ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and -Mediated Adhesion

This information is current as Seung Hee Jung, Eun Hye Yoo, Mi Jin Yu, Hyeon Myeong of September 25, 2021. Song, Hee Yoon Kang, Je-Yoel Cho and Jong Ran Lee J Immunol 2016; 197:942-952; Prepublished online 22 June 2016; doi: 10.4049/jimmunol.1501913

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Supplementary http://www.jimmunol.org/content/suppl/2016/06/22/jimmunol.150191 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 47 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/197/3/942.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and Integrin-Mediated Adhesion

Seung Hee Jung,* Eun Hye Yoo,*,† Mi Jin Yu,*,† Hyeon Myeong Song,* Hee Yoon Kang,* Je-Yoel Cho,‡ and Jong Ran Lee*,†

A novel adaptor protein was identified by analyzing phosphotyrosine proteomes from membrane rafts of activated T cells. This protein showed sequence similarity to a well-known T cell adaptor protein, adhesion and degranulation-promoting adaptor protein (ADAP); therefore, the novel protein was designated activation-dependent, raft-recruited ADAP-like phosphoprotein (ARAP). Sup- pression of ARAP impaired the major signaling pathways downstream of the TCR. ARAP associated with the Src homology 2 domain of Src homology 2–containing leukocyte protein of 76 kDa via the phosphorylation of two YDDV motifs in response to TCR stimulation. ARAP also mediated integrin activation but was not involved in actin polymerization. The results of this study indicate that a novel T cell adaptor protein, ARAP, plays a unique role in T cells as a part of both the proximal activation signaling Downloaded from and inside–out signaling pathways that result in integrin activation and T cell adhesion. The Journal of Immunology, 2016, 197: 942–952.

