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DOI 10.1002/pmic.201400194

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RESEARCH ARTICLE

Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics Nerea Osinalde, Virginia Sanchez-Quiles, Vyacheslav Akimov, Barbara Guerra, Blagoy Blagoev and Irina Kratchmarova Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark

Common ␥-chain family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, where IL stands for interleukin) are key regulators of the immune homeostasis that exhibit pleiotropic biological activities and even sometimes redundant roles as a result of the utilization of the same receptor subunit. However, they also exert distinct functions that make each of them to be indispensable. For instance, all family members can act as T-cell growth factors; however, we found that IL-15 but not IL-7 can replace IL-2 to promote and sustain the proliferation of Kit225T cells. In addition to the ␥-chain, IL-2 and IL-15 share the ␤-chain, which creates the paradox of how they can trigger diverse phenotypes despite signaling through the same receptors. To investigate this paradigm, we combined SILAC with enrichment of tyrosine-phosphorylated proteins and peptides followed by mass spectrometric analysis to quantitatively assess the signaling networks triggered downstream IL-2/IL-2R and IL-15/IL-15R. This study confirmed that the transduction pathways initiated by both cytokines are highly similar and revealed that the main signaling branches, JAK/STAT, RAS/MAPK and PI3K/AKT, were nearly equivalently activated in response to both ILs. Despite that, our study revealed that receptor internalization rates differ in IL-2- and IL-15-treated cells indicating a discrete modulation of cytokine signaling. All MS data have been deposited in the ProteomeXchange with identifier PXD001129 (http://proteomecentral.proteomexchange.org/dataset/PXD001129).

Received: May 6, 2014 Revised: July 28, 2014 Accepted: August 14, 2014

Keywords: Cell biology / IL-2 / IL-15 / Quantitative phosphoproteomics / Signaling / SILAC



Additional supporting information may be found in the online version of this article at the publisher’s web-site

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Correspondence: Dr. Irina Kratchmarova, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark E-mail: [email protected] Fax: +45-6593-3018 Abbreviations: APC, allophycocyanin; CCV, clathrin-coated vesicle; ␥c, common ␥-chain; EGF, epidermal growth factor; GAB, GRB-associated binders; IL, interleukin; IL-2R, IL-2 receptor; JAK, Janus kinase; NK, natural killer; PI3K, phosphatidylinositol 3kinase; PTPN11, tyrosine-protein phosphatase nonreceptor type 11; pY, phosphotyrosine; Q-MS, quantitative MS; STAT, signal transducer and activators of transcription

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Introduction

Interleukin-2 (IL-2) is the best characterized cytokine of a family comprising at least five additional members (IL-4, IL-7, IL-9, IL-15, and IL-21) that initiate signaling cascades through a common ␥-chain (␥c ). As a consequence of sharing a receptor subunit, there is a considerable overlap between the actions of the distinct family members; however, none of them is fully replaceable [1]. The functional overlapping is even more dramatic between IL-2 and IL-15 since they also share the ␤ subunit of the receptor. Both cytokines can stimulate proliferation and cytotoxicity of T, B, and natural killer Colour Online: See the article online to view Fig. 2 in colour.

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(NK) cells and it has recently been demonstrated that they modulate essentially overlapping sets of genes in CD4+ and CD8+ T cells [2,3]. Nonetheless, each cytokine evokes distinct and even sometimes opposing actions in the modulation of several immune responses depending on the specific cell type. For example, both cytokines stimulate diverse lymphocyte and NK cell subsets, where IL-2 favors the homeostasis of regulatory T cells and the regulation of the differentiation of helper T cells, whereas IL-15 favors the population expansion of CD8+ memory cells, NK cells, and NK T cells [4, 5]. Moreover, IL-2 plays a unique role in activation-induced cell death, whereas IL-15 has an antiapoptotic effect in a number of systems [6, 7]. The distinct phenotype of mice deficient in IL-2 and IL-15 corroborates previous ex vivo data indicating that despite sharing receptor subunits, IL-2 and IL15 are not completely replaceable or functionally redundant [8]. Within the last decade, several attempts have been made to unveil the paradox of how IL-2 and IL-15 signal through IL-2R␤ (where IL-2R stands for IL-2 receptor) and IL-2R␥ resulting in divergent outcomes. It has been proposed that differences in the expression of their distinct ␣-subunits, which intriguingly lack signaling activity, or the cis versus trans presentation of IL-2 and IL-15 by the ␣-chain could account for the differences observed between these cytokines [9]. Another conjecture relies on the possibility that signaling complexes have alternative topologies that affect the types of signals generated. However, recent comparison of the IL2/IL-2R and IL-15/IL-15R quaternary structures has emphasized their similarities without entirely decoding the still remaining paradigm [3]. The complete understanding of the actions of IL-2 and IL-15 turns even more crucial considering the relevance of both systems in the development of a wide variety of diseases [10, 11]. Consequently, components of the IL-2/IL-2R and IL-15/IL-15R complexes have become potential targets for immunotherapy. The use of IL-2 for the treatment of distinct types of cancer was first approved in the 1990s, whereas IL-15 still remains a preclinical cytokine that has not been administered to human yet. However, IL-15 holds a great promise for exploiting the immune system to treat cancer and other diseases due to its unique role in inhibiting IL-2mediated activation-induced cell death and stimulating the development of CD8+ T cells [12, 13]. Hence, it is evident that a better understanding of the effects of IL-2 and IL-15 in modulating the immune system is essential to expand the horizons in the development of more efficient and less harmful therapies. In this regard, a more exhaustive knowledge of the molecular details of signaling by the two cytokines is imperative. Early signaling networks initiated by cytokines are finetuned modulated by transient tyrosine phosphorylation events [14]. The substoichiometric nature of phosphorylated proteins together with the minor prevalence of phosphorylation occurring at tyrosine residues are the fundamental challenges to overcome when studying cell signaling. Con C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

sequently, various methodologies have been developed to efficiently enrich phosphorylated peptides or proteins from biological samples [15]. Quantitative MS (Q-MS) based strategies have also greatly advanced becoming the fundamental pillar of most large-scale proteomic studies at present [16, 17]. Indeed, enrichment of phosphopeptides/proteins followed by Q-MS analysis has arisen as one of the most successful strategies to dissect global signaling cascades as evident from studies decoding epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) cascades [18–21]. Following a SILAC-based Q-MS strategy, we have previously dissected the IL-2 signaling pathways in Kit225 T cells, which mimic IL-2-mediated signaling events in activated human peripheral blood lymphocytes [22]. In the present work, aiming to compare in detail the complex signaling networks triggered by IL-2 and IL-15, we followed a triple SILAC-based workflow. Our data support previous observations that the signaling response to IL-2 and IL-15 in T cells is highly similar [23, 24]. A broad and varied representation of JAK/STAT (where JAK and STAT stand for Janus kinase and signal transducer and activators of transcription, respectively), RAS/MAPK, and PI3K/AKT (where PI3K stands for phosphatidylinositol 3-kinase) pathway effectors was found similarly enriched in the phosphotyrosine (pY) immunocomplexes isolated upon 5-min stimulation with either of the ILs. Additional enrichment of phosphopeptides using TiO2 beads led to the identification of specifically regulated pY sites, allowing further understanding of the fine-tune modulation of signals triggered by IL-2 and IL-15. Intriguingly, we detected that several proteins involved in receptor endocytosis were more enriched in the pY immunocomplexes following IL-2 treatment. Further analysis confirmed that receptor internalization was accelerated in cells stimulated with IL-2 with respect to the IL-15-treated ones. In summary, despite being highly similar, IL-2- and IL-15-triggered signaling networks are not identical and combination of discrete differences in their transduction cascades may explain the functional dichotomy existing between these cytokines.

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Materials and methods

2.1 Reagents and antibodies IL-2 was kindly provided by AIDS Research and Reference Reagent Program, Division of AIDS, NIAD, NIH, USA. IL-15 and IL-7 were purchased from PeproTech. The following antibodies were used in this study: sepharoseconjugated pY100 (#9419), phospho-STAT5 (#9351), STAT5 (#9352), phospho-p44/42 MAPK (#9101), p44/p42 MAPK (#9102), pAKT (#9271), AKT (#9272), phospho-JAK3 (#5031), and JAK3 (#3775) were obtained from Cell Signaling. SHC (610082) and JAK1 (J24320) were obtained from BD Transduction, EPS15 (sc-1840) and tyrosine-protein www.proteomics-journal.com

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phosphatase nonreceptor type 11 (PTPN11) (sc-7384) from Santa Cruz Biotechnology, and agarose-conjugated 4G10 anti-pY (#16–101) from Millipore. Immunocomplexes were visualized with ECLT-HRP conjugated anti-mouse (NA931, GE Healthcare) or anti-rabbit (NA934, GE Healthcare) secondary antibodies, followed by ECL Western blotting detection (PRN2106, GE Healthcare) and scanning on a Kodak Image Station 1000.

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antibodies. Eluted immune complexes were equally split and run in two parallel lanes of a precast gradient NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and visualized with Colloidal Blue (Invitrogen). Both gel lanes were separately cut into slices and subjected to in-gel reduction, alkylation, and trypsin digestion as previously described [25]. Peptides derived from one of the lanes were directly concentrated and desalted using C18 stage Tips to further analyze by LC-MS/MS, whereas peptides derived from the other line were subjected to TiO2 enrichment [26] prior MS analysis.

2.2 Cell culture and stimulation The human IL-2-dependent T lymphoma cell line Kit225 was maintained in RPMI 1640 medium supplemented with 10% FBS, 1% Glutamax, 1% penicillin/streptomycin, 1% sodium pyruvate, and 16 U/mL of recombinant human IL-2 at a density of 1 × 106 cells/mL. For SILAC experiments, the RPMI was custom-made and deficient for L-Arg and L-Lys (GibcoInvitrogen, Carlsbad, CA, USA). L-Arginine (Arg0), L-lysine (Lys0), L-arginine-13 C6 (Arg6), L-lysine-2 H4 (Lys4), L-arginine13 C6 15 N4 (Arg10), and L-Lysine-13 C6 15 N2 (Lys8) were from Sigma–Aldrich, and dialyzed serum from Gibco-Invitrogen. The IL-2-independent human T-cell lymphoma cell line Hut102 was purchased from ATCC. Hut102 cells were maintained in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin at a density of 2 × 105 cell/mL. Prior stimulation Kit225 cells were cytokine starved for 48 h, whereas Hut102 cells were serum starved for 16 h in order to obtain homogeneous cell populations synchronized at the G1 phase of the cell cycle that resemble true resting T cells. Signaling cascades were induced by incubating Kit225 and Hut102 cells with 200 U/mL and 5 × 103 U/mL of ILs, respectively, for 5 min at 37⬚C. The treatment was quenched by keeping cells on ice for 5 min and rapidly proceeding with cell lysis using ice-cold modified radioimmunoprecipitation assay buffer (RIPA) (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 % NP-40, 1 mM EDTA, 0.25 % sodium deoxycholate, 1 mM sodium pervanadate, 5 mM beta-glycerophosphate, 5 mM NaF, complete protease inhibitor cocktail (complete tablets, Roche)).

2.3 SILAC experiment: Immunoprecipitation, in-gel digestion, and phosphopeptide enrichment For SILAC experiments, Kit225 cells were grown in media containing Arg0/Lys0, Arg6/Lys4, or Arg10/Lys8 for 2 weeks prior treatment. After 48 h deprivation with IL-2, cells were kept unstimulated (Arg0/Lys0) and stimulated with IL2 (Arg6/Lys4) or IL-15 (Arg10/Lys8). Immunoprecipitation of tyrosine-phosphorylated proteins was performed as previously described [22]. Briefly, the three differentially labeled protein lysates were combined in 1:1:1 ratio according to their protein concentration, precleared with A-sepharose beads, and immunoprecipitated using two complementary anti-pY  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.4 MS and data analysis Acidified peptide mixtures were separated on a 75 ␮m inner diameter and 20-cm long column packed in-house with ReproSil-Pur C18-AQ 3 ␮m resin (Dr. Maisch GmbH). Reverse-phase chromatography was performed with an EASY-nLC 1000 ultra-high pressure system (Thermo Fisher Scientific), which was coupled to the Q Exactive mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Peptides were loaded in solvent A (0.5% acetic acid) and eluted with a nonlinear 140min gradient of 8–45% solvent B (0.5% acetic acid, 80% ACN) at a flow rate of 250 nL/min. MS instrument was operated to automatically switch between full-scan MS (m/z range, 300– 1750; maximum injection time 120 ms; resolution 70 000 at m/z 400; target of 106 ions) and up to ten data-dependent MS/MS scans (maximum injection time 124 ms; resolution 35 000 at m/z 400; target of 104 ions). Precursors were fragmented by higher energy C-trap dissociation with normalized collision energy of 25 eV. Repeat sequencing of peptides was minimized by excluding the selected peptide candidates for 45 s. Acquired raw mass spectra data from both biological replicates were combined and processed using the MaxQuant software v1.3.0.5 applying the following parameters: SILAC triplets with medium label Arg6/Lys4 and heavy label Arg10/Lys8; maximum of three labeled AA; maximum deviation allowed for precursor ion was 7 ppm and 0.5 Da for MS/MS events. A maximum of two missed cleavages were allowed and enzyme specificity was set to trypsin, allowing for cleavage N-terminal to protein and between aspartic acid and proline. Carbamidomethyl (C) was set as fixed modification and variable modifications, which included oxidation (M), acetyl (protein N-term), deamidation (NQ), and phosphoSTY (STY). Minimal peptide length was seven amino acids. For protein identification, at least two peptides, including one unique, were required. Both razor and unique peptides, except phospho-STY-modified peptides, were considered for protein group quantification. A minimum of two ratio counts were required for confident protein quantification. For the analysis of pY-containing peptides, 1% false discovery rate (FDR), a minimum localization probability of 0.75, score difference of 5, and MaxQuant score of 50 were demanded [27, 28]. www.proteomics-journal.com

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The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD001129.

2.5 Proliferation assay For proliferation experiments, IL-2-deprived Kit225 cells were reintroduced in the cell cycle by the addition of IL-2, IL-7, or IL-15 every 48 h. The number of cells was estimated every 24 h using NucleoCounter (ChemoMetec).

2.6 Receptor endocytosis Following stimulation with IL-2 or IL-15, Kit225 cells were stained with allophycocyanin (APC) anti-human IL-2R␤ antibody (339007 Biolegend) for 15 min at 4⬚C, washed twice with fluorescence-activated cell sorting (FACS) buffer (2 % FBS, 0.1 % sodium azide in PBS), and fixed with 1 % paraformaldehyde. Receptor subunits remaining at the cell surface were detected and analyzed using a FACSCalibur (Becton & Dickinson) flow cytometer. The percentage of IL-2R␤ in the cell surface was calculated by normalizing the values with respect to the initial value corresponding to IL-2R␤ present in nonstimulated Kit225 cells.

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Figure 1. IL-2-, IL-7-, and IL-15-induced response in Kit225 T cells. (A) Cell numbers over time for Kit225 cells cultured in continuous presence of indicated concentration of ILs or culture media alone. (B) Western blot detection of global pY pattern and phosphorylated STAT5, ERK1/2, and AKT in starved cells and cells stimulated for 5 min with various concentrations of ILs. (C) Immunodetection of phosphorylated STAT5, ERK1/2, and AKT in Kit225 cells stimulated with 2 ng of IL-2 and IL-15 per 1 × 106 cells for indicated times. Detection of total STAT5, ERK1/2, and AKT served as loading controls.

Results and discussion

3.1 IL-15, but not IL-7, can mimic the proliferative effect of IL-2 in Kit225 T cells The ␥c family members IL-2, IL-7, and IL-15 share the ability to induce proliferation of T lymphocytes; therefore, we aimed to determine whether IL-7 and/or IL-15 could replace IL-2 and promote proliferation of Kit225 T cells [29]. For that purpose, Kit225 cells were synchronized at G1 phase of the cell cycle by depriving with IL-2 for 48 h followed by reintroduction of each cytokine alone at the indicated concentration every 48 h. As expected, nonproliferating starved cells reentered the cell cycle and started growing in the presence of IL-2 (Fig. 1A). Although the Kit225 cells express the IL-7R␣ (data not shown), IL-7 did not succeed in rescuing Kit225 cells from cytokine starvation, even when using five times higher concentration than IL-2. In contrast, as it was previously observed by Zambricki and colleagues [24], we detected that IL-15 efficiently induced proliferation of Kit225 cells to a similar level as IL-2, demonstrating that IL-15 could replace IL-2 and ensure growth of the CD4+ CD3+ CD8– T cell line Kit225. Yamada and co-workers had also reported that IL-15 could supplant IL-2 signal in four different IL-2-dependent adult T-cell leukemia cell lines, which unlike Kit225 are positive for human T lymphotropic virus-I [30].  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

In agreement with the ability of IL-15 to promote proliferation of Kit225 cells, 5-min incubation with IL-15 induced global tyrosine phosphorylation in Kit225 cells similar to IL-2, whereas stimulation with IL-7 had no effect on the total pY pattern of these cells (Fig. 1B). Further detection by Western blotting of phosphorylated STAT5, ERK1/2, and AKT, the core representative effectors of JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, respectively, confirmed that IL15 could mimic IL-2-induced phosphorylation whereas IL7 was not capable of inducing phosphorylation-dependent changes (Fig. 1B). Similarly, Johnston and co-workers had reported that IL-2 and IL-15, but not IL-4, can induce tyrosine phosphorylation of STAT5 in peripheral T lymphocytes [31]. All together, we can note that the sole signaling through the ␥c of the receptor is not sufficient to induce activation of the entire signaling networks downstream the receptors. To further characterize the response of Kit225 cells to IL-2 and IL-15 stimulation, we studied the activation dynamics of JAK/STAT, MAPK/ERK, and PI3K/AKT signaling pathways upon IL-2 and IL-15 treatment. Starved T cells were incubated with each cytokine for the indicated times, and the levels of phosphorylated STAT5, ERK1/2, and AKT were detected by immunoblotting. As demonstrated in Fig. 1A and www.proteomics-journal.com

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Figure 2. (A) Schematic diagram of the experimental workflow utilized to decipher IL-2 and IL-15 signaling cascades. (B) Venn diagram showing the overlap of confidently quantified proteins between the two SILAC experiments and overall fold change for the tyrosinephosphorylated proteome of IL2- and IL-15-treated cells as a function of protein intensity in the MS. (C) Correlation between combined normalized ratios from proteins significantly enriched (ratio > 1.5; p < 0.01) in pY immunocomplexes upon IL-2 and IL-15 stimulation.

1B, the time-course activation profiles following treatment with IL-2 and IL-15 are highly similar. In addition, as shown in Fig. 1C, phosphorylation of the three main signaling effectors was already pronounced after 5-min stimulation with both cytokines and increased gradually with longer incubation times reaching the maximum after 1 h. The signal transduction pathways initiated by the IL-2and IL-15 are dependent on series of phosphorylation events, and the immediate phosphorylation of tyrosine residues following acute cytokine stimulation is the main trigger of the downstream signaling processes. Here, we aimed to decipher and compare the early phosphotyrosine-dependent signaling events that occur upon cytokine stimulation; therefore, we focused on the characterization of the molecular snapshot of changes at 5 min of IL-2 and IL-15 treatment.

3.2 Simultaneous dissection of IL-2 and IL-15 signaling pathways by SILAC-based quantitative phosphoproteomics To identify and compare on a global scale the complex network of tyrosine-phosphorylated proteins involved in IL-2and IL-15-mediated signaling cascades, we followed a triple SILAC-based quantitative phosphoproteomic approach that combined enrichment of tyrosine-phosphorylated proteins and peptides with high-resolution MS (Fig. 2A). Two biological replicas of the experiment were performed confidently quantifying a total of 1255 proteins in both experiments (Supporting Information Table 1) that showed a Pearson correlation coefficient of 0.7 and 0.65 for IL-2- and IL-15-induced  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

changes, respectively. As expected, most of the proteins did not change their abundance in the tyrosine-phosphorylated complexes in response to the cytokines following pY IP (Fig. 2B). As an indicative of efficient activation of the system, the obtained ratios did not follow a normal distribution, since there is a higher number of values along the right side of the scatter plot corresponding to proteins that were enriched in the pY immunocomplexes following cytokine stimulation. A total of 41 proteins were found to be enriched by at least 1.5-fold and with a statistically significance value p < 0.01 in both replicates in response to the cytokines, comprising the group of identified IL-2 and IL-15 signaling effectors (Table 1). A wide variety of proteins such as receptor subunits, kinases, phosphatases, and adaptor proteins that we have already reported to participate in the propagation of IL-2 signaling in Kit225 cells [22] were also enriched in the pY immunocomplexes of the present work not only following IL-2 stimulation but also in response to IL-15 treatment. Moreover, a comparison of the combined ratios of the 41 effectors obtained for IL-2 and IL-15 treatments with respect to untreated cells showed very strong correlation (Pearson coefficient of 0.9; Fig. 2C), which is in agreement with previously published data pinpointing that signaling networks activated by IL-2 and IL-15 are greatly similar in T cells [3, 23, 24]. In addition, we performed further pY IPs followed by Western blotting detection of a number of proteins that were enriched in the pY immunocomplexes upon IL-2 and IL-15 stimulation. We had previously reported that the adaptor proteins SHC1 and EPS15, JAK1 kinase, and PTPN11 phosphatase are bona fide effectors of the IL-2 signaling cascades [22]. Besides corroborating that, we confirm that all of

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Proteomics 2015, 15, 520–531 Table 1. Effector proteins enriched in pY immunocomplexes of Kit225 cells treated with IL-2 and IL-15 Gene name

Uniprot

Peptides (unique)

Protein group IL-2/Ctr

Signal transducer IL-2RG FAM59A JAK3

P31785 Q9H706 P52333

SHC1 GAB3

P29353-6 Q8WWW8-2

pY site IL-15/Ctr

Position

Ratio

Sign. B

Ratio

Sign. B

11 (11) 16 (16) 49 (49)

23.6 20.9 20.8

5.70E-142 4.05E-113 5.68E-131

10.4 19.9 19.7

1.40E-121 3.27E-157 1.32E-195

24 (6) 18 (18)

17.6 14.7

2.5E-117 5.132E-89

17.0 13.8

3.14E-177 3.08E-121

IL-2RB JAK1 CDK7 GAB2

P14784 P23458 P50613 Q9UQC2

17 (17) 56 (54) 2 (2) 15 (15)

14.4 13.6 9.1 8.7

1.40E-101 2.67E-97 9.67E-61 2.40E-58

13.6 13.5 7.8 8.4

3.58E-150 1.16E-149 3.63E-75 3.78E-80

PIK3C2B MAPK3 PTPN11

O00750 P27361 Q06124-2

12 (12) 15 (12) 29 (1)

8.3 6.1 5.5

2.76E-35 6.85E-48 7.06E-43

6.2 8.6 5.3

7.73E-24 4.36E-103 6.49E-63

PIK3CA STAT5B SHIP1 MAPK1 SOS2 SHIP2 SOS1 PIK3R2 PIK3R3 PIK3CB STAT3 PIK3R1 PIK3CD GRB2

P42336 P51692 Q92835 P28482 Q07890 O15357 Q07889 O00459 F6TDL0 P42338 P40763 P27986-4 Q5SR50 P62993

4 (4) 17 (5) 13 (11) 17 (14) 18 (13) 25 (23) 22 (17) 22 (21) 10 (7) 12 (12) 21 (21) 27 (23) 11 (11) 15 (15)

5.4 5.0 5.0 4.9 4.8 4.1 3.9 3.5 3.2 3.1 2.7 2.7 2.4 2.3

2.81E-23 5.07E-33 4.59E-21 4.59E-21 2.35E-20 4.65E-26 1.89E-24 4.38E-21 6.93E-12 3.66E-17 4.05E-14 1.58E-15 6.88E-11 1.02E-11

6.2 4.3 3.1 7.5 5.4 3.6 4.3 3.5 4.1 2.7 2.7 2.8 2.1 2.2

8.16E-24 1.08E-38 6.56E-10 1.76E-90 1.51E-20 1.10E-30 2.63E-38 3.16E-29 1.35E-14 9.63E-19 6.87E-19 3.08E-25 1.64E-11 3.29E-15

Ubiquitination (endocytosis and protein trafficking) STAM2 O75886 14 (12)

7.2

2.01E-56

3.6

8.56E-30

HGS AGFG1 EPS15 CBL STAM EPS15L1 CLTA CLTC

O14964 E9PHX7 P42566 P22681 Q92783 Q9UBC2-2 P09496 Q00610

22 (22) 8 (8) 25 825) 15 (14) 15 (13) 48 (48) 4 (4) 84 (65)

5.0 4.8 4.7 4.7 4.1 2.7 2.6 2.6

6.86E-38 1.28E-31 4.77E-31 6.16E-31 7.03E-30 5.57E-16 1.32E-08 2.29E-14

2.8 4.6 4.0 4.0 2.6 3.2 3.4 3.1

1.04E-24 1.59E-42 1.65E-35 5.82E-35 4.05E-21 2.50E-31 1.69E-11 4.51E-29

Cytoskeleton ARHGAP32 LIMD1 LPP

A7KAX9 Q9UGP4 Q93052

23 (22) 8 (8) 8 88)

9.5 8.0 4.5

9.82E-63 4.22E-54 1.61E-18

6.9 8.2 4.8

3.14E-66 7.14E-79 4.26E-18

Others TJP2 SRI TBC1D5

F5H301 P30626 Q92609-2

20 (20) 5 (5) 2 (2)

2.7 2.0 2.0

3.39E-09 2.06E-08 0.00006

3.1 2.3 1.6

5.43E-10 3.83E-16 0.0094

them are also enriched upon IL-15 treatment and therefore participate in IL-15 signaling pathways (Fig. 3A). Interestingly, we observed very similar response in Hut102T-cells, a human T lymphotropic virus-I (+) T cell line that unlike Kit225 does not depend exclusively on IL-2 for growing (Fig. 3A and Supporting Information Fig. 1). These data further indicate that IL-2 and IL-15 similarities are not restricted

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IL-2/Ctr

IL-15/Ctr

Ratio

Ratio

980 981 980; 981 428 507 534 561 536

84.5 39.9 6.1 38.7

70.9 33.0 6.0 31.1

27.5 31.2

34.6 24.3

293 324 228 204 546 584

15.5 1.6 5.9 9.8 22.0 22.0

17.3 2.1 4.2 21.0 12.9 20.9

699

52.2

50.4

187

13.7

31.2

192 374

5.2 18.1

3.5 6.2

1557

11.7

5.8

300

14.7

12.5

209

to a single T cell line with respect to their downstream signaling events. In addition to our generic phosphoproteomics strategy, we also enriched phosphopeptides from the pY immunocomplexes using TiO2 beads, which resulted in the identification of 41 unique tyrosine phosphorylation sites (Supporting Information Table 2). It is worth noting that two of those

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Figure 3. Validation of selected IL-2 and IL-15 effectors (A) and specific regulated phosphosites (B) by immunoblotting.

phosphorylation sites are novel and have not been previously reported according to the PhosphoSitePlus database [32]. Manual annotation of all identified phosphopeptides containing pY is shown in Supporting Information Fig. 2. Within the identified 41 pY sites, 20 correspond to 14 different proteins that were enriched in our SILAC approach following IL-2 and IL-15 stimulation (Table 1). Using tyrosine phospho-specific antibodies, we managed to validate IL-2- and IL-15-induced phosphorylation of STAT5 Y694, ERK1/2 T202/Y204, and JAK3 Y980/Y981 not only in Kit225, but also in the Hut102 T cells (Fig. 3B). As it was previously observed by Zhang and co-workers, we also detected that Hut102 cells show constitutive phosphorylation of JAK3, which was dramatically increased following both IL-2 and IL-15 stimulation [33].

3.3 Deciphering IL-2- and IL-15-induced signaling pathways The 41 proteins that were enriched in the immunoprecipitated pY complexes (Table 1) represent a wide range of known effectors that orchestrate signal transduction right after IL-2 and IL-15 engage with their respective receptors (Fig. 4). All of them have previously been shown to participate in IL-2-triggered signaling networks in Kit225 T cells [22], whereas several have never been connected to IL-15 signaling before. As mentioned in Section 1, IL-2 and IL-15 are the only members of the IL-2 cytokine family sharing the ␤ and ␥ subunits of the receptor. Upon ligand engagement, receptor subunits oligomerize activating JAK kinase family members and initiating a tangle of complex signaling networks  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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that culminate in gene transcription regulation [31, 34]. In agreement with that, the ubiquitously expressed JAK1 and the hematopoietic lineage-specific JAK3 were highly enriched in the pY immunocomplexes showing ratios of 13 and 20, respectively, in response to both cytokines. It is worth noting that as it has previously been reported for YT NK cells [35], we also observed a higher enrichment of JAK3 compared with JAK1, suggesting that JAK3 is the predominant tyrosinephosphorylated JAK kinase involved in IL-2 and IL-15 signaling. Three different peptides belonging to JAK3, containing Y980, Y981, or both residues phosphorylated, were also found to be highly enriched with ratios of 75, 35, and 6, respectively, in response to both cytokines. The two adjacent tyrosines are localized in the activation loop of JAK3 and regulate its activity [36]. The pY980, which showed the highest ratio in our SILAC experiment, positively regulates the catalytic activity of JAK3, whereas pY981 has the opposite effect. Overall, our data implicate that although both antagonist sites are phosphorylated in response to IL-2 and IL-15, JAK3 is catalytically activated following cytokine stimulation. However, these data also suggest that negative feedback loop regulation occurs already within 5 min of cytokine addition. Upon activation, JAK1 and JAK3, which are constitutively associated with IL-2R␤ and IL-2R␥, respectively, phosphorylate specific tyrosine residues in the receptor, which serve as docking sites for proteins containing Src-homology 2 or pY-binding domains. Consistently, both receptor subunits were found highly enriched displaying ratios above 10 upon 5 min of IL-2 and IL-15 stimulation. Most interestingly, we detected phosphorylation on Y536 of IL-2R␤, which is known to act as an anchoring site of the transcription factor STAT5 and mediate its activation [37, 38]. Since Y536 is localized at the C-terminal end of IL-2R␤ and the tryptic peptide baring this site does not contain Arg or Lys residues, it was not possible to obtain quantitative information using our approach. Nonetheless, combining iTRAQ peptide labeling with tyrosine phosphopeptide enrichment strategies, Arneja and co-workers have recently confirmed that IL-2 and IL-15 stimulation results in a similar induction of IL-2R␤ Y536 phosphorylation [23]. In addition, we found that STAT5B was equally enriched in Kit225 upon IL-2 and IL-15 treatment, implying that its docking site may be similarly phosphorylated after stimulation of Kit225 cells with IL-2 and IL-15. Once recruited to the receptor, the STAT proteins are phosphorylated by JAK kinases. Accordingly and in agreement with recently published data [23], we found that pY699 of STAT5B was dramatically induced following 5 min incubation with either of the ILs. In fact, this specific residue, which is also conserved in STAT5A, regulates the DNAbinding activity of STAT5 proteins [39]. Therefore, we can predict that upon JAK-mediated phosphorylation of STAT5, they are released from their docking sites on the receptors and dimerize to translocate to the nucleus where they specifically bind discrete promoter regions of target genes. We also observed an enrichment of 2.7-fold of STAT3, an additional STAT family member that is known to associate with www.proteomics-journal.com

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Figure 4. Reconstruction of IL-2 and IL-15 signaling pathways. The two sequential red bars close to each effector represent the log2 ratio of IL-2/Ctr and IL-15/Ctr, respectively.

IL-2R␤ under IL-2 stimulation condition [40]. Notably, we found that STAT5B, showing a SILAC ratio of around 5, responded stronger than STAT3 to either of the cytokines in agreement with previously published data showing that IL-2 activates STAT5 more efficiently than STAT3 in Kit225 cells [40]. Apart from the C-terminal anchoring site of IL-2R␤ mentioned above, the active form of the receptor contains additional phosphotyrosines but only the most membrane-proximal residue (Y388) appears to be involved in signal transduction [41]. pY388 serves as a docking site for the pY-binding domains present in proteins such as SHC1 [42], an adapter molecule that showed a SILAC ratio of 17 in IL-2- and IL-15-treated T cells. Upon recruitment to the receptor, SHC1 is similarly tyrosine phosphorylated on Y428 displaying ratio of 38.7 and 31.1 in response to IL-2 and IL-15, respectively, which is in line with recently published data by Arneja and co-workers [23]. This phosphorylation site is known to participate in the recruitment of the adaptor GRB2 through its Src-homology 2 domain [43]. Through its Src-homology 3 domains, GRB2 also interacts with the Ras guanine nucleotide exchange factor SOS inducing the Ras-Raf-MAPK pathway [44]. GRB2, SOS1, and SOS2 were consistently enriched in Kit225 pY immunocomplexes following IL-2 and IL-15 stimulation displaying ratios between 2 and 5. In addition, Y209 of GRB2 was also found to be phosphorylated in our study by both cytokines. Interestingly, it has been reported that EGF-induced phosphorylation of this specific residue serves as a negative feedback loop regulation by disrupting the association with SOS1/2 [28,45]. Consequently, our data further indicate that already following 5-min stimulation with IL-2 or IL-15 mechanisms involved in signal attenuation are activated. As mentioned above, upon ligand engagement, the receptor complex is coupled via SHC1/GRB2/SOS to the MAPK pathway. Accordingly, the two main kinases of this cascade,  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

MAPK1 and MAPK3, were highly enriched in response to IL-2 and IL-15. Moreover, we observed that cytokine treatment specifically induced phosphorylation in MAPK1 pY187 and MAPK3 pY204, which are required for the enzyme activation. It has recently been described that the MAPK pathway can also be activated by the FAM59A/PTPN11/GRB2 complex in response to EGF stimulation [43]. FAM59A was enriched 20 times following IL stimulation, confirming our previous finding that FAM59A is involved in IL-2 signaling networks [22], and more interestingly suggesting that this adaptor protein is a novel IL-15 effector. PTPN11 was also enriched in the pY immunocomplexes in response to both cytokines, suggesting that together with FAM59A it could be involved in the activation of the MAPK pathway in IL-2- and IL-15-treated Kit225 T cells. Notably, we found that PTPN11 pY546/pY584, the residues that mediate the association between GRB2 and PTPN11 [44], were strongly induced upon both treatments. PTPN11 can also activate the MAPK pathway via the formation of a complex with the scaffold protein GAB2 (where GAB stands for GRB-associated binders) and its constitutive partner GRB2 [46]. GAB2 is tyrosine phosphorylated in response to IL-2 and IL-15 and, accordingly, we found it enriched over eight times upon both cytokine treatments [47]. Y293 of GAB2 was strongly phosphorylated upon both stimuli, whereas pY324 was not induced suggesting that only the first site is implicated in the signal transduction initiated by either of the cytokines. The most recently described GAB family member GAB3 is mainly expressed in lymphoid tissue and also associates with PTPN11 presumably through its two tyrosine motifs at amino acid positions 533 and 560. This study provides the first evidence that Y533 and Y560 of GAB3 are highly phosphorylated in response to IL-2 and IL-15, suggesting that GAB3 could interact with PTPN11 and play a role in the signaling networks triggered by both cytokines.

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The relevance of the PI3K pathway in IL-2-induced activation of T cells is well appreciated [48]. From the four existing classes of PI3Ks (IA, IB , II, and III), two were found enriched in our large-scale phosphoproteomic data set. All catalytic subunits (PIK3CA, PIK3CB, and PIK3CD) as well as three of the five regulatory subunits (PIK3R1, PIK3R2, and PIK3R3) comprising the Class IA of PI3K family were similarly enriched in response to both cytokines displaying ratios between 2 and 6. Class II PI3K member PIK3C2B was even more enriched than Class I subunits in response to IL-2 and IL-15. Overall, our data suggest that the less characterized of the PI3K classes may play a preferential role in propagating the signal initiated by both ILs. Upon stimulation, the PI3K molecules are recruited to activated receptors in close proximity with their lipid targets located in the plasma membrane, which results in an increase of the PtdIns(3,4,5)P3 and activation of the pleiotropic kinase AKT. Western blot analysis of both Kit225 and Hut102 cell lysates showed that AKT is phosphorylated and consequently activated in response to IL-2 and IL-15 (Supporting Information Fig. 1). The signaling mediated by the PI3K is antagonized among others by the action of the lipid phosphatases SHIP1 and SHIP2. Both enzymes were enriched in the pY immunocomplexes following IL-2 and IL-15 treatments with ratios between 3 and 5. Once again, these results suggest that mechanisms involved in signal attenuation are readily induced following 5-min stimulation with IL-2 and IL-15. Directed reorganization of the actin cytoskeleton is also decisive for multiple aspects of T-cell function including signaling [49]. Accordingly, several proteins involved in cytoskeleton rearrangement such as LPP, ARGHAP32, and LIMD were enriched upon IL-2 and IL-15 treatment implying that mechanisms modifying T-cell shape are activated in response to the ILs. To our knowledge, this is the first study demonstrating that they may also have a role in the signal transduction initiated by IL-15.

3.4 Endocytosis The group of enriched proteins following IL-2 and IL-15 treatment is also highly represented by molecules involved in receptor internalization and signal attenuation (Table 1). Although IL-2R subunits are constitutively internalized, this process is enhanced in the presence of the ligand [50]. Based on our phosphoproteomics data, this observation could be extended to IL-15/IL-15R complex as well. Internalization of ␤ and ␥ receptor chains is believed to follow mainly clathrin-independent endocytic routes [51]. Nonetheless, our data strongly suggest that clathrin-dependent pathways are also relevant in IL-2- and IL-15-mediated receptor internalization since several components of clathrin-coated vesicles (CCV) and other factors involved in their assembly were found consistently enriched in the pY immunocomplexes  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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upon stimulation with both cytokines (Fig. 5A and Table 1) [52]. Corroborating our previously published data [22], we observed that clathrin heavy and light chains were increased in abundance following 5-min incubation with IL-2. For the first time, we report that IL-15 treatment results in a similar enrichment of clathrin heavy and light chains, suggesting that clathrin-dependent mechanisms may also play a prominent role in IL-15R internalization in Kit225 cells. Stimulation with either cytokines resulted in a discrete enrichment of TBC1D5, a putative GTPase-activating protein for Rab family proteins that has recently been described to associate with AP2 complex, another major component of the CCV [53]. Two additional constitutive units of the CCV and essential components of the endocytic pathway, EPS15 and EPS15L1 [54], were also enriched in response to IL-2 and IL-15. Both molecules contain three N-terminal Eps15 homology domains that mediate interaction with proteins such as AGFG1, an endocytic molecule that we originally described to be involved in IL-2triggered signaling cascades [22]. We demonstrate here that it also participates downstream the IL-15/IL-15R complex in Kit225 T cells [55]. EPS15 also associates with HGS, a protein that regulates intracellular trafficking to early and late endosomes by recruiting clathrin [56]. Although HGS was enriched in the pY immunocomplexes upon both treatments, for the first time we show that IL-2 stimulation results in approximately twofold enrichment with respect to IL-15 treatment. The HGS-binding proteins STAM and STAM2 were found two times more enriched in response to IL-2 as well. Nevertheless, in agreement with recently published data [23], we observed that IL-2 stimulation results in about threefold increase in phosphorylation of STAM2 Y374 relative to IL-15, a modification that regulates the affinity of STAM2 for ubiquitin. In addition, the E3-ubiquitin ligase protein CBL, a negative regulator of several signaling pathways that can promote receptor endocytosis, was more abundant in IL-2-induced pY immunocomplexes, which to our knowledge has never been reported before. Altogether, our data suggest that the endocytic machinery responsible for attenuating the signal initiated by the ligands is engaged to a higher degree after 5-min stimulation with IL-2 in comparison with IL-15. To test if receptor internalization rate differs between IL-2- and IL-15-treated cells, cytokine-starved Kit225 were kept unstimulated or were stimulated for 5 min with IL-2 or IL-15 and were allowed to internalize the receptors. The noninternalized receptors remaining in the surface were detected by incubating cells with an anti-IL-2R␤ antibody coupled to APