Dynamic Interaction- and Phospho-Proteomics Reveal Lck as a Major Signaling Hub of CD147 in T Cells

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 1, 2017, doi:10.4049/jimmunol.1600355 The Journal of Immunology

Dynamic Interaction- and Phospho-Proteomics Reveal Lck as a Major Signaling Hub of CD147 in T Cells

Verena Supper,* Ingrid Hartl,* Cyril Boule`gue,† Anna Ohradanova-Repic,* and Hannes Stockinger*

Numerous publications have addressed CD147 as a tumor marker and regulator of cytoskeleton, cell growth, stress response, or im- mune cell function; however, the molecular functionality of CD147 remains incompletely understood. Using affinity purification, mass spectrometry, and phosphopeptide enrichment of isotope-labeled peptides, we examined the dynamic of the CD147 microenvironment and the CD147-dependent phosphoproteome in the Jurkat T cell line upon treatment with T cell stimulating agents. We identified novel dynamic interaction partners of CD147 such as CD45, CD47, GNAI2, Lck, RAP1B, and VAT1 and, furthermore, found 76 CD147- dependent phosphorylation sites on 57 . Using the STRING network database, a network between the CD147 micro-

environment and the CD147-dependent phosphoproteins was generated and led to the identification of key signaling hubs around the G Downloaded from proteins RAP1B and GNB1, the kinases PKCb, PAK2, Lck, and CDK1, and the chaperone HSPA5. ontology biological process term analysis revealed that wound healing–, cytoskeleton-, immune system–, stress response–, phosphorylation- and protein mod- ification–, defense response to virus–, and TNF production–associated terms are enriched within the microenvironment and the phosphoproteins of CD147. With the generated signaling network and biological process term grouping, we identify potential signaling routes of CD147 affecting T cell growth and function. The Journal of Immunology, 2017, 198: 000–000. http://www.jimmunol.org/ D147 is a ubiquitously expressed type 1 transmembrane the amino acid transporter CD98 H chain (CD98) (54, 55), has glycoprotein belonging to the Ig superfamily. It is therefore been shown to affect cell growth and survival by modulating in- C not surprising that it is involved in a plethora of cell tracellular lactate or amino acid concentrations and stimulating functions such as cell proliferation (1–15), cell death (11, 12, 16– PI3K/PKB (3, 55). The activation of PI3K/PKB and subsequent 22), stress response (17, 23), chemosensitivity (10, 17, 21, 24, 25), activation of the MAPK pathway upon engagement of CD147 with chemotaxis (9, 26–34), migration (1, 2, 10–13, 18, 33, 35–41), cyclophilins has also been observed to support cell proliferation (56, adhesion (1, 6, 14, 33, 35, 42–45), and metabolism (3, 5, 16). 57), stress protection (23–25), and chemotactic processes (9, 26–32, Concomitantly, the effect of CD147 on these cellular functions has 43, 58). Moreover, activation of PI3K (59) and ERK (36) were found implications in the regulation of T cell activity (1, 4, 6–9, 14, 15, to be prerequisites for the CD147/b1-integrin–dependent modulation by guest on September 23, 2021 34, 46–48), cancer (5, 13, 49–51), and metastasis (37–41, 52), and of FAK activation and the adhesive capacities of cells. Activation of makes CD147 an ideal disease marker and therapy target (49, 53). FAK and MAPKs and the cytoskeletal regulators vinculin and pax- In using CD147 as a therapy target, it is essential to understand illin were shown to be involved in subsequent CD147-dependent the possible signaling outcomes, and several attempts have been cytoskeletal changes in actin, microtubule, and vimentin fila- made to understand CD147-dependent molecular mechanisms. ments (60, 61). In addition to cytoskeletal processes, CD147 The interaction of CD147 with transporters, such as the mono- has also been observed to protect cells from by carboxylate transporters 1 and 4 (MCT1 and MCT4) (3, 5, 16) or regulating caspase-3 (19, 20, 22), BIM (20, 22), Bcl-2 (20),

*Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Cen- The online version of this article contains supplemental material. ter of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Abbreviations used in this article: ACIN1, apoptotic chromatin condensation inducer 1; 1090 Vienna, Austria; and †Microchemistry Core Facility, Max Planck Institute of AP-MS, affinity-purification mass spectrometry; ARHGEF2, Rho/Rac guanine nu- Biochemistry, 82152 Martinsried, Germany cleotide exchange factor 2; ATP1A1, Na+/K+ ATPase a-1 subunit; B2M, b-2-micro- ORCIDs: 0000-0003-2831-600X (V.S.); 0000-0002-8005-8522 (A.O.-R.); 0000- globulin; CD226, platelet and T cell activation antigen 1; CD43, sialophorin; CD45, 0001-6404-4430 (H.S.). protein tyrosine phosphatase receptor type C; CD47, integrin-associated signal trans- ducer CD47; CD98, amino acid transporter CD98 H chain; CDK1, -dependent Received for publication February 29, 2016. Accepted for publication January 6, kinase 1; CFL1, cofilin 1; DOCK2, dedicator of cytokinesis 2; GNAI2, a 2017. inhibiting activity polypeptide 2; GNB1, G protein b polypeptide 1; GOBP, gene This work was supported by the GEN-AU-Program of the Austrian Federal Ministry ontology biological process; G protein, guanine nucleotide binding protein; HA, of Science and Research (FA644A0103), the Austrian Research Promotion Agency hemagglutinin; HACD147etc, HA-tagged RNAi-resistant CD147; HSPA5, heat (Mobility Stipendium 831947), the Seventh Framework Program (FP7/2007-2013) shock 70 kDa protein 5; Lck, lymphocyte-specific protein tyrosine kinase; MAP1A, under Grant Agreement NMP4-LA-2009-228827 NANOFOL and the European microtubule-associated protein 1A; MCT1, monocarboxylate transporter 1; MCT4, Union’s Horizon 2020 Research and Innovation Program under Grant Agreement monocarboxylate transporter 4; MYH10, myosin-10; NCOR1, nuclear receptor co- 683356. repressor 1; PAK2, p21-activated kinase 2; PKCb, C b; PMCA4, plasma membrane calcium ATPase isoform 4; RAP1B, Ras family small GTP bind- H.S. initiated the project; V.S. and H.S. designed the experiments; V.S., A.O.-R., and 13 ing protein; RB1, retinoblastoma 1; R6K4, [ C6L-arginine/D4 L-lysine; R10K8, H.S. wrote the paper; V.S., I.H., C.B., and A.O.-R. performed the experiments; V.S., 13 15 13 15 [ C , N ]L-arginine/[ C , N ]L-lysine; RNAi, RNA interference; RPS6, ribosomal I.H., C.B., and A.O.-R. analyzed data; all authors read and approved the manuscript. 6 4 6 2 protein S6; shControl, short hairpin RNA control; shRNA, short hairpin RNA; The mass spectrometry proteomics data presented in this article have been deposited SILAC, stable isotope labeling with amino acids in cell culture; SPTBN1, spectrin to the ProteomeXchange Consortium via the PRIDE partner repository (https://www. b nonerythrocytic 1; STIM1, stromal interaction molecule 1; TiO2, titanium oxide; ebi.ac.uk/pride/archive/) with the dataset identifier PXD002055. VCP, valosin-containing protein.

Address correspondence and reprint requests to Prof. Hannes Stockinger, Molecular Ó Immunology Unit, Institute for Hygiene and Applied Immunology, Kinderspitalgasse Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 15, 1090 Vienna, Austria. E-mail address: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600355 2 MICROENVIRONMENT AND SIGNALING DYNAMICS OF CD147 and XIAP (21), an effect that is at least partially dependent on (shCD147) containing an shRNA construct specific for human CD147 and the MAPK activation (22). A possible link between CD147 and pBMN-IRES-GFP-HACD147etc containing the RNA interference (RNAi)- these pro- and anti-apoptotic regulators might lie in the CD147- resistant full-length CD147 with a hemagglutinin (HA)-tag (HACD147etc) were cloned as described previously (64). dependent activation of FAK and Src, which enhances heat shock 70 kDa protein 5 (HSPA5) promoter activity via the phosphorylation Gene transfer of TFII-I (17). Viral transduction was used for gene delivery, as documented elsewhere (66). In addition, CD147 also plays a crucial role in T cell biology: it has been shown to affect thymocyte development (62) and T cell pro- T cell stimulation liferation (1, 4, 6–8, 14, 15), which was ascribed to modulated Jurkat T cells were stimulated with either 16.2 nM PMA plus 1 mM ion- expression levels of IL-2 (7) and its receptor CD25 (1, 7, 47, 63). omycin or a 1:100 diluted hybridoma supernatant containing mAb C305 Further, two studies have reported reduced phosphorylation of early against the T cell Ag receptor at 37˚C in 5% CO2 and a humidified atmosphere. TCR signaling components and reduced calcium mobilization upon CD147 silencing (34) or CD147 Ab treatment (7), whereas in an- Pull-down and peptide preparation other study enhanced JNK and p21-activated kinase 1 activation and Cells were lysed in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM thus higher NFAT transcriptional activity in CD147-silenced cells PMSF, 1 mM sodium orthovanadate, 50 mM NaF, 0.5% lauryl-maltoside, has been reported (48). We showed recently that interaction of and protease inhibitor mixture). The lysate was incubated with agarose CD147 with the plasma membrane calcium ATPase isoform 4 coated with anti-HA mAb (Sigma-Aldrich). The proteins were eluted with (PMCA4) bypasses TCR proximal signaling and inhibits IL-2 ex- urea buffer (6 M urea, 2 M thiourea, 10 mM HEPES, pH 8), reduced with 1 mM DTT, alkylated with 5.5 mM iodoacetamide and digested in-solution pression (64). Nevertheless, the understanding of the molecular for 3 h with LysC (1 mg/50 mg of protein). After four times dilution with Downloaded from functionality of CD147 is still incomplete. In particular, not all the 50 mM ammonium bicarbonate, the proteins were digested overnight with direct interaction partners and first-line signal transducers are un- 1 mg trypsin/50 mg protein (Promega, Fitchburg, WI) at room temperature. derstood, which might generate pitfalls for several therapeutic The next day samples were desalted on a C18 stage tip. strategies. In addition, a huge bias has been generated in earlier Peptide preparation using the filter-aided sample preparation studies, because major findings are limited and have focused on method signaling routes for which specific Abs for Western blot analysis http://www.jimmunol.org/ For the phosphopeptide enrichment experiments, cells were lysed in ex- were available. The present study combines two proteomic ap- traction buffer (4% SDS, 0.1 M Tris/HCl, pH 7.6, protease and phosphatase proaches and analysis of the in silico protein network database to inhibitor mixture), and the lysate was boiled for 5 min at 95˚C and soni- find hidden molecules in the CD147-dependent signaling system cated. The protein concentration was estimated by measuring the trypto- and to scrutinize the major signaling hubs. phan content with the Infinite 200 (Tecan, Ma¨nnedorf, Switzerland) at 280 nm and the same protein amounts of the shControl and shCD147 lysate were pooled. Proteins (5–6 mg) were loaded with 8 M urea on to an Amicon Materials and Methods Ultracell 30k by centrifugation for 30 min at 2500 3 g. The proteins Abs were then washed three times with8Mureaandincubatedwith0.05M iodoacetamide in 8 M urea for 20 min. Thereafter, the proteins were CD147 mAb MEM-M6/1 was provided by Vaclav Horejsi (Institute of washed twice with 8 M urea and twice with digest buffer (10% aceto- by guest on September 23, 2021 Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, nitrile, 0.1 M Tris/HCl, pH 8.5). Proteins were digested with 40 mg Czech Republic); mAb C305 to the TCR was a gift from Arthur Weiss trypsin in digest buffer overnight at 37˚C in a wet chamber. The next day, (University of California, San Francisco, CA). The rabbit anti-phospho-Src peptides were collected by centrifugation and the remaining protein in pan-Tyr416, the rabbit anti–phospho–lymphocyte-specific protein tyrosine the Amicon cell was subjected to further digestion with 20 mg trypsin in kinase (Lck) Tyr505, the rabbit anti-phospho–protein kinase C (PKC) pan- digest buffer for 150 min before residual peptides were collected again b a b II Ser660, the rabbit anti-phospho-PKC / II Thr638/641, the rabbit by centrifugation. Lastly, 0.1% trifluoroacetic acid was added to the d u anti-phospho-PKC / Ser643/676, the mouse anti-rabbit mAb (clone eluate and the peptide concentration was determined at 280 nm by the 4H1), and the rabbit anti-GAPDH mAb (clone 14c10) were purchased NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). from Cell Signaling Technology (Danvers, MA). Anti-Lck polyclonal rabbit Ab (clone H-95) was from Santa Cruz Biotechnology (Dallas, TX). Titanium dioxide phosphopeptide enrichment The goat anti-rabbit IgG-HRP conjugates were purchased from Cell Sig- naling Technology and Rockland (Limerick, PA) and the goat anti-mouse The phosphopeptides were enriched with titanium oxide (TiO2) beads as IgG-HRP conjugate was from Sigma-Aldrich (St. Louis, MO). described by Sharma et al. (67). In short, phosphopeptides were isolated in six sequential isolation steps with the TiO2 beads using the supernatant of Reagents the preceding pull-down for the subsequent pull-down as illustrated in Fig. 2A. For the first to fourth pull-downs, a peptide-to-bead mass ratio of PMA and ionomycin calcium salt (ionomycin) from Streptomyces conglobatus 1:0.25 was used and for the fifth and sixth pull-downs the ratio was 1:4. were purchased from Sigma-Aldrich. The protease inhibitor mixture Complete, Mini, EDTA-free and the phosphatase inhibitor mixture PhosSTOP were from Mass spectrometry Roche (Basel, Switzerland). N-Dodecyl b-D-maltoside (lauryl-maltoside) and Nonidet P-40 were from Thermo Fisher Scientific (Waltham, MA). The peptides were desalted on a C18 stage tip. A chromatographic sepa- ration was performed using an Agilent 1200 nanoflow system (Thermo Cell culture and stable isotope labeling Electron, Bremen, Germany), coupled with a reversed-phase ReproSil-Pur C18-AQ 3 mm resin (Dr. Maisch, Ammerbuch-Entringen, Germany) in a The human leukemic T cell line Jurkat E6.1 and the human embryonic 15-cm silica emitter (75 mm inner diameter; Proxeon Biosystems, Odense, kidney cell line HEK 293T were obtained from the American Type Culture Denmark). The flow rate of injection was set to 500 nl per min and pep- Collection (Manassas, VA). Cells were cultivated as previously described tides were eluted with a flow rate of 250 nl per min using a 100 min (65). For stable isotope labeling with amino acids in cell culture (SILAC), 13 gradient of 2–40% acetonitrile in 0.5% acetic acid. Peptides were then cells were fed with RPMI 1640 containing [ C6]L-arginine/D4 L-lysine 13 15 13 15 analyzed with the LTQ Orbitrap mass spectrometer (Thermo Electron) (R6K4) or [ C , N ]L-arginine/[ C , N ]L-lysine (R10K8), respectively 6 4 6 2 containing an electrospray ion source (Proxeon Biosystems). The settings (Cambridge Isotope Laboratories, Tewksbury, MA), and supplemented for the precursor ion analysis were: m/z 300–1800, a resolution of 60,000, with 10% dialyzed FCS (Invitrogen, Carlsbad, CA). and an ion accumulation to a target value of 1,000,000. The 10 most Plasmids abundant ions were further fragmented and recorded in the ion trap and then dynamically excluded for 60 s. A lock mass option was enabled. The lentiviral packaging plasmids psPAX and pMD2.G were from Addgene The data were processed with MaxQuant version 1.3.0.5 (68) with the (Cambridge, MA); the pLKO.1-puro-nonmammalian short hairpin RNA integrated Andromeda search engine (69) and the UNIPROT protein index (shRNA) control (shControl) was supplied by the German Science Center database for Homo sapiens with common contaminants added. Search for Genome Research (Berlin, Germany). The pLKO.1-puro-CD147_333 parameters set to #3 allowed missed cleavages for the affinity-purification The Journal of Immunology 3 mass spectrometry (AP-MS) experiments, and #2 allowed missed cleavages (GNB1), and valosin-containing protein (VCP) were identified be- for the phosphopeptide analysis, with cystein carbamidomethylation as the 13 ing part of the CD147 microenvironment. Upon stimulation, we fixed modification and N-acetyl protein, oxidized methionine, [ C6]L-arginine, 13 15 13 15 found significant interaction of CD147 with the D4 L-lysine, [ C6, N4]L-arginine, and [ C6, N2]L-lysine as variable modi- a fications. For the AP-MS experiments, the mass tolerance was set to 7 ppm tyrosine receptor type C (CD45), the G protein inhibiting activity for precursor ion peaks and to 0.5 kDa for the product ion peaks; for the polypeptide 2 (GNAI2), HSPA5, Lck, VAT1, the integrin-associated phosphopeptide analysis, the mass tolerance was 6 and 20 ppm for the pre- signal transducer CD47 (CD47), and the Ras family small GTP cursor and product ion peaks respectively. The false discovery rate was de- binding protein (RAP1B) (Fig. 1). The numeric data are provided in termined by searching a reverse database and the false discovery rate of 0.01 on peptide and protein level. A minimum peptide length of five residues was Supplemental Table I. In Supplemental Fig. 1 we compare the in- set for the AP-MS experiments and a minimum of six residues for the tensity values of the coisolated molecules of the different stimula- phosphopeptide analysis and one unique and a second identified peptide was tion conditions with each other side-by-side. Most interactions of set as the prerequisite for protein identification. The match between runs CD147 were intensified after stimulation, and PMA plus ionomycin option with a time window of 2 min between replicates was enabled. enforced the interactions with all interaction partners, except Quantification, random error issues, and estimates of uncertainty were performed as detailed in Cox and Mann (68). ATP1A1, calnexin, and moesin: ATP1A1 showed reduced interac- The mass spectrometry proteomics data have been deposited to the tion with CD147 upon stimulation with both mAb C305 and PMA ProteomeXchange Consortium via the PRIDE partner repository (70) with plus ionomycin, and calnexin and moesin were reduced associated the dataset identifier PXD002055 (https://www.ebi.ac.uk/pride/archive/). upon short-term stimulation with the mAb C305. Immunoblotting Analysis of the CD147-dependent phosphoproteome in resting The procedure described by Pfisterer et al. (71) was used for Western blot

and activated T cells Downloaded from analysis. For analysis of protein phosphorylation, the cells were stimulated as described above or left untreated for the indicated time, then quickly washed To elucidate CD147-dependent signaling pathways in unstimulated with ice-cold stopping buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, and stimulated Jurkat T cells, we followed the strategy illustrated in 0.1 mM sodium orthovanadate), and lysed in lysis buffer (20 mM Tris-HCl, pH Fig. 2A. In short, Jurkat T cells were labeled using SILAC, either m 7.5, 140 mM NaCl, 1 M PMSF, 1 mM sodium orthovanadate, 20 mM NaF, with R10K8 or with R6K4. After 10 d, the label incorporation 1% (v/v) Nonidet P-40, protease inhibitor mixture) on ice for 30 min. Lysates were precleared by brief centrifugation in a table-top centrifuge for 20 s and tested by electrospray ionization-liquid chromatography–tandem the protein concentration was determined using the Pierce BCA Protein mass spectrometry was above 95% for 99% of the identified http://www.jimmunol.org/ Assay Kit (Thermo Fisher Scientific). Samples of 10 mg were separated peptides (Fig. 2B). Next, shRNA constructs targeting CD147 or a by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, nontarget control construct were lentivirally transferred to the blocked, and probed with the respective phospho-specific Abs, and processed further as already described (71). To prevent protein dephos- heavy R10K8-labeled or the light R6K4-labeled cells, respec- phorylation, the blocking buffer, Ab solutions, and the wash buffer were tively, and after 1 wk the silencing efficiency was estimated by supplemented with 0.1 mM sodium orthovanadate. After the detection of Western blot analysis (Fig. 2C). the chemiluminescent signal, Abs were stripped off the membrane with Then the CD147-silenced and shControl Jurkat T cells were either acidic stripping buffer [0.2 M glycine, 0.1% (w/v) SDS, 1% (v/v) Tween- left unstimulated or separately stimulated for 1 min with TCR mAb 20, pH 2.2], washed extensively, then blocked and reprobed using mAbs recognizing phosphorylation-independent epitopes. C305, or for 5 and 30 min with PMA plus ionomycin. After lysis, equal protein amounts from the shControl and shCD147 samples were by guest on September 23, 2021 Statistics pooled and subsequently digested with trypsin. Phosphopeptides were VolcanoplotsbasedonunpairedStudentt tests were generated for isolated, prefractionated using the sequential phosphopeptide en- statistical evaluation of interaction partners identified in AP-MS ex- richment method with TiO2 beads (67), and analyzed by electrospray periments from three biological replicates with the Perseus software ionization-liquid chromatography–tandem mass spectrometry. We package 1.5.2.6 (http://ww.perseus-framework.org/). The plots show the difference (the fold-intensity difference as LogN) of two conditions on thereby identified 1374 different phosphopeptide pairs of CD147- the x-axis and the p value (as 2log10) on the y-axis. Western blotting silenced cells and shControl cells from which a SILAC ratio was data were analyzed by two-way ANOVA with Bonferroni posttest using generated. Using the Perseus software package 1.5.2.6, we correlated Prism 5 (GraphPad Software, La Jolla, CA). Significance was accepted the SILAC ratios of two independent experiments and assigned 76 p , at 0.05. phosphopeptides from 57 proteins that were consistently enriched or decreased by at least one-third in response to CD147 silencing Results (Fig. 3). The numeric data are provided in Supplemental Table II. Analysis of the dynamic of the CD147 microenvironment upon To determine which of the phosphopeptides were differentially T cell stimulation phosphorylated in response to T cell stimulating agents and which To examine how the microenvironment of CD147 changes during changes are stimulation independent, we used the Perseus software T cell stimulation, we employed AP-MS in the presence or absence of package 1.5.2.6 Euclidean clustering function to generate a heat map T cell stimulating agents. We retrovirally transferred HACD147etc as showing the ratios of the 76 phosphopeptides (Fig. 4). Most CD147- we described earlier (64) into CD147-silenced Jurkat T cells. Then dependent changes in phosphorylation were unaffected by T cell we treated the cells with T cell stimulating agents for the indi- stimulation and thus stimulation independent. For instance, over all cated time points. The anti-HA Sepharose was used for affinity different stimulations, phosphosites associated with cell cycle regu- purification from HACD147etc-expressing cells, and wild-type lations, such as T373, S788, S811, T821, T823, and T826 in the Jurkat T cells were used as negative control. Purified proteins retinoblastoma 1 (RB1) protein and T14 in cyclin-dependent kinase 1 were subjected to LysC and trypsin digest in-solution and ana- (CDK1) were phosphorylated to a greater degree in CD147-silenced lyzed using electrospray ionization-liquid chromatography–tandem cells. Cytoskeleton-associated molecules, such as the dedicator of mass spectrometry. Volcano plots based on unpaired Student t tests cytokinesis 2 (DOCK2) at S1784, erythrocyte membrane protein showed significant (p # 0.05) and strongly enriched (difference band 4.1 (EPB41) at S85, microtubule-associated protein 1A .2) CD147 interaction partners that have been previously described (MAP1A) at S2056, and septin 2 (SEPT2) at S218 were less phos- by us and others: MCT1 (54, 72, 73), CD98 (43, 54), MHC class 1A phorylated upon CD147 silencing. Similarly, Lck, which we found to (HLA-A), the Na+/K+ ATPase a-1 subunit (ATP1A1) (54), PMCA4, associate with CD147 upon stimulation (Fig. 1), and protein kinase and moesin (64). Moreover, calnexin, b-2-microglobulin (B2M), C b (PKCb) were persistently less phosphorylated at Y394 and S660/ the guanine nucleotide binding protein (G protein) b polypeptide 1 T641, respectively. 4 MICROENVIRONMENT AND SIGNALING DYNAMICS OF CD147 Downloaded from http://www.jimmunol.org/

FIGURE 1. Dynamic of the CD147 microenvironment upon T cell stimulation. Jurkat T cells expressing HACD147etc and Jurkat wild type cells (negative control) were (A) used unstimulated or (B) stimulated for 5 min with TCR mAb C305, or (C) for 5 min or (D) 30 min with PMA plus ionomycin. The lysates were subjected to HA pull-down and subsequent mass spectrometric analysis. Normalized intensity values of detected proteins from three independent experiments were analyzed using volcano plots with Perseus software package 1.5.2.6. As detection of ATP1A1, a known interaction partner of by guest on September 23, 2021 CD147 (64), failed in one experiment in the unstimulated condition, this experiment was excluded for calculations of the ATP1A1 values in unstimulated cells. Proteins significant (p # 0.05 and difference .2) in at least one condition are labeled and the significance area is indicated by the gray-shaded box. The numeric data are provided in Supplemental Table I.

In contrast to the stimulation-independent changes, we also de- Similarly, phosphorylation of PKC a/bII at T638/641 was lower tected CD147-dependent phosphopeptides only at specific time in shCD147 cells. We also tested phosphorylation of PKCu, points of stimulation (Fig. 4, highlighted in gray). These dynamic which is an important player in TCR-downstream signaling stimulation-dependent phosphopeptides were further analyzed for events and is involved in CD43 phosphorylation (74). However, peptide intensity levels and those with intensities .106 are indi- using the phospho-PKCd/u Ab recognizing S643/676, we found cated with asterisks: phosphorylation of reticulon 4 (RTN4) at S184 no significant effect caused by CD147 silencing on this phos- was only enhanced in unstimulated CD147-silenced cells and was phorylation site, though the level of phosphorylation was also not detected any more upon stimulation. Dynamic phosphorylations mildly decreased compared with the levels found in shControl at S291 in sialophorin (CD43) and at S2358 in spectrin b non- cells (Fig. 5). Moreover, it should be noted that stimulation with erythrocytic 1 (SPTBN1) were gradually enhanced by PMA plus PMA did not affect phosphorylation of PKCb at S660 and T641 ionomycin stimulation in CD147-silenced cells. Similarly, the or PKCu at S676 in general, although the activity of both of phosphosite T679 on the Rho/Rac guanine nucleotide exchange these PKC isoforms was described to be induced by PMA (75). factor 2 (ARHGEF2) was increased after 30 min PMA plus ion- Interestingly, we found decreased phosphorylation at the in- omycin treatment. Phosphorylation at S575 in the stromal interaction hibitory Y505 site of Lck as well as reduced protein levels of molecule 1 (STIM1) was decreased in unstimulated CD147-silenced total Lck in CD147-silenced cells. Further, the protein levels of cells and was not detected upon short-term stimulation. The de- RB1 were rather increased (Fig. 5), which might give rise to the creased phosphorylations at S1938 in myosin-10 (MYH10) and at higher levels of its phosphorylated versions. Thus, some of the T241 in the ribosomal protein S6 (RPS6) were only reproducibly differentially phosphorylated peptides in CD147-silenced ver- detected upon 5 min PMA plus ionomycin stimulation. sus shControl cells might be caused by downregulation or up- To confirm CD147-dependent changes in protein phosphoryla- regulation of the specific proteins in the CD147-silenced cells. tion by an independent approach, we stimulated CD147-silenced and shControl Jurkat T cells with PMA plus ionomycin for 5 Analysis of the molecular network of CD147 by linking the and 30 min (or left them untreated), and analyzed some of the CD147 microenvironment with the CD147-dependent detected phosphosites by Western blotting using specific Abs phosphoproteome (Fig. 5). We consistently detected lower phosphorylation of Lck To get a comprehensive picture of the interaction and signaling at Y394 and of PKCb at S660 in CD147-silenced cells (Fig. 5). routes of CD147, we linked the microenvironment of CD147 with The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Workflow and quality controls for phosphopeptide enrichment. (A) Visualization of the workflow for the phosphopeptide enrichment ex- periments showing SILAC labeling, silencing, stimulation conditions, lysis and sample pooling, digest, sequential phosphopeptide isolation, and proteomic analysis. (B) SILAC incorporation efficiency distribution of all detected peptides. Peptides originate from shControl Jurkat T cells propagated for five doublings in medium containing R6K4. (C) CD147 immunoblot showing the CD147 silencing efficiency. Actin was used as the loading control. the CD147-dependent phosphoproteome using STRING (76) RAP1B, GNB1, Lck and HSPA5 with p21-activated kinase 2 analysis (Fig. 6A). Direct connections of proven protein-protein (PAK2); GNB1 and Lck with PKCb; HSPA5 with SPTBN1 and interactions indicated by magenta lines between interaction part- MYH10; moesin with CD43; VCP and HSPA5 with CDK1; VCP ners and signaling targets of CD147 were found with the fol- with the ubiquitin fusion degradation protein 1 homolog lowing molecules: RAP1B with the GTPase activating (UFD1L); PMCA4 with STIM1 and ATP1A1 with cofilin 1 protein 2 (RAP1GAP2); RAP1B and GNAI2 with the leucine-rich (CFL1). Thus, the G proteins RAP1B and GNB1, the kinases repeats and WD repeat domain containing protein 1 (LRWD1); PKCb, PAK2, Lck, and CDK1, and the chaperone HSPA5 appear 6 MICROENVIRONMENT AND SIGNALING DYNAMICS OF CD147 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Identification of CD147-dependent phosphopeptides. The SILAC ratios (shCD147/shControl) of two separate experiments were plotted against each other and are shown in a dot plot diagram with logarithmic scale. Labeling indicates phosphopeptides enriched or decreased repeatedly byat least 33% after CD147 silencing. For convenience, only the names of the corresponding proteins are given. Phosphopeptides of (A) unstimulated cells, (B) cells stimulated for 1 min with mAb C305, or (C) stimulated for 5 min or (D) 30 min with PMA plus ionomycin are shown. The numeric SILAC peptide ratios are provided in Supplemental Table II. as major signaling hubs connecting several of the identified in- cytoskeleton-associated terms (green) are the CD147 interaction teraction partners and signaling targets of CD147. partners CD47, Lck, CD45, B2M, moesin, CD98, and MCT1, and On the basis of STRING database gene ontology biological the CD147-dependent phosphoproteins reticulon 4 (RTN4), process (GOBP) term enrichment analysis (shown in the table of DOCK2, MYH10, PAK2, SPTBN1, RPS6, the bridging integrator Fig. 6B and indicated in Fig. 6A by the colored circles), we found 2 (BIN2), CDK1, CD43, the nuclear receptor corepressor 1 the terms associated with wound healing as the highest enriched (NCOR1), and CFL1. The STRING database also found connec- terms in the combined analysis of CD147 interaction partners tions to the GOBP terms associated with the immune system and CD147-dependent phosphoproteins. Other enriched GOBP (violet) for the CD147 interaction partners CD47, Lck, CD45, terms include cytoskeleton-, immune system–, stress response–, B2M, and HLA-A, and the CD147-dependent phosphoproteins phosphorylation- and protein modification–, defense response– MAP1A, the platelet and T cell activation Ag 1 (CD226), the (particularly to virus), and TNF production–associated terms. The SAM and SH3 domain containing protein 3 (SASH3), the apo- links between proteins in the same GOBP term group indicate ptotic chromatin condensation inducer 1 (ACIN1), RPS6, possible CD147-dependent signaling routes for the respective DOCK2, PAK2, PKCb, the heterogeneous nuclear ribonucleo- biological processes. For instance, interaction partners GNAI2, protein K (HNRNPK), CDK1, RB1, CD43, NCOR1, and CFL1. RAP1B, GNB1, Lck, CD47, HSPA5, CD98, MCT1, and PMCA4, Proteins linked with stress response-associated terms (gray) were and phosphoproteins PKCb, MYH10, CD43, CFL1, and STIM1 the CD147 interaction partners RAP1B, GNAI2, CD47, HSPA5, of CD147 are all linked according to the STRING database to the Lck, CD45, B2M, HLA-A, VCP, CD98, MCT1, and PMCA4, GOBP term wound healing (orange). Proteins enriched in the and the CD147-dependent phosphoproteins MAP1A, ARHGEF2, The Journal of Immunology 7

ACIN1, the transformer 2 b homolog (Drosophila) (TRA2B), the sperm associated Ag 9 (SPAG9), RCSD domain containing protein 1 (RCSD1), RAD18 E3 ubiquitin protein ligase, CD226, the cerebral cavernous malformations 2 protein (CCM2), DOCK2, the pro- grammed cell death 4 (PDCD4), MYH10, PAK2, PKCb, CDK1, CD43, NCOR1, STIM1, and CFL1. Phosphorylation- and protein modification–associated terms (red) were found with the CD147 in- teraction partners RAP1B, GNAI2, GNB1, HSPA5, Lck, CD45, B2M, VCP, CD98, and PMCA4, and the CD147-dependent phos- phoproteins ARHGEF2, Sorbin and SH3 domain containing protein 3 (SORBS3), PDCD4, PAK2, RPS6, HNRNPK, CDK1, SPAG9, RB1, CD43, and NCOR1. Proteins assigned to the defense response (blue) include the interaction partnersLck,HLA-A,B2M,andCD47,and the CD147 phosphoproteins MAP1A, ACIN1, PAK2, DOCK2, CDK1, CD43, and CD226. The TNF production-associated terms (yellow) involve the CD147-dependent phosphoproteins ARHGEF2, SASH3, nucleolin, and CD43.

Discussion Downloaded from A number of reports and data indicate that the Ig family member CD147 fulfills a plethora of functions in immune cells, particularly in T cells; functions that are typically hijacked by cancer cells. However, until now few direct links between function and phys- ically or functionally associated signaling partners of CD147 have

been recognized. Using AP-MS and various T cell stimulation http://www.jimmunol.org/ strategies, we found the previously identified interaction partners ATP1A1 (54), CD98 (43, 54), HLA-A (54), MCT1 (54, 72, 73), moesin, and PMCA4 (64), furthermore novel interaction partners in B2M, calnexin, GNB1, and VCP, and CD45, CD47, GNAI2, HSPA5, Lck, RAP1B, and VAT1 as novel interaction partners that dynamically interact with CD147 upon T cell activation. With the exception of HSP5A, all interactions were enforced or decreased with T cell stimulation: in particular, the increased interaction between PMCA4 and CD147 is interesting, as we reported re- by guest on September 23, 2021 cently that via this interaction CD147 inhibits IL-2 production and the calcium-exporting function of PMCA4 (64). Interestingly, we also found a CD147- and stimulation-dependent phosphorylation site in STIM1. Based on our findings and as CD147 translocates to the T cell synapse (7), and STIM1 and PMCA4 were shown to regulate localized calcium fluxes there (77, 78), we hypothesize that the dynamic interaction with CD147 plays a role in the timely regulation of the localized activity of PMCA4. Additionally, both CD147 and PMCA4 possess an ezrin/radixin/moesin binding site, the potential association site with the CD147 interaction partner moesin, and PMCA4 also possesses cytoskeleton regulatory ac- tivity (79–81). Thus, CD147 might directly and indirectly affect cytoskeletal mechanisms and localized signaling. Using phosphopeptide enrichment in CD147-silenced cells, we found 76 CD147-dependent phosphosites. The majority of the detected CD147-dependent phosphorylation sites are not func- tionally characterized and phospho-specific Abs are not yet available; however, there are some exceptions: the phosphorylation of Lck at Y394 is essential for its kinase activity (82), and T641 and S660 of PKCb are necessary for its kinase activity and subcellular FIGURE 4. Dynamic of CD147-dependent phosphorylations during T localization respectively (83, 84). All three phosphosites were cell stimulation. Heat map showing SILAC ratio values (shCD147/ constantly reduced in CD147-silenced T cells, and thus we con- shControl) from phosphopeptides increased or decreased by 33% upon clude that CD147 promotes the activation of PKCb and Lck. The CD147 in two separate experiments in at least one of the four indicated T cell stimulation conditions. Gene names and phosphorylation sites are impact of CD147 on phosphorylation of Lck at Y394 and on Src at given. Red fields indicate phosphopeptides that were increased; blue fields Y416 was also reported recently by others (34, 85). We further confirm show those that were decreased; white fields indicate same phosphoryla- these data using Western blotting by finding the hypophosphorylated tion; dark gray fields indicate that no phosphopeptides were detected in shControl and shCD147 samples. Perseus software package 1.5.2.6 was used for pattern and intensity-dependent Euclidean clustering. Phospho- in gray. Asterisks within gray boxes indicate phosphopeptides derived from peptides reproducibly detected only at specific time points are highlighted peptide intensities .106 from a range of 105–109. 8 MICROENVIRONMENT AND SIGNALING DYNAMICS OF CD147 Downloaded from

FIGURE 5. Western blotting analysis of key proteins affected by CD147 silencing. CD147-silenced and shControl Jurkat T cells were mock-stimulated or treated with PMA (16.2 nM) plus ionomycin (1 mM) for 5 or 30 min. Lysates were then analyzed for the presence of the indicated (phospho) proteins by Western blotting. GAPDH was used as a loading control. For quantification, the specific signal for each (phospho) protein was normalized to the respective GAPDH signal and then compared with the value obtained from shControl cells mock-stimulated for 5 min, which was set to one. One representative experiment (A) and the quantification of three independent experiments (B) are shown. Data represent mean 6 SD. Significance was determined by two- http://www.jimmunol.org/ way ANOVA with Bonferroni posttest. *p , 0.05, **p , 0.01, ***p , 0.001.

Lck Y394 residue in CD147-silenced cells. Moreover, we also CD147-dependent phosphorylations of cell cycle regulating sites found that the phosphorylation of Y505 and the total Lck levels T14 in CDK1 and T373, S788, S811, T821, T823, T826, and S855 were decreased in CD147-silenced cells. These new data about in RB1. Thus, these phosphosites might be important in another Lck and PKC support our previous report (64), and that from major function of CD147: the regulation of cell growth. Guo et al. (34), showing that early TCR signaling components GOBP term analysis by the STRING database revealed a strong are not upregulated in CD147-silenced Jurkat T cells but are enrichment of wound healing–, immune system–, stress response–, by guest on September 23, 2021 rather reduced. We speculate that the stimulation-induced in- phosphorylation- and protein modification–, defense response–, par- teraction of CD147 with both Lck and Lck-regulating phos- ticularly virus defense response–, and TNF production–associated phatase CD45 might have localized functional consequences in terms in the combined dataset of identified CD147 interaction part- the phosphorylation status and thus activity of Lck. ners and CD147-dependent phosphoproteins. CD147 is known to Another functionally studied phosphosite is T679 on ARHGEF2, play a role in all these biological processes and the reported analysis an activating phosphorylation (86) leading to binding of Rho and for known and predicted protein-protein interactions describes pos- inducing cytoskeletal rearrangements (87). ARHGEF2 is associ- sible CD147-dependent signaling routes in these processes. A role for ated with microtubules and is released and activated after their CD147 in the wound-healing process has been already described, but depolymerization, inducing stress fiber formation via the RhoA/ the interaction of CD147 with matrix metalloproteinases has been ROCK/MLC signaling module (88). In contrast to the previously made primarily responsible for this function (91, 92). Based on the discussed phosphosites, the phosphorylation of ARHGEF2 at STRING database analysis and the lack of matrix metalloproteinases T679 depends on PMA plus ionomycin stimulation, just as for the in the AP-MS approach, we hypothesize that CD147 is involved in functionally unknown phosphorylation sites S291 in CD43 or the wound-healing process through additional interaction partners. S2358 in SPTBN1. These three phosphorylation events show The most important signaling hubs in the CD147 signal network similar dynamics; therefore, we speculate that they act in concert appear to be the G proteins RAP1B and GNB1, the kinases PKCb, to transduce CD147-dependent signals to the cytoskeleton, PAK2, Lck, and CDK1, and the chaperone HSPA5. Notably, among thereby modulating the localization of signaling complexes during those, Lck might be the central hub because it was detected as a CD147 T cell synapse formation. Another CD147-dependent and PMA interaction partner, on CD147 silencing showed not only reduced plus ionomycin stimulation–dependent phosphorylation was found phosphorylation on both its activating site Y394 and inhibitory site in the site S1938 in MYH10. This site, in concert with neighboring Y505 but also reduced protein expression, was strongly molecularly sites, was recently described to regulate polarization of migrating interlinked by the STRING database, and was associated with nearly cells (89) and might be also important for CD147-dependent cy- all of the GOBP terms enriched in the CD147 microenvironment and toskeletal reorganizations in response to T cell stimulation. signaling network. Thus, Lck might be of great importance in CD147 In addition to phosphorylations regulating cytoskeletal mecha- signaling and a good candidate for further detailed analysis. nisms, we also found CD147-dependent phosphorylations of cell In summary, we connected known interaction partners of CD147 growth regulating molecules. We observed CD147-dependent and several novel ones that dynamically interact upon T cell stim- phosphorylation of RPS6 at serines 235 and 236, which are sites ulation as well as proteins whose phosphorylation depend on CD147 regulating cap-dependent translation (90). Moreover, we found into a molecular network. We also found signaling groups by GOBP dynamic CD147-dependent phosphorylation of RPS6 at the func- term analysis and correlated trends in association and phosphory- tionally uncharacterized site T241. Further there were increased lation dynamics to generate evidence for their functional association. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. The molecular network of CD147. (A) The STRING database was used to identify functional associations between the proteins found in the microenvironment of CD147 by AP-MS and the phosphoproteins found by proteomic phosphopeptide analysis to respond to CD147 silencing. The CD147 microenvironment is separated from CD147-dependent phosphoproteins by a dashed line; Lck, which is both interaction partner and CD147-dependent phosphoprotein, is highlighted by a red circle. The color code of the connecting lines indicating functional partners is as follows: experimentally proven interactions (magenta lines), association predictions based on gene neighborhood (dark green lines), gene co-occurrence (dark blue lines), coexpression experiments (black lines), on databases (blue lines), on text mining (green lines), and on homology research (gray lines). The small, colored circles indicate the GOBP term groups shown in (B). Orange: wound healing-associated terms; green: cytoskeleton-associated terms; violet: immune system-associated terms; gray: stress response-associated terms; red: phosphorylation- and protein modification-associated terms; blue: defense response-associated terms; yellow: TNF production-associated terms; white: other terms. (B) Significantly enriched GOBP terms within the CD147 microenvironment and CD147- dependent phosphoproteins shown in (A). A significance threshold for the false discovery rate was set at ,0.01. 10 MICROENVIRONMENT AND SIGNALING DYNAMICS OF CD147

Thereby we provide a great deal of interesting CD147-dependent 16. Baba, M., M. Inoue, K. Itoh, and Y. Nishizawa. 2008. Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism. proteins and phosphorylation sites, most of those to date unde- Biochem. Biophys. Res. Commun. 374: 111–116. scribed, as starting point for further detailed analysis. 17. Tang, J., Y. S. Guo, Y. Zhang, X. L. Yu, L. Li, W. Huang, Y. Li, B. Chen, J. L. Jiang, and Z. N. Chen. 2012. CD147 induces UPR to inhibit apoptosis and chemosensitivity by increasing the transcription of Bip in hepatocellular carci- Acknowledgments noma. Cell Death Differ. 19: 1779–1790. We thank Herbert Schiller, Rochelle D’Souza, and Kirti Sharma (Department 18. Jin, A., H. Chen, C. Wang, L. L. Tsang, X. Jiang, Z. Cai, H. C. Chan, and X. Zhou. of Proteomics and , Max Planck Institute of Biochem- 2014. Elevated expression of CD147 in patients with endometriosis and its role in regulating apoptosis and migration of human endometrial cells. Fertil. Steril. 101: istry, Martinsried, Germany) for valuable technical advice and discussion; 1681–1687.e1. Reinhard Fa¨ssler (Department of Molecular Medicine, Max Planck Institute 19. Chen, H., K. L. Fok, X. Jiang, J. Jiang, Z. Chen, Y. Gui, H. C. Chan, and Z. Cai. of Biochemistry, Martinsried, Germany) for experimental and intellectual 2012. CD147 regulates apoptosis in mouse spermatocytes but not spermatogo- support with mass spectrometric analysis; Vaclav Horejsi (Institute of Mo- nia. Hum. Reprod. 27: 1568–1576. 20. Zhou, X., J. Y. Lv, and B. L. Gong. 2010. Down-regulation of CD147 expression lecular Genetics, Academy of Sciences of the Czech Republic, Prague, induces SiHa cell apoptosis through the Bcl-2 pathway. Nan Fang Yi Ke Da Xue Czech Republic) for providing the CD147 mAb MEM-M6/1; Arthur Weiss Xue Bao 30: 1695–1698. (University of California, San Francisco, CA) for providing the TCR mAb 21.Kuang,Y.H.,X.Chen,J.Su,L.S.Wu,L.Q.Liao,D.Li,Z.S.Chen,and C305; Sheila Stewart (Washington University School of Medicine, St. Louis, T. Kanekura. 2009. RNA interference targeting the CD147 induces apoptosis of multi- drug resistant cancer cells related to XIAP depletion. Cancer Lett. 276: 189–195. MO) for providing the lentiviral shRNA expression vector pLKO.1-puro; 22. Yang, J. M., P. O’Neill, W. Jin, R. Foty, D. J. Medina, Z. Xu, M. Lomas, Giulio Superti-Furga (Center for Molecular Medicine, Austrian Academy G. M. Arndt, Y. Tang, M. Nakada, et al. 2006. Extracellular matrix metal- of Sciences, Vienna, Austria) for providing the pfMSCV Strep3xHA plas- loproteinase inducer (CD147) confers resistance of breast cancer cells to Anoikis mid; and Gary Nolan (Stanford University School of Medicine, Stanford, through inhibition of Bim. J. Biol. Chem. 281: 9719–9727. 23. Kim, K., H. Kim, K. Jeong, M. H. Jung, B. S. Hahn, K. S. Yoon, B. K. Jin, CA) for providing the retroviral expression vector pBMN-IRES-GFP. G. H. Jahng, I. Kang, J. Ha, and W. Choe. 2012. Release of overexpressed CypB Downloaded from activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress. Apoptosis 17: 784–796. Disclosures 24. Kuang, Y. H., X. Chen, J. Su, L. S. Wu, J. Li, J. Chang, Y. Qiu, Z. S. Chen, and The authors have no financial conflicts of interest. T. Kanekura. 2008. Proteome analysis of multidrug resistance of human oral squamous carcinoma cells using CD147 silencing. J. Proteome Res. 7: 4784–4791. 25. Boulos, S., B. P. Meloni, P. G. Arthur, B. Majda, C. Bojarski, and N. W. Knuckey. 2007. Evidence that intracellular cyclophilin A and cyclophilin

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