ntigen recognition by T cells occurs at a junction of a (18–21). Upon TCR stimulation, ZAP-70 translocates from the cy- T cell and an APC known as the immunological synapse toplasm to membrane rafts; there, it phosphorylates signaling mole- A (IS) and leads to signaling events in T cells (1–5). After cules that contain several phosphotyrosine sites and protein–protein http://www.jimmunol.org/ triggering TCR signaling, actin polymerization and inside–out interaction domains, resulting in the recruitment of other cytoplasmic signaling follow at the T cell contact site (6, 7). Adhesion is signaling molecules to the rafts (18–21). mediated by the binding of LFA-1 to a family of adhesion mol- To analyze the phosphotyrosine proteins that are concentrated in ecules, including ICAM-1, by an F-actin–dependent mechanism and recruited to membrane rafts at a TCR-mediated activation state, (8–11), and therefore T cells become more adhesive. rafts were isolated by the ultracentrifugation of Jurkat T cell lysates Previous studies have demonstrated the roles of membrane micro- in a sucrose density gradient and the subsequent collection of domains, also called glycolipid-enriched microdomains or detergent- floating fractions (18). Phosphotyrosine proteins were then iso- insoluble rafts, in the initiation of TCR-mediated signaling (12–17). lated by an immunoprecipitation (IP) of the raft fractions using IS formation occurs at rafts, and a variety of signaling molecules are anti-phosphotyrosine Abs. Further separation and identification of by guest on September 25, 2021 concentrated within rafts; these signaling molecules include the Src the proteins were accomplished using liquid chromatography– family kinase Lck, the TCR, the CD4 and CD8 coreceptors, and the tandem mass spectrometry (LC-MS/MS) (22, 23). transmembrane adaptor protein linker for activation of T cells (LAT) In this study, we report a novel adaptor protein that is recruited to membrane rafts and is involved in early T cell activation after TCR stimulation. This 83-kDa phosphotyrosine protein shares sequence *Department of Life Science, College of Natural Sciences, Ewha Womans Univer- sity, Seoul 03760, Korea; †Research Center for Cellular Homeostasis, Ewha Womans similarity with the adhesion and degranulation-promoting adaptor University, Seoul 03760, Korea; and ‡Department of Veterinary Biochemistry, Col- protein (ADAP) (24, 25); therefore, this novel adaptor protein was lege of Veterinary Medicine, Seoul National University, Seoul 08826, Korea designated activation-dependent, raft-recruited ADAP-like phospho- Received for publication August 27, 2015. Accepted for publication May 19, 2016. protein (ARAP). ARAP contains several modular domains, such as a This work was supported by the National Research Foundation of Korea funded by the proline-rich (PR) domain, a lysine-rich domain, and an Src homology Korean Government (Grants 2009-0074129, 2011-0016543, 2012R1A5A1051354, and 2015R1A2A2A01004209 to J.R.L.). S.H.J. and H.M.S. were supported in part (SH)3 domain. It also contains multiple phosphotyrosine sites, in- by the Brain Korea 21 Project of the Korean Ministry of Education. cluding tyrosine motifs (YDDV) (see Fig. 1A). We show that ARAP The human and mouse cDNA clones presented in this article have been submitted to associates with the SH2 domain of SH2-containing leukocyte protein GenBank under accession numbers NM_001004303 and NM_001162980. of 76 kDa (SLP-76) via the phosphorylation of specific tyrosine Address correspondence and reprint requests to Prof. Jong Ran Lee, Department of motifs in ARAP. We also demonstrate that ARAP is not involved in Life Science, College of Natural Sciences, Ewha Womans University, Science Build- ing C-407, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Korea. E-mail address: actin polymerization but is required for T cell activation via the TCR [email protected] and for inside–out signaling during integrin activation. Based on The online version of this article contains supplemental material. these results, we propose that ARAP is an important new com- Abbreviations used in this article: ADAP, adhesion and degranulation–promoting ponent of TCR-mediated signaling and plays a positive regulatory adaptor protein; ARAP, activation-dependent, raft-recruited adhesion and degran- role in T cell activation. ulation–promoting adaptor protein–like phosphoprotein; CMTMR, 5-(and-6)-(((4- chloromethyl)benzoyl)amino)tetramethylrhodamine; CT, C-terminal region; IB, immunoblotting; IP, immunoprecipitation; IS, immunological synapse; LAT, linker Materials and Methods for activation of T cells; LC, liquid chromatography; LC-MS/MS, liquid chromatog- raphy–tandem mass spectrometry; MS, mass spectrometry; NT, N-terminal region; Abs and reagents ORF, open reading frame; PLC, phospholipase C; PR, proline-rich; PV, pervanadate; SEE, staphylococcal enterotoxin E; SH, Src homology; shRNA, short hairpin RNA; Anti-human CD3 mAb (UCHT1; BD Pharmingen, San Diego, CA) was SKAP, Src kinase–associated phosphoprotein; SLP-76, Src homology 2 domain– used for cell stimulation. For IP and immunoblotting (IB), we used anti-LAT containing leukocyte protein of 76 kDa; WT, wild-type. polyclonal Ab, anti–phospholipase C (PLC) g1, and anti-phosphotyrosine (4G10) mAbs (Upstate Biotechnology, Lake Placid, NY), anti–SLP-76, Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 anti-Akt, anti-ERK2, anti-Rac1, anti-Rap1, anti-GST, anti-Lck, anti-Fyn, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501913 The Journal of Immunology 943 anti–b-actin, and anti–phospho-PLCg1 (pPLCg1, Y783) polyclonal Abs Cell lines, cell culture, and T cell activation (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA and anti-FLAG (M2) mAbs, peroxidase-conjugated cholera toxin B subunit for detection Human Jurkat T leukemia cells, additional Jurkat derivatives created of ganglioside GM1, and BSA (Sigma-Aldrich, St Louis, MO), anti–Src using the pSuperior vector suppressing ARAP and pCMS4 suppression/ kinase–associated phosphoprotein (SKAP) 1 polyclonal Ab (BD Trans- reexpression vector suppressing ARAP/reexpressing various mutant forms duction Laboratories, Franklin Lakes, NJ), and anti–phospho-Akt1 (pAkt, of ARAP, and Raji B cells were cultured in RPMI 1640 medium containing S473), anti–phospho-ERK1/2 (pERK, T202/Y204), and anti-phosphotyrosine 10% heat-inactivated FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, (p-Tyr100) mAbs (Cell Signaling Technology, Danvers, MA). We generated and 20 mM glutamine. Cell lines were maintained at 37˚C with 5% CO2/ a mouse polyclonal anti-ARAP antisera using purified GST–ARAP–C– 95% O2. For activation, Jurkat T cells and derivatives were washed and 3 7 terminal region (ARAP-CT, aa 546–728) as the immunogen. Secondary resuspended in RPMI 1640 medium at 1 10 cells/ml and incubated with Abs, anti-mouse IgG, anti-rabbit IgG conjugated with HRP from Bio-Rad either control or anti-CD3 Ab for indicated times at 37˚C. (Hercules, CA), and anti-Armenian hamster IgG from Jackson Immuno- Research Laboratories (West Grove, PA) were used. Expression of plasmid DNA Ficoll-Paque Plus, ECL reagents, and GammaBind G Sepharose To generate stable cell lines, Jurkat T cells were electroporated using a were purchased from Amersham Pharmacia Biotech (Arlington Heights, Pulser (Bio-Rad) at a setting of 250 V and 950 mFd in cuvettes IL). CellTracker Orange 5-(and-6)-(((4-chloromethyl)benzoyl)amino) containing 2 3 107 cells in 0.4 ml of cytomix intracellular buffer (27) and tetramethylrhodamine (CMTMR) and fura-2 acetoxymethyl ester (fura- 40 mg of plasmid. After 7 d in culture, cells were sorted for expression of 2 AM) were purchased from Molecular Probes (Eugene, OR). FITC- or tet- GFP using a FACSAria (BD Biosciences, Mountain View, CA). After an ramethylrhodamine isothiocyanate–phalloidin, polyethylenimine branched, additional 2 wk in culture, cells were sorted again to obtain stable trans- laminin, poly-L-lysine, polyoxyethylene (20) oleyl ether (Brij 98), octyl-b-D- fectants showing GFP fluorescence. Similar levels of surface TCR and glucopyranoside, glutathione-agarose, and luciferin were obtained from LFA-1 expression were confirmed. Transient expression of plasmid DNA Sigma-Aldrich. Superantigen staphylococcal enterotoxin E (SEE) from in HEK 293T cells was previously described (28). HEK 293T cells were Toxin Technology (Sarasota, FL), human fibronectin from BD Biosciences transfected with a mixture of each of the various expression plasmids with Downloaded from (Franklin Lakes, NJ), and ICAM-1–Fc chimera from R&D Systems (Min- polyethylenimine solution. neapolis, MN) were also used. A pan T cell isolation kit II from Miltenyi Biotec (Bergisch Gladbach, Germany) was used for depletion of non–T cells. Human T cell purification and activation Plasmids and cloning Peripheral blood samples were collected from normal healthy donors after written informed consent was obtained from all donors. All studies were To clone the ARAP cDNA, a full ARAP open reading frame (ORF) clone approved by the Institutional Human Ethics Review Board of Ewha Womans (DKFZp686O04253Q) was purchased from ImaGenes (Berlin, Germany). University Medical Center. The isolation of mononuclear cells and T cell http://www.jimmunol.org/ The human ARAP cDNA (ARAP–wild-type [WT]) was generated using a fraction using Ficoll-Paque Plus density centrifugation followed by deple- PCR with oligonucleotide primers 59-CGCGGATCCATGAAGGGGAA- tion of non–T cells with superparamagnetic microbeads was described pre- GGG-39 and 59-CCGCTCGAGCTAAGGTGACCAACTTTG-39 and ligated viously (29). Isolated T cells were resuspended in RPMI 1640 medium and into pCMV-Tag2B (Stratagene, La Jolla, CA) at BamH1 and XhoI sites. rested at 37˚C. For activation, cells (1 3 107/ml) were incubated with either PCR products were verified with DNA sequencing. The N-terminal region control Ab or anti-CD3 Ab (5 mg/ml) for 5 min at 37˚C. of ARAP (ARAP-NT, aa 1–545) and the CT of ARAP (ARAP-CT, aa 546– 728) were created from ARAP-WT by restriction digests using BamHI/ IP and IB PstI and PstI/XhoI, respectively, and cloned between the respective sites in the vector pCMV-Tag2B. The GFP-conjugated plasmids for ARAP-WT, T cell stimulation was terminated by adding an equal volume of ice-cold -NT, and -CT were generated by restriction digests of pCMV-Tag2B/ medium, and cell lysates were prepared in 1% Nonidet P-40 lysis buffer ARAP-WT, -NT, and -CT followed by ligation at BglII and SalI sites in- containing protease and phosphatase inhibitors, as described previously by guest on September 25, 2021 to the mammalian GFP expression plasmid pEGFP-C1 (Clontech Labo- (30). For IB, lysates from 1 3 106 cells were mixed with 23 Laemmli’s ratories, Palo Alto, CA). sample buffer, boiled, and subjected to 8, 10, or 14% SDS-PAGE. For IP, Short hairpin RNA (shRNA) for ARAP (shARAP) was generated by lysates from 10 3 106 cells were tumbled with GammaBind G Sepharose cloning 19 nucleotides of human ARAP (nucleotides 979–997, 59-CCA- beads conjugated with individual Ab as indicated. The immune complexes CATAATTACGAGGCAA-39) into the pSuperior vector (Oligoengine, were then subjected to SDS-PAGE, followed by IB. Seattle, WA) according to the manufacturer’s instructions. The same nu- cleotides were also cloned into the pCMS4-H1-pEGFP vector (a gift from Measurements of intracellular calcium Dr. D.D. Billadeau, Mayo Clinic, Rochester, MN) (pCMS4-shARAP), and Jurkat T cells (2 3 106) were washed, suspended in RPMI 1640 medium, and this pCMS4 suppression/re-expression vector (26) was used for shRNA si- loaded with 5 mM fura-2 AM for 1 h at 37˚C. After two washes with Ca2+ lencing and re-expression of resistant cDNA. A mutant form of ARAP re- loading buffer (140 mM NaCl, 2 mM KCl, 2.5 mM CaCl ,1mMMgCl, sistant to shRNA (rARAP) was created using a QuickChange site-directed 2 2 10 mM HEPES, 10 mM glucose, 40 mM sucrose, 0.05% BSA [pH 7.4]), the mutagenesis kit (Stratagene). The mutation did not confer translational cells were resuspended in Ca2+ loading buffer (1 3 106 cells/ml). After in- changes (59-ccGcaCaaCtaTgaggcaa-39; uppercase letters indicate mutations cubating for 20 min at room temperature, baseline Ca2+ levels were measured introduced to the targeting sequences). The FLAG-tagged WT (rARAP), the for 50 s. The cytosolic free Ca2+ concentration was monitored by the addition proline-rich region (PR, aa 1–310), and the SH3 domain (aa 591–728) of of an anti-CD3ε mAb (2 mg/ml) followed by crosslinking with a goat anti- ARAP were cloned into the pCMS4-shARAP plasmid at the MluI and SalI Armenian hamster IgG (10 mg/ml) with an RF-5301 PC spectrofluorophoto- restriction sites. Point mutants of single (Y491F, Y521F, Y587F), double meter (Shimadzu, Kyoto, Japan). Ionomycin (2 mM) was added to induce the (Y2F: Y491, 587F), and triple tyrosine residues (Y3F: Y491, 521, 587F) maximum Ca2+ increase as a control for the fura-2 AM dye loading. were generated from pCMS4-shARAP-rARAP using a QuickChange mu- tagenesis kit. All of the constructs were verified by sequencing. Luciferase assay Plasmids of IL-2 enhancer region NF-AT–luciferase, CMV–b-galactosi- dase, FLAG-ADAP, HA– or FLAG–SLP-76 (WT), and the SH2 domain The cells were transfected by electroporation with a luciferase construct of mutant FLAG–SLP-76 (RK) were provided by Dr. G.A. Koretzky (Weill NF-AT and a pCMV/b-galactosidase plasmid. After 24 h, the cells were Cornell Medical College, New York, NY). The active form of Lck (Y505F) harvested, plated in 24-well plates precoated with 2 mg/ml anti-CD3 mAb and Fyn (Y528F) as well as HA-SKAP1 plasmids were gifts from Drs. Y.D. at 5 3 105 cells per well, and incubated for 16 h. The cells were also Yun (Ewha Womans University), A. Veillette (Institute de Recherches incubated with medium alone or with 50 ng/ml PMA and 1 mM ion- Cliniques de Montreal, Montreal, Canada), and B. Schraven (Otto von omycin. The cells were lysed, and luciferase activity was measured with an Guericke University, Magdeburg, Germany), respectively. The GST-RalGDS LB953 luminometer (Berthold, Bad Wildbad, Germany). To control for and GST-PAK1 plasmids were also provided by Drs. D.D. Billadeau and Y.S. transfection efficiency, luciferase activities were normalized to the activity Bae (Ewha Womans University), respectively. of b-galactosidase. The experiments were performed twice in triplicate. Polymerase chain reaction Adhesion assay TheARAPcDNAinamultipletissuepanelandanimmunesystem Flat-bottom 96-well plates were coated with fibronectin (3 mg/ml), laminin panel (Clontech Laboratories) was detected with a set of primers (59- (10 mg/ml), ICAM-1–Fc (1 mg/ml), or 2.5% (w/v) BSA overnight at 4˚C CGCGGATCCATGAAGGGGAAGGG-39 plus 59-CCGCTCGAGCTAAGGT- and blocked with 2.5% BSA for 2 h at 37˚C. T cells (2.5 3 105), either GACCAACTTTG-39). unstimulated or stimulated with anti-CD3 (5 mg/ml) and anti-hamster IgG 944 A NOVEL ADAPTOR PROTEIN IN T CELL ACTIVATION

(10 mg/ml) together for 30 min at 37˚C, were added in triplicate to the Nonidet P-40, 10% [v/v] glycerol, 1 mM PMSF, 10 mg/ml leupeptin, 5 mg/ml prepared wells. The plates were incubated for 30 min at 37˚C, and adherent aprotinin, and 1 mM Na3VO4) and centrifuged at 18,000 3 g for 10 min. cells were removed using EDTA (50 mM) and counted. Cleared cell lysates (500 ml) were transferred to glutathione agarose beads conjugated either with PAK1 PBD (GST-PBD) for the Rac1 activation assay Rafts isolation or with RalGDS RBD (GST-RBD) for the Rap1 activation assay. After in- 3 The Brij-insoluble rafts fractions were separated from the cell lysates by cubation with the lysates for 10 min at 4˚C, the beads were mixed with 2 sucrose density gradient ultracentrifugation as described previously (18, Laemmli’s reducing buffer, boiled for 5 min at 96˚C, and subjected to 14% 29). Jurkat T cells (1 3 108) were stimulated with either control Ig or with PAGE. After being transferred to a nitrocellulose membrane, IB was per- anti-CD3 Ab (2 mg/ml) for 5 min at 37˚C. Cell lysates were prepared in formed for Rac1 or Rap1. Loading controls for the IB were shown using an anti-GST Ab. 1% Brij-98 lysis buffer containing protease and phosphatase inhibitors. Brij-insoluble fractions were separated by sucrose density gradient cen- Analyses of phosphotyrosine proteomes trifugation. Fractions (1 ml) were collected sequentially from the top of the gradient and membrane rafts were analyzed by detection of GM1. The raft fractions were collected and concentrated using n-octyl-b-D- glucoside. IP was performed on the concentrated rafts using a mixture of IS formation and immunocytochemistry anti-phosphotyrosine Abs (4G10 and p-Tyr100), and the immune complex was separated using 10% SDS-PAGE. The gel was stained with a Coo- Conjugation of Jurkat T cells and Raji B cells as APCs, followed by im- munocytochemistry, was described previously (29). Jurkat T cells were massie brilliant blue solution and excised into 15 pieces. In-gel tryptic transfected with expression plasmids encoding GFP-ARAP constructs, and digestion and LC-MS/MS analysis using a Thermo Finnigan ProteomeX Raji B cells were stained with CellTracker Orange CMTMR. workstation LTQ linear ion trap MS (Thermo Electron, San Jose, CA) equipped with electrospray ionization sources (San Jose, CA) were per- Actin polymerization formed on each of the excised protein bands, as previously reported (22). Tandem mass spectra were extracted, and the charge state was deconvo- Jurkat T cells conjugated with CMTMR-stained Raji B cells were prepared luted and deisotoped by Sorcerer 3.4 beta2 (Sorcerer software 3.10.4, Downloaded from on poly-L-lysine–coated slides. Slides were fixed in 3.7% paraformalde- Sorcerer Web interface 2.2.0 r334 and Trans-Proteomic Pipeline 2.9.5) as hyde for 20 min at room temperature, permeabilized in 0.1% Triton X-100, previously reported (23). All MS/MS samples were analyzed using Sequest blocked, and stained with FITC- or tetramethylrhodamine isothiocyanate– (Thermo Finnigan, San Jose, CA; version 27, revision 11). Scaffold (ver- conjugated phalloidin. F-actin was imaged using a Zeiss Axiovert 200 sion Scaffold-01_07_00, Proteome Software, Portland, OR) was used to fluorescence microscope equipped with a 340 objective lens (Carl Zeiss, validate MS/MS-based peptide and protein identifications. Oberkochen, Germany). Background fluorescence levels in T cells that

expressed vector-associated GFP were minimal. Correction of background http://www.jimmunol.org/ fluorescence was used for T cell fluorescence measurements. All images Results were acquired and analyzed with Zeiss software. Identification and cloning of a novel adaptor protein in the TCR signaling pathway GTPase activation assay We analyzed the phosphotyrosine proteomes that are concentrated Jurkat T cells (5 3 106) were either unstimulated or stimulated with an anti-CD3 Ab (2 mg/ml) at 37˚C for the indicated durations. At each time in and recruited to membrane rafts following TCR cross-linking. point after stimulation, cells were lysed in 600 ml of GTPase activation Raft fractions were isolated from either resting or TCR-activated buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 5 mM MgCl2, 0.5% [v/v] Jurkat T cells using centrifugation of sucrose density gradients by guest on September 25, 2021

FIGURE 1. Amino acid sequence, domain structure, and expression of human ARAP. (A) The predicted amino acid sequences of human (Hs) and mouse (Mm) ARAP. The putative modular domains are indicated by thick, thin, and dotted lines above the respective sequences. The conserved tyrosine motifs YD(E)DV are indicated by square boxes, and tyrosine residues are indicated by asterisks. (B) Schematic presentation of the putative domains of ARAP and ADAP. The PR [thick line in (A)], lysine-rich [thin line in (A)], and SH3 [dotted line in (A)] domains are shown with their corresponding numbers in the amino acid sequence of ARAP. The sequences for the two putative nuclear localization sites, an internal SH3 domain, and a binding site for enabled/ vasodilator-stimulated phosphoprotein homology 1 are exclusive to ADAP. The putative tyrosine (Y)-based motifs and sequence homologies (—, ∼ 50%; …, ∼28%) are also indicated. (C and D) The presence of ARAP mRNA in various tissues as detected by PCR of the multiple tissue cDNA panels (Clontech Laboratories) is shown. (E) Western blots of various human cell lines (ERK2 is a loading control). (F) Western blots of lysates from Jurkat T cells (50 and 100 mg), purified primary human T cells (80 mg), and HEK 293T cells transfected with ARAP cDNA (5 mg). The Journal of Immunology 945 as described in Materials and Methods. Phosphotyrosine proteomes polyclonal anti-ARAP antiserum was generated as described in were separated from the rafts using IPs with anti-phosphotyrosine Materials and Methods. ARAP was widely expressed in the various Abs. The immune complexes were separated on a gel, and in-gel human tissues and cell lines tested (Fig. 1C–E). ARAP transcripts tryptic digestion and LC-MS/MS analyses were carried out as were detected by PCR in cDNA prepared from heart, liver, kidney, described in Materials and Methods. pancreas, and lymphoid tissues, including thymus, lymph node, In this study, we identify one protein that is recruited to and tonsil (Fig. 1C, 1D). ARAP expression levels varied in dif- membrane rafts and undergoes tyrosine phosphorylation follow- ferent batches of cDNA panels. ARAP expression was not de- ing TCR activation. The corresponding human cDNA, containing tected in bone marrow in any batch of cDNA panels. ARAP 1 ORF 168 (NC_000001.11, http://www.ncbi.nlm.nih. expression in spleen, thymus, lymph node, and peripheral blood gov/gene/199920), encoded a novel protein. The human cDNA leukocyte was observed, although the expression level varied in clone (GenBank accession no. NM_001004303) contained an different batches of cDNA panels. The expression of ARAP pro- ORF encoding a protein of 728 aa with a predicted molecular tein was verified in purified primary human T cells as shown in mass of 83 kDa. The homologous mouse cDNA clone (GenBank Fig. 1F. Although we only showed the band that matches with the accession no. NM_001162980) showed a 70% overall identity expression of full gene sequence in HEK 293T cells, four shorter with the human cDNA clone and contained a chromosome 4 ORF variant forms known in the database could be detected by a mouse encoding a 90-kDa protein of 793 aa (Fig. 1A; alignment from the polyclonal anti-human ARAP antiserum that was generated by gene database is shown). This newly identified protein also shares using the ARAP C-terminal immunogenic peptides. with a known T cell adaptor protein, ADAP

(24, 25), and we therefore named the novel protein ARAP. As a ARAP is tyrosine phosphorylated and is recruited to membrane Downloaded from novel adaptor protein, ARAP contains modular domains, such as rafts and the IS after TCR stimulation N-terminal PR sequences (228–286), a lysine-rich sequence (527– We confirmed the tyrosine phosphorylation of ARAP with an IP 662), and an SH3 domain (667–728) near the C terminus (Fig. 1A, of Jurkat T cell lysates after stimulation of the TCR for 5 min 1B). Several putative tyrosine motifs are also found in ARAP, and followed by IB for phosphotyrosine proteins (Fig. 2A). To clearly some of these motifs are homologous to those (EVYDDV) in demonstrate ARAP phosphorylation, primary human T cells were

ADAP that mediate its interaction with the SH2 domain of SLP-76 stimulated with anti-CD3 Ab for 5 min, and proteins were immu- http://www.jimmunol.org/ (31–34) (Fig. 1A, 1B). noprecipitated using anti-phosphotyrosine Abs followed by West- The human ARAP cDNA (ARAP-WT) was cloned by PCR using a ern blotting with anti-ARAP antiserum, anti-ADAP, and anti-LAT clone containing the full ORF of ARAP as a template, and a mouse Abs (Fig. 2B). A low level of tyrosine phosphorylation of ARAP by guest on September 25, 2021

FIGURE 2. Induction of ARAP tyrosine phosphorylation and recruitment to membrane rafts. (A) ARAP tyrosine phosphorylation after TCR stimulation in Jurkat T cells. Cells were unstimulated (2) or stimulated for 5 min via CD3 (+). ARAP was immunoprecipitated from lysates and analyzed by IB to visualize tyrosine-phosphorylated ARAP (top) and total ARAP (bottom). (B) ARAP tyrosine phosphorylation after TCR stimulation in purified primary human T cells. Cells were unstimulated (control) or stimulated for 5 min with anti-CD3 Ab. IP was performed using a mixture of anti-phosphotyrosine (pY) Abs (4G10 and p-Tyr100) and analyzed by IB to visualize tyrosine-phosphorylated ARAP (top), ADAP (middle), and LAT (bottom). (C) ARAP recruitment to membrane rafts after TCR stimulation. Membrane rafts were isolated from Jurkat T cell lysates after stimulation with control or anti-CD3 Abs for 5 min and resolved by SDS-PAGE. IB for ARAP, SLP-76, LAT, and GM1 followed. (D–F) Localization of ARAP to the IS. ARAP-WT, NT-, and -CT were conjugated with GFP (D). Jurkat T cells expressing GFP-ARAP-WT, -NT, or -CT were incubated with CMTMR-labeled Raji B cells in the absence (2)or presence (+) of the superantigen SEE for 15 min, and localization of GFP to the IS was visualized (original magnification 3400). Fluorescence and phase- contrast micrographs are shown for each GFP-ARAP construct expressed (E). Maximum fluorescence intensity at the IS in the presence of SEE was quantified, and the intensity values were compared between T cells expressing GFP-ARAP-WT, -NT, or –CT. *p , 0.05, unpaired t test (F). All data shown are representative of two (primary T cells) or three (Jurkat T cells) separate experiments. 946 A NOVEL ADAPTOR PROTEIN IN T CELL ACTIVATION and ADAP was observed in resting T cells, which was enhanced Several major proteins that are known to be tyrosine phosphory- after TCR stimulation. In this experiment, tyrosine phosphoryla- lated and involved in TCR-mediated signaling pathways were tion of LAT was examined to determine whether isolated human examined after the stimulation of TCRs in ARAP-suppressed T cells responded properly to TCR stimulation. We also confirmed Jurkat T cells using IB with a protein-specific anti-phosphotyrosine that ARAP translocated to the fractions of membrane rafts that Ab. TCR-mediated tyrosine phosphorylation of the enzymes include proteins such as GM1 and LAT (Fig. 2C). Similarly to PLCg1, ERK2, and Akt was dramatically reduced in ARAP- SLP-76, the translocation of ARAP was TCR stimulation-dependent suppressed Jurkat T cells (Fig. 3C). Other cellular functions (Fig. 2C). To examine the structural requirements of ARAP in that require the tyrosine phosphorylation and activation of TCR-mediated T cell activation, the following GFP-tagged con- these enzymes were also examined. A dramatic reduction in the structs were prepared: ARAP-WT, the NT of ARAP (ARAP-NT, cytosolic Ca2+ level following TCR stimulation in ARAP- aa 1–545), and the CT of ARAP (ARAP-CT, aa 546–728) (Fig. 2D). suppressed Jurkat T cells was observed (Fig. 3D); conse- The recruitment of ARAP to the IS of T cell–APC (Raji B cells) quently, the NF-AT luciferase activity, which is the readout of conjugates was visualized using fluorescence microscopy with GFP- TCR signaling (35), was significantly reduced in Jurkat T cells tagged constructs expressed in Jurkat T cells prior to conjugation when the ARAP gene was suppressed (Fig. 3E). In contrast, with APCs (Fig. 2E). In the presence of the superantigen (SEE), ARAP-suppressed cells had similar responses to treatment with green fluorescence from T cells expressing GFP-ARAP-WT and ionomycin or a combination of PMA and ionomycin because GFP-ARAP-NT, but not GFP-ARAP-CT, localized to the IS; these treatments induce signaling mechanisms that bypass the moreover, yellow fluorescence overlapped with red fluorescence TCR (Fig. 3D, 3E). in APCs only when GFP-ARAP and GFP-ARAP-NT constructs Downloaded from ARAP is required for the activation of integrin, but not for the were expressed (Fig. 2E). Maximum fluorescence intensity to the polymerization of actin IS in each T cell–APC conjugate in the presence of SEE was quantified and the intensity values were compared between T cells The initiation of TCR signaling at the IS by cross-linking of the Ag expressing GFP-ARAP-WT, GFP-ARAP-NT, and GFP-ARAP-CT affects cytoskeletal properties, such as actin polymerization and (Fig. 2F). integrin signaling, which further strengthen TCR activation (6–11).

Therefore, we also examined cytoskeletal rearrangements after http://www.jimmunol.org/ ARAP is required for TCR-mediated activation of signaling TCR stimulation in ARAP-suppressed Jurkat T cells. F-actin was pathways labeled using FITC-conjugated phalloidin, and fluorescence was The functional significance of ARAP was examined in Jurkat compared between control and ARAP-suppressed T cells both T cells by suppressing ARAP through expression of a shRNA for before and after conjugation with the Ag (SEE)–APC (Raji ARAP (Fig. 3A). Suppression of ARAP did not seem to affect B cells) complex. The results demonstrated that actin polymeri- expression of ADAP and SKAP55 (Fig. 3A). In ARAP-suppressed zation was not influenced by the suppression of ARAP in T cells Jurkat T cells, tyrosine phosphorylation of proteins was remark- (Fig. 4A). These results were quantitatively compared by counting ably reduced following TCR stimulation compared with the re- the number of conjugates that showed F-actin staining in the IS sponse in cells treated with a scrambled control shRNA (Fig. 3B). (Fig. 4B). In agreement with these results, the activation kinetics of by guest on September 25, 2021

FIGURE 3. Suppression of the ARAP gene and inhibition of TCR signal transduction. (A) The suppression of the ARAP gene by the expression of an shRNA for ARAP was visualized by Western blotting. The expression of ADAP and SKAP1 was determined in ARAP-suppressed Jurkat T cells. Loading control is shown by IB with anti–b-actin. (B and C) Inhibition of protein tyrosine phosphorylation in ARAP-suppressed Jurkat T cells. Control or ARAP- suppressed Jurkat T cells were stimulated with an anti-CD3 Ab for the indicated durations. The lysates were resolved by SDS-PAGE, and tyrosine- phosphorylated proteins were detected by IB (B). The same blot was used to analyze the tyrosine phosphorylation of PLCg1, ERK2, and Akt. IB for tyrosine-phosphorylated protein (top) and total protein (bottom) (C) is shown. Data shown are representative of three separate experiments. (D) Reduced calcium response in ARAP-suppressed Jurkat T cells. Control or ARAP-suppressed Jurkat T cells were loaded with fura-2, and intracellular calcium elevation after TCR stimulation (anti-CD3) or ionomycin treatment was detected by spectrofluorometry. Calcium measurements were repeated with similar responses. (E) Inhibition of NF-AT luciferase activity by ARAP suppression. Control or ARAP-suppressed Jurkat T cells were transfected with an NF-AT– luciferase reporter plasmid. Cells were either unstimulated (control) or stimulated as indicated and subsequently assayed for luciferase activity. The results are shown as fold stimulation compared with activity in unstimulated cells and are normalized by b-galactosidase activity from the cotransfected pCMV–b-gal plasmid. The values are expressed as the mean 6 SD of three independent experiments. ***p , 0.001 versus control Ig, ###p , 0.001. The Journal of Immunology 947

Rac1 GTPases was comparable in both control and ARAP-suppressed NF-AT activity was analyzed in ARAP-suppressed Jurkat T cells T cells following stimulation of the TCR (Fig. 4C). In contrast, re-expressing ARAP-WT, ARAP-PR, or ARAP-SH3. The results cell adhesion to matrix proteins such as fibronectin and laminin demonstrated that neither the PR nor the SH3 domain of ARAP is (Fig. 4D) and integrin-mediated adhesion to ICAM-1 (Fig. 4E) sufficient for the full recovery of NF-AT activity in T cells (Fig. were significantly reduced in ARAP-suppressed Jurkat T cells. Cell 5C). These results also suggested that other regions in addition to adhesion occurring via TCR-bypassed signaling using PMA was the PR and SH3 domains are required for ARAP to transduce ac- normal in ARAP-suppressed cells. These defects in TCR-mediated tivation signals after TCR stimulation. The region between the PR cell adhesion correspond with the inhibition of TCR-induced Rap1 and SH3 domains includes many tyrosine residues, and the con- GTPase activity in ARAP-suppressed T cells (Fig. 4F). served tyrosine motif YD(E)DV was found at Y491, Y521, and Y587. These specific tyrosine motifs were also conserved in ADAP ARAP function in T cell signaling is mediated by the and were previously shown to mediate ADAP association with SLP- phosphorylation of specific tyrosine motifs that associate with 76 (33, 34). To examine the functional significance of these three the SH2 domain of SLP-76 tyrosine motifs in ARAP, single tyrosines or combinations of ty- To further examine the mechanisms by which ARAP functions in rosines were mutated, and these mutants were re-expressed in T cell activation, the recovery of ARAP-suppressed T cell function ARAP-suppressed Jurkat T cells. Single tyrosine mutants at either in Jurkat T cells was monitored following reconstitution with Y491 or Y587 did not fully restore the TCR-mediated NF-AT several ARAP mutants. As described, a vector system was used to activity, but the single tyrosine mutant at Y521 fully recovered suppress ARAP while also re-expressing the mutant ARAP. The activity in control T cells (Fig. 5D). Furthermore, ARAP-suppressed re-expressed ARAP constructs contained both a FLAG epitope Jurkat T cells that re-expressed double tyrosine ARAP mutants at Downloaded from and mutations in the targeting sequences for shARAP (Fig. 5A). Y491 and Y587 (Y2F) did not respond to TCR stimulation and did Re-expression constructs of ARAP included WT, truncation mu- not induce NF-AT activation (Fig. 5D). tants, ARAP-PR and ARAP-SH3, and tyrosine mutants of ARAP The TCR-activated cytosolic Ca2+ response was restored in at Y491, Y521, and Y587. All of the tyrosine mutants were located ARAP-suppressed Jurkat T cells that re-expressed WT ARAP or within YD(E)DV motifs (Fig. 5A). The expression of these con- the ARAP Y521F single tyrosine mutant (Fig. 6A). In contrast, re-

structs in ARAP-suppressed Jurkat T cells is shown by IB with an expression with neither the Y491F nor the Y587F mutant restored http://www.jimmunol.org/ anti-FLAG Ab (Fig. 5B). the TCR-mediated Ca2+ response to the same extent as the re- by guest on September 25, 2021

FIGURE 4. Suppression of the ARAP gene and impaired integrin activation result in impaired cell adhesion. (A and B) Actin polymerization is not affected by ARAP suppression. Control or ARAP-suppressed Jurkat T cells were incubated with CMTMR-labeled Raji B cells in the absence (2) and presence (+) of the superantigen SEE. The cells were transferred to poly-L-lysine–coated slides, fixed, permeabilized, and stained with FITC-conjugated phalloidin. Images obtained using fluorescence microscopy are shown (original magnification 3400), and the localization of F-actin to the IS was visu- alized using fluorescence microscopy (A). The F-actin localized in the IS from conjugates (.60) was quantified in three independent experiments. The values are expressed as the mean 6 SD (B). (C) Rac1 GTPase activation was assayed using the GST-PBD fusion protein. Control or ARAP-suppressed Jurkat T cells were stimulated with the anti-CD3 Ab for the indicated times, and the lysates were incubated with GST-PBD beads for 10 min. The proteins from the beads were resolved by SDS-PAGE and were subject to IB for Rac1 and GST for equal amounts of each fusion protein. ERK2 IB is shown for total protein. Data shown are representative of three separate experiments. (D and E) Cell adhesion is inhibited by ARAP suppression. Control or ARAP- suppressed Jurkat T cells were stimulated for 30 min as indicated and added to the wells that were coated with BSA, fibronectin, and laminin. After 30 min incubation, adherent cells were removed and counted. The values are expressed as the mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 versus control Ig; ##p , 0.01, ###p , 0.001 (D). Control or ARAP-suppressed Jurkat T cells that were stimulated as in (D) were incubated in ICAM-1–coated wells for 30 min, and adherent cells were removed and counted. The values are expressed as the mean 6 SD of three in- dependent experiments. ***p , 0.001 versus control Ig, ###p , 0.001 (E). (F) Rap1 GTPase activation was assayed using the GST-RBD fusion protein as in (C). IB was performed for Rap1 and GST for equal amounts of each fusion protein. Rap1 IB is shown for total protein. Data shown are representative of three separate experiments. 948 A NOVEL ADAPTOR PROTEIN IN T CELL ACTIVATION sponse in ARAP WT re-expressed cells (Fig. 6A). As expected, a (Fig. 7A). When we induced tyrosine phosphorylation in the Ca2+ response was not observed after TCR stimulation in ARAP- transfected cells by pervanadate (PV) stimulation, SLP-76 protein suppressed Jurkat T cells; moreover, it was not observed in cells was immunoprecipitated with both ARAP and ADAP (Fig. 7A). with the double tyrosine mutants of re-expressed ARAP (Fig. 6A). We also tested for the association of the endogenous proteins by Similar results were also observed for ICAM-1 binding after TCR performing IP in Jurkat T cells. As shown in Fig. 8A and 8B, TCR stimulation (Fig. 6B). stimulation with anti-CD3 Ab induced protein tyrosine phos- These experiments demonstrated that tyrosine phosphorylation of phorylation, and IP with anti–SLP-76 Ab immunoprecipitated ARAP, especially at Y491 and Y587, is required for ARAP function both ADAP and ARAP. A basal level of interaction was observed, in T cell activation after TCR stimulation. Therefore, the level of and the level of interaction increased after TCR stimulation tyrosine phosphorylation was examined in ARAP-suppressed Jurkat (Fig. 8B). T cells where one of WT ARAP or a tyrosine mutant of ARAP was SKAP1 plays a central role in ADAP-mediated signaling to re-expressed. Tyrosine phosphorylation of re-expressed WT ARAP , and the identical motifs of the ARAP PR region occur in or mutant ARAPs was detected by IP with an anti-FLAG Ab fol- the primary site of mouse and human ADAP that binds to SKAP1 lowed by IB with the anti-phosphotyrosine Ab 4G10. Reduced ty- (36, 37). Therefore, we examined whether ARAP also binds to rosine phosphorylation in the Y491F and Y587F mutants and an SKAP1 through the ARAP PR region. FLAG-tagged ADAP or even further reduction in the double tyrosine mutants were observed ARAP was transiently overexpressed in HEK 293T cells in (Fig. 6C). The patterns of tyrosine phosphorylation in ARAP cor- combination with HA-tagged SKAP1, and tyrosine phosphorylation roborated the patterns of ARAP association with SLP-76. SLP-76 in the transfected cells was induced by PV stimulation (Fig. 7B). association was also reduced in the Y491F and Y587F mutants of SKAP1 protein was immunoprecipitated with both ARAP and Downloaded from ARAP, and it was almost blocked in the double tyrosine mutants of ADAP regardless of PV stimulation (Fig. 7B). IP with anti-SKAP1 ARAP (Fig. 6C). Thus, the association of ARAP with SLP-76 may Ab in Jurkat T cells detected both ADAP and ARAP (Fig. 8A, 8C). have been mediated by the ARAP phosphotyrosines at Y491 and The association was not changed after TCR stimulation with anti- Y587 and by the SH2 domain of SLP-76. This association was CD3 Ab (Fig. 8C). To confirm that SKAP1 binding to ARAP is further confirmed by an experiment where SLP-76–deficient Jurkat mediated by the ARAP PR region but not by the ARAP tyrosine

T cells were reconstituted with WT SLP-76 or with a SLP-76 SH2 motifs, we overexpressed FLAG-tagged ARAP mutants, ARAP- http://www.jimmunol.org/ mutant (RK) (Fig. 6D). ARAP association with WT SLP-76 was PR, or ARAP-Y3F in HEK 293T cells together with HA-tagged strongly induced after TCR stimulation, but the association of SKAP1 (Fig. 7C). Irrespective of the tyrosine phosphorylation ARAP with SLP-76 RK was not changed by TCR stimulation, al- induced by the PV stimulation of the transfected cells, SKAP1 though tyrosine phosphorylation of WT and SH2 mutant (RK) SLP- protein was immunoprecipitated with both ARAP-PR and ARAP- 76 was induced by TCR stimulation (Fig. 6D). Y3F (Fig. 7C). To clearly demonstrate ARAP interaction with SLP-76 and ARAP directly associates with SLP-76, SKAP1, Lck, and Fyn SKAP1, primary human T cells were stimulated with anti-CD3 Ab To determine whether the interaction of ARAP with SLP-76 occurs for 5 min, and cell lysates were immunoprecipitated using anti- directly, we transiently overexpressed FLAG-tagged ADAP or ARAP antiserum followed by Western blotting with anti-ARAP by guest on September 25, 2021 ARAP in combination with HA-tagged SLP-76 in HEK 293T cells antiserum, anti–SLP-76, and anti-SKAP1 Abs (Fig. 8D, 8E). The

FIGURE 5. Functional analysis of ARAP mutants in T cell signaling. (A) Schematic presentation of FLAG epitope-tagged ARAP mutants. The mu- tations in the targeting sequences of shARAP are indicated (^^^^). (B) IB of re-expression constructs in ARAP-suppressed Jurkat T cells using an anti-FLAG mAb (SLP-76 is a loading control). (C) NF-AT luciferase activity was assayed in control cells (EV), ARAP-suppressed Jurkat T cells (shARAP), or ARAP- suppressed cells that re-expressed ARAP-WT, ARAP-PR, or ARAP-SH3. These cells transfected with an NF-AT–luciferase reporter plasmid were placed to the wells coated with control Ig (unstimulated) or anti-CD3 Ab. Luciferase activity was subsequently assayed and the results are shown as fold stimulation compared with activity in unstimulated cells and are normalized by b-galactosidase activity from the cotransfected pCMV–b-gal plasmid. The data are the means 6 SD of three independent experiments performed in triplicate. Statistical significance was analyzed by two-way ANOVAwith Bonferroni multiple comparison tests. *p , 0.05, ***p , 0.001 versus each control Ig; ###p , 0.001 versus shARAP (anti-CD3). (D) NF-AT luciferase activity assay in control cells (EV), ARAP-suppressed Jurkat T cells (shARAP), or ARAP-suppressed cells that re-expressed various tyrosine mutants of ARAP. The data are the means 6 SD of three independent experiments performed in triplicate. Statistical significance was analyzed by two-way ANOVAwith Bonferroni multiple comparison tests. ***p , 0.001 versus each control Ig, ###p , 0.001 versus EV (anti-CD3). The Journal of Immunology 949 Downloaded from

FIGURE 6. Functional analysis of ARAP tyrosine mutants in TCR signal transduction. (A) Intracellular calcium elevation after TCR stimulation (anti- CD3) in fura-2–loaded control cells (EV), ARAP-suppressed Jurkat T cells (shARAP), or ARAP-suppressed cells that re-expressed various tyrosine mutants of ARAP. The data are representative of three separate experiments. (B) Adhesion assay in ICAM-1–coated wells. After TCR stimulation (anti- CD3), control cells (EV), ARAP-suppressed Jurkat T cells (shARAP), and ARAP-suppressed cells that re-expressed various tyrosine mutants of ARAP were incubated in ICAM-1–coated wells for 30 min, and adherent cells were removed and counted. The data are the means 6 SD of three independent experiments performed in triplicate. Statistical significance was analyzed by two-way ANOVAwith Bonferroni multiple comparison tests. **p , 0.01, ***p , http://www.jimmunol.org/ 0.001 versus each control Ig; #p , 0.01, ###p , 0.001 versus EV (anti-CD3). (C) ARAP associates with SLP-76 by the ARAP phosphotyrosines at Y491 and Y587. Control or ARAP-suppressed Jurkat T cells that re-expressed various tyrosine mutants of ARAP were unstimulated or stimulated via CD3 for 5 min. Re- expressed ARAP WT and tyrosine mutants were immunoprecipitated from lysates with an anti-FLAG mAb and analyzed by IB to visualize SLP-76 (top), tyrosine-phosphorylated ARAP (middle), and total ARAP (bottom). The intensity of the bands was quantified, and the normalized values were calculated based on the intensity of the total ARAP bands. The SLP-76 interaction and tyrosine phosphorylation of ARAP tyrosine mutants after CD3 stimulation were compared with those of the ARAP WT. The data are representative of three separate experiments. (D) Association of ARAP with SLP-76 is mediated by the SH2 domain of SLP-76. SLP-76–deficient J14 cells that were reconstituted with the FLAG-tagged WT or SH2 mutant (RK) of SLP-76 were unstimulated or stimulated via CD3 for 5 min. The reconstituted SLP-76 WT and RK mutant were immunoprecipitated from lysates with an anti-FLAG mAb and analyzed by IB to visualize ARAP (top), tyrosine-phosphorylated SLP-76 (middle), and total SLP-76 (bottom). As in (C), the ARAP interaction was normalized and compared with values of unstimulated SLP-76 WT. The data are representative of two separate experiments. by guest on September 25, 2021

IP with anti-ARAP Ab revealed constitutive binding of ARAP and tyrosine-phosphorylated after TCR stimulation, such as the TCR SKAP1 as well as tyrosine phosphorylation-dependent binding of z-chain, ZAP-70, PLCg1, LAT, and SLP-76 (18–21) were among ARAP and SLP-76 (Fig. 8D, 8E). the proteins identified from the phosphotyrosine proteome analy- We next examined whether ARAP also binds to Lck or Fyn. sis in TCR-stimulated Jurkat cells. These results confirmed that FLAG-tagged ADAP or ARAP was overexpressed in combination the methods used for the analysis of TCR signaling complexes with constitutively active form of Lck (Y505F) or Fyn (Y528F) in were effective and that novel candidate proteins can be found to HEK 293T cells (Fig. 7D). Both ARAP and ADAP proteins were better understand the mechanisms of T cell activation. immunoprecipitated with a constitutively active from of Lck ARAP, the novel protein that we identified by analysis of the (Y505F) or Fyn (Y528F) (Fig. 7D). These interactions were phosphotyrosine proteome in activated Jurkat T cells, shares se- irrespective of the tyrosine phosphorylation induced by a consti- quence homology (Supplemental Fig. 1) with the T cell adaptor tutively active form of kinases, because both ARAP and ADAP protein, ADAP (∼50%), and also with promyelocytic leukemia– also immunoprecipitated with an inactive form of Lck or Fyn (data retinoic acid receptor a–regulated adaptor molecule-1 (∼40%) not shown). that is mainly expressed in myeloid cells (38, 39). Following TCR stimulation, ADAP is well known to mediate specific signaling Discussion pathways for inside–out activation of the integrin molecule LFA-1 To better understand the initial stages of TCR signaling and the (24, 25). Similar to ADAP, ARAP contains a PR region at the N subsequent mechanisms of T cell activation, we analyzed the terminus, an SH3 domain at the C terminus, and several tyrosine proteins involved in the TCR signaling complex formed at the IS motifs between these domains (Fig. 1A, 1B). However, the se- after T cell activation. To isolate signaling complexes localized to quences for the two putative nuclear localization sites, an internal the IS, membrane rafts were purified from activated Jurkat T cells SH3 domain, and a binding site for an enabled/vasodilator- using sucrose density gradient centrifugation. The tyrosine- stimulated phosphoprotein homology 1 domain are exclusive to phosphorylated proteins from the isolated membrane rafts were ADAP (39, 40). The absence of these domain sequences may collected by IP using anti-phosphotyrosine Abs and were separated explain the differential regulation of F-actin organization between with SDS-PAGE. In-gel tryptic digestion was applied to each ADAP and ARAP. ADAP regulates TCR-mediated F-actin orga- excised protein band, and peptides were identified by LC-MS/MS nization by mediating interactions between SLP-76 and Wiskott– as described in Materials and Methods. To exclude endogenous Aldrich syndrome protein together with Nck adaptor protein (41). phosphotyrosine proteins, the same procedures were also applied Actin polymerization and Rac1 activation (the small GTPase to an analysis of the phosphotyrosine proteomes from Jurkat cells that regulates actin polymerization) were unaffected in ARAP- subjected to mock stimulation. Numerous proteins known to be suppressed T cells after TCR stimulation (Fig. 4A–C). It remains 950 A NOVEL ADAPTOR PROTEIN IN T CELL ACTIVATION Downloaded from http://www.jimmunol.org/

FIGURE 7. Direct interaction of ARAP with SLP-76, SKAP1, Lck, and Fyn. Flag-tagged ADAP or ARAP in combination with HA-tagged SLP-76 (A) or SKAP1 (B) were overexpressed in HEK 293T cells, and tyrosine phosphorylation was induced by PV stimulation for 5 min. Protein expression was detected by IB with anti-HA or anti-FLAG Ab. The amounts of IP from untreated (control) or PV-treated lysates were determined by IB with anti-HA Ab. The interaction between SLP-76, SKAP1, ARAP, and ADAP was detected by IB using anti-FLAG Ab. (C) HEK 293T cells were transfected with plasmids carrying ARAP mutants, either ARAP-Y3F (FLAG) or ARAP-PR (FLAG), along with SKAP1 (HA). Protein expression was detected by IB with anti-HA or anti-FLAG Ab. The amounts of IP from untreated (control) or PV-treated lysates were determined by IB with anti-HA Ab. The interaction between SLP- 76 and ARAP mutants was detected by IB using anti-FLAG Ab. (D) ARAP (FLAG) or ADAP (FLAG) was expressed in HEK 293T cells in the presence or by guest on September 25, 2021 absence of Lck (Y505F) or Fyn (Y528F). Protein expression was detected by IB using anti-Lck, anti-Fyn, or anti-FLAG Ab. Equal amounts of lysates were determined by IB with anti–b-actin. Tyrosine phosphorylation induced by Lck or Fyn expression was determined by IB with 4G10. The amounts of IP were determined by IB with anti-FLAG Ab. The interaction of ARAP or ADAP with Lck or Fyn was detected by IB using anti-Lck or anti-Fyn Abs. to be determined whether TCR-driven assembly of the Bcl-10– TCR signaling. A notable similarity between the two adaptors is the CARMA1 complex upstream of NF-kB activation and consequent phosphotyrosine motifs that are conserved in both ARAP and IkB degradation regulated by ADAP (42) is due to these unique ADAP. ARAP contains specific tyrosine residues, which are lo- ADAP domains, and whether ARAP may not participate in these cated within YDDV motifs at Y491 and Y587 (Fig. 1A, 1B). TCR- TCR-mediated physiological functions due to the absence of these mediated phosphorylation of these motifs is required for the unique domains. function of ARAP (Figs. 5, 6). Similar to ADAP, ARAP associates The ARAP tissue distribution is much broader than that of ADAP with the SH2 domain of SLP-76 following TCR stimulation via (Fig. 1C–E) whereas ADAP expression is restricted to T and the phosphorylation of tyrosines Y491 and Y587 (Fig. 6C). myeloid cells (42). Further studies are needed to determine the Considering the results shown in Figs. 2D–F and 6A, Y491 ap- biological significance of ARAP for integrin activation in distinct pears to be strongly required for the TCR-mediated proximal cell types and to clarify the difference in upstream and down- signaling event as compared with Y587. These results may sug- stream signaling pathways for ARAP and ADAP in T cells. A gest that ARAP participates with Y491 phosphorylation in the comparison of ARAP expression levels in the immune system proximal signaling event by other mechanisms in addition to SLP- shows almost no ARAP in the bone marrow, but a strong ex- 76 binding. The intervening phosphorylation site Y521 in ARAP pression in the thymus, lymph node, and tonsil. It remains to be may have a different tyrosine consensus sequence, perhaps more determined whether ARAP expression is developmentally regu- similar to the YDGI in ADAP that was previously identified to lated or enhanced by TCR signaling. Four shorter variant forms mediate the association of ADAP with the Src kinase Fyn (43). are often identified in the database, and the expression patterns of This might explain why Y521 was essentially irrelevant for SLP- alternatively spliced isoforms in different tissues may contribute to 76 binding in the present studies. Using the HEK 293T cell ex- cell-specific functional regulation. Other important questions are: pression system, we confirmed that ARAP directly binds Fyn why do two homologous adaptor proteins (ADAP and ARAP) and Lck, which subsequently phosphorylate tyrosine residues in exist in T cells, if the two proteins cross-regulate each other, and ARAP (Fig. 7D). Another similarity is that the N-terminal ARAP whether ADAP expression increases in response to ARAP sup- PR region appears to show almost complete identity with the pression, or vice versa. primary ADAP site responsible for SKAP1 binding (36). As The similarities and differences between ARAP and ADAP may shown in Fig. 7B and 7G, both ARAP and ADAP constitutively indicate that they have similar yet critically distinct functions in interact with the adaptor protein SKAP1. Although SKAP1 plays a The Journal of Immunology 951 Downloaded from

FIGURE 8. Association of ARAP with SLP-76 and SKAP1 in T cells. (A–C) Jurkat T cells were unstimulated or TCRstimulated with anti-CD3 Ab for 5 min at 37˚C. Lysates were immunoprecipitated with Ab against either SLP-76 (B) or SKAP1 (C). Tyrosine phosphorylation of lysates was detected by IB with 4G10 and equal loading amounts were shown by IB with anti–b-actin (A). Equal levels of IP and the association of SLP-76 (B) or SKAP1 (C) with http://www.jimmunol.org/ ADAP and ARAP were determined by IB with Abs for each protein. All data shown are representative of three separate experiments. (D and E) purified primary human T cells were unstimulated (control) or stimulated for 5 min with anti-CD3 Ab for 5 min at 37˚C. Tyrosine phosphorylation of lysates was detected by IB with 4G10 and loading control was shown by IB with anti–b-actin (D). IP was performed using anti-ARAP Ab and ARAP interaction proteins were analyzed by IB with anti–SLP-76 or anti-SKAP1 (E). Data shown are representative of two separate experiments. key role for the regulation of LFA-1 adhesion (37), ARAP re- may explain why Rap1 activation is defective in ARAP-suppressed cruitment to the IS only occurs on interaction of T cells with SEE- cells, whereas ADAP deficiency has only been associated with loaded APCs (Fig. 2F). Thus, constitutive interaction of SKAP1 defects in Rap1 membrane targeting and not with GTPase activa- with ARAP could be a prerequisite for the function of T cell tion. ARAP is therefore an additional molecular adaptor that cou- by guest on September 25, 2021 adhesion. Previous studies reported the loss of SKAP1 expres- ples TCR signaling to integrin activation, inside–out signaling, and sion in the absence of ADAP, and they suggested that ADAP could T cell adhesion. Further studies are needed to understand the exact maintain SKAP1 stability (44, 45). Questions remain whether mechanisms by which ARAP regulates Rap1 activation and links ARAP also regulates SKAP1 stability or helps to generate a TCR stimulation to changes in integrin avidity. complex including RapL and Rap1 for LFA-1 adhesion. Given the The novel protein ARAP identified in the present study is first broad tissue distribution reported for SKAP2 (46), which also described in the context of T cells. ARAP has a role in the major binds ADAP in the same manner as SKAP1 (47), ARAP also may signaling pathways downstream of the TCR and couples TCR functionally bind SKAP2 in many cell types. signaling with integrin activation, inside–out signaling, and Although both ADAP and ARAP associate with the SH2 domain T cell adhesion. Considering the similarities in domain sequences of SLP-76 in a TCR stimulation–dependent manner, each adaptor and binding partners of ARAP and ADAP, a well-known adaptor molecule plays a distinct role in TCR signaling. ADAP-deficient protein that mediates inside–out integrin activation, additional T cells exhibited early signaling pathways downstream of the TCR studies are needed to understand how these two homologous similar to those of control cells (24, 25). In contrast, ARAP is re- adaptor proteins function in T cells. As ARAP binds to ADAP- quired for the major signaling pathways downstream of TCR binding proteins such as Fyn, Lck, SKAP1, and SLP-76, it would stimulation and T cell activation as shown in cells where ARAP was be useful to examine the affinity of these proteins to ARAP and suppressed by shRNA (Fig. 3). Stimulation of ARAP-suppressed ADAP. It will be necessary to investigate whether ARAP or ADAP T cells with an anti-CD3 Ab revealed differences in total tyro- expression is upregulated when one or the other is suppressed. sine phosphorylation and activation of PLCg1, ERK2, and Akt Considering that the loss of ADAP or SLP-76 does not affect the compared with control T cells. Consequently, the calcium flux and most upstream TCR-mediated signaling event (tyrosine phosphory- NF-AT activation after anti-CD3 cross-linking was defective in lation of proteins), ARAP may function as an immediate adaptor to ARAP-suppressed T cells. Further studies are required to elucidate couple TCR signaling with downstream effector molecules that how ARAP deficiency affects TCR-induced tyrosine phosphoryla- are yet to be discovered. Further studies are needed to better un- tion of many proteins, and whether this also affects TCR-induced derstand the specific roles and possible relationships of these mole- tyrosine phosphorylation of specific signaling proteins such as cules and to fully elucidate how TCR signals are coupled to Vav1, SLP-76, LAT, TCRz-chain, and ZAP-70. ARAP and ADAP integrin activation and T cell adhesion. have contrasting roles in F-actin formation; however, they are re- quired for integrin-mediated adhesion in response to TCR stimu- lation. The activation of the small GTPase Rap1, which regulates Acknowledgments integrin clustering, was defective after TCR stimulation in ARAP- We thank Drs. D.D. Billadeau, G.A. Koretzky, B. Schraven, A. Veillette, suppressed T cells (Fig. 4F). The role of ARAP in TCR signaling Y.D. Yun, and Y.S. Bae for providing valuable plasmids. 952 A NOVEL ADAPTOR PROTEIN IN T CELL ACTIVATION

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FEBS Lett. 435: 55–60. 26. Nolz, J. C., L. P. Nacusi, C. M. Segovis, R. B. Medeiros, J. S. Mitchell, 47. Liu, J., H. Kang, M. Raab, A. J. da Silva, S. K. Kraeft, and C. E. Rudd. 1998. Y. Shimizu, and D. D. Billadeau. 2008. The WAVE2 complex regulates T cell FYB (FYN binding protein) serves as a binding partner for lymphoid protein and receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells. Proc. Rap1. J. Cell Biol. 182: 1231–1244. Natl. Acad. Sci. USA 95: 8779–8784. 1 ------MEGE-GVRNFKELRAKFQNLDAP-PLPGPIKFPAGVSP 36 Q5VWT5 CA168_HUMAN 1 MAKYNTGGNPTEDVSVNSRPFRVTGPNSSSGIQARKNLFNNQGNASPPAGPSNVPKFGSP 60 O15117 FYB_HUMAN 1 MAHH------LPAAME---SHQDFRSIKAKFQ------A- 24 Q96QH2 PRAM_HUMAN *

37 KGDIGGTQ------STQILANGKPLSSNHKQRTPYCS--SSESQPLQPQ 77 Q5VWT5 CA168_HUMAN 61 KPPVAVKPSSEEKPDKEPKPPFLKPTGAGQRFGTPASLT--TRDPEAKVG--FLKPVGPK 116 O15117 FYB_HUMAN 25 -----SQPEPSDLPKKPPKPEFGK----LKKFSQPELSEHPKKAPLPEFGAVSLKPPQPQ 75 Q96QH2 PRAM_HUMAN * * * *

78 KIKLAQKSEIPKCS------91 Q5VWT5 CA168_HUMAN 117 PINLPKEDSKPTFPWPPGNKP------SLHSVNQ------DHDL--KPLG-- 152 O15117 FYB_HUMAN 76 FTDLPKKPPPPEVTDLPKKPPPPEVTDLPKKPPPPEVTDLPKKPPPPEVTDLPKKPPPPE 135 Q96QH2 PRAM_HUMAN * *

92 ------91 Q5VWT5 CA168_HUMAN 153 ----PKSGP------TPP-TSENEQKQAFPKLTGVKGKFMSASQDLEPKPLFPKP 196 O15117 FYB_HUMAN 136 VTDLPKKPPPPEVTDLPKKPPPPEVTDLPKKPSKLELSDLSKKFPQLG------AT 185 Q96QH2 PRAM_HUMAN

92 ------NSPGPLGKSTVCSATSSQKASLLLEVTQSNVEI 124 Q5VWT5 CA168_HUMAN 197 AFGQKPPLSTENSHEDESPMKNVSSSKGSPAPLGVRSKSGP------LKPAREDSEN 247 O15117 FYB_HUMAN 186 PFPRKP-LQ---PEVGEAPLKASLPEPGAPARK------P------LQPDELS-HP 224 Q96QH2 PRAM_HUMAN * *

125 ITKEKVMVANSFRNKLWNWEKVSSQKSEMSSALLLANYGSKAIHLEGQKGMGLTPEEPRK 184 Q5VWT5 CA168_HUMAN 248 KDHAGEISSLPFPGVVLKPAAS------RGGPGLSKNGE-- 280 O15117 FYB_HUMAN 225 ARPPSEPKSGAFPRKLWQPEAG------EATPRSP------253 Q96QH2 PRAM_HUMAN *

185 KLETKGAQTLPSQKHVVAPKILHNVSEDPSFVISQHIRKSWENPPPERSPAS----SPCQ 240 Q5VWT5 CA168_HUMAN 281 --EKKEDRKIDAAKNTFQSKI-----NQEELASG-TPPARFPKAPSKLTVGG------324 O15117 FYB_HUMAN 254 ------QPELSTFPKKP-----AQPEF------NVYPKKPPQPQVGGLPKKSVPQ 291 Q96QH2 PRAM_HUMAN * *

241 PIY----ECELASQAPEKQPDVRHHHLPKTKPLPSIDSLGPPPPKPSRPPIVNLQAFQRQ 296 Q5VWT5 CA168_HUMAN 325 ------PWGQSQEKEKGDKNSATPKQKPLPPLFTLGPPPPKPNRPPNVDLTKFHKT 374 O15117 FYB_HUMAN 292 PEFSEAAQTPLWKPQSS-EPKRDSSAFPKKASQPPLSDFPK------KPPQPELGDLTRT 344 Q96QH2 PRAM_HUMAN ** * ** *

297 PAAVPKTQGEVTVEEGSLSPERLF-NAEFEE---PHNYEATISYLRH--SGNSINLCTA- 349 Q5VWT5 CA168_HUMAN 375 SSGNSTSKGQTSYSTTSLPPPPPSHPASQPPLPASHPSQPPVPSLPPRNIKPPFDLKSPV 434 O15117 FYB_HUMAN 345 SSEPEVS------VLPKRP--RPAEFKA-LSKKPPQPELGGLPRTSSEPEFNSLPR- 391 Q96QH2 PRAM_HUMAN * * *

350 --KEIADPTY----EVGIEELQKPGKNFPYPEPSAKHEDKKMKEKQP-CELKPKN-TEKE 401 Q5VWT5 CA168_HUMAN 435 NEDNQDGVTHSDGAGNLDEEQDSEGETYEDIEASKEREKKREKEEKKRLELEKKEQKEKE 494 O15117 FYB_HUMAN 392 ------KLLQPERRGPPRKFSQPEPS------AVLKRHPQPE-- 421 Q96QH2 PRAM_HUMAN * * * * *

402 PYSNH------VFKVDACEGTPEKIQMTNVHTGRRNMLAGKQEAMID------442 Q5VWT5 CA168_HUMAN 495 KKEQEIKKKFKLTGPIQVIHLAKACCDV------KGGKNELSFKQGEQIE------538 O15117 FYB_HUMAN 422 -FFGDLPRKPPLPSSASESSLPAAVAGFSSRHPLSPG-FGAAGTPRWRSGGLVHSGGARP 479 Q96QH2 PRAM_HUMAN * *

443 IIQTN------PCPEGPKLARHSQGHCGHLEVLESTKETPDLGVSKTSSISEEIYDDVEY 496 Q5VWT5 CA168_HUMAN 539 IIRITDNPE------GKWLGRTA------RGSYGY 561 O15117 FYB_HUMAN 480 GLRPSHPPRRRPLPPASSLGHPP------AKPPLP------PGPVDMQSF 517 Q96QH2 PRAM_HUMAN *

497 SRKEVPKLNYSS------SLASSSEENRELYEDVYKTKNNYPKIDLDGKEA 541 Q5VWT5 CA168_HUMAN 562 IKTTAVEIDYDSL------KLKKDSLGAPSRPIEDDQEVYDDVAEQDDISSHSQSGSG-- 613 O15117 FYB_HUMAN 518 RRPSAASIDLRRTRSAAGLHFQDRQPEDIPQVPDEIYELYDDVEPRDDSSP------568 Q96QH2 PRAM_HUMAN * * **

542 LKRLQQFFKKEKDRFKIKKTKSKENLSAFSILLPDLELKSQEVIIYDDVDLSEKESKDED 601 Q5VWT5 CA168_HUMAN 614 -----GIFPPPPDDDIYDGIEEEDADDGFPA--PP-KQLDMGDEVYDDVDTSDFPVSSA- 664 O15117 FYB_HUMAN 569 ------S---P-KGRDEAPSVQQAA---RRPPQDP- 590 Q96QH2 PRAM_HUMAN

602 KLKMWKPKFLTPKEKKEKNGAEESESFSPRNFFKTKKQNL-EKNRMKREEKLFRERFKYD 660 Q5VWT5 CA168_HUMAN 665 ------EMSQ---GTNV------GKAKTEEKDLKKLKKQEKEEKDFRKKFKYD 702 O15117 FYB_HUMAN 591 ------ALRKEKDPQPQ------QLPPMDPKLLKQLRKAEKAEREFRKKFKFE 631 Q96QH2 PRAM_HUMAN * * ** **

661 KEIIVINTAVAC--SNNSRNGIFDLPISPGEELEVIDTTEQNLVICRNSKGKYGYVLIEH 718 Q5VWT5 CA168_HUMAN 703 GEIRVLYSTKVTTSITSKKWGTRDLQVKPGESLEVIQTTDDTKVLCRNEEGKYGYVLRSY 762 O15117 FYB_HUMAN 632 GEIVVHTKMMIDPNAKTRRGGGKHLGIRRGEILEVIEFTSNEEMLCRDPKGKYGYVPRTA 691 Q96QH2 PRAM_HUMAN ** * * * ** **** * ** ******

719 LDFKHQ-SWSP------728 Q5VWT5 CA168_HUMAN 763 LADNDGEIYDDIADGCIYDND------783 O15117 FYB_HUMAN 692 LLPLETEVYDDVDFCDPLENQPLPLGR 718 Q96QH2 PRAM_HUMAN *

Supplementary Fig. 1. Alignment of the predicted protein sequence of ARAP (CA168), ADAP (FYB), and PRAM