. Some genes of these families have been previously described to be translated in poliovirus-infected cells, featuring a general inhibition of capdependent translation (21). In addition to a translational control by rapamycin, rapamycin affected transcript levels of several genes within a short time period. Most genes were growth-related and may explain the strong inhibition of proliferation observed in rapamycintreated cells. We observed an up-regulation of several negative regulators of cell proliferation such as cyclin G2 (47), MAD1like 1, bridging integrator 1, a Myc-interacting protein (48), BTG1 (49), and the Syk tyrosine kinase (50). CENPE function is required for the transition from metaphase to anaphase and accumulates in the G2 phase of the cell cycle (51). A concomitant decrease in genes promoting cell growth was observed. These genes included cyclin D2 (52), the Cdc7-related kinase, a regulator of the G1/S phase transition, and/or DNA replication in mammalian cells (53) and the initiation factor eIF4E (54). Polyadenylation of mRNA requires multiple protein factors, including three cleavage stimulation factors. Reduction of CSTF2 causes reversible cell cycle arrest in G0/G1 phase, whereas depletion results in apoptotic cell death (55). Phosphatidylinositol 3-kinase activity is implicated in diverse cellular response triggered by mammalian cell surface receptors. Using mice deficient in phosphatidylinositol 3-kinase ␥, it has been demonstrated that phosphatidylinositol 3-kinase ␥ controls thymocyte survival and activation of mature T cells (56 –58). Up-regulation of I-␬B-like 1, Fas, and tumor necrosis factor receptor was also observed. Remarkably, expression of three subunits of the 26 S proteasome was also decreased, suggesting an inhibition of the proteasome at both transcriptional and translational control. Similarly, cyclin F, ETS2 repressor factor, Fas/Apo-1, and tumor necrosis factor receptor were both transcriptionally and translationally up-regulated by rapamycin. Proteomic analysis of the same populations did validate the

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microarray data. Intensities of 8% of the [35S]methionine-labeled protein spots increased, and intensities of 5% decreased. In addition, microarray and proteomic analysis were similar for 15 regulated proteins identified. The oligonucleotide array and proteomics analyses undertaken in this study have uncovered novel genes and proteins with potential roles in the immunosuppressive response effect of rapamycin. Microarray analysis has identified important changes in genes involved in immune response and growth control as well as in the degradation pathway. This study also demonstrates that close to 7% of cellular mRNAs are still translated in a context of a general shut-off of protein synthesis. Acknowledgments—We thank David Misek, Pascal Lescure, Sophie Girard, and Robert Hinderer for help. REFERENCES 1. Sehgal, S. N., Baker, H., and Vezina, C. (1975) J. Antibiot. (Tokyo) 38, 727–732 2. Saunders, R. N., Metcalfe, M. S., and Nicholson, M. L. (2001) Kidney Int. 59, 3–16 3. 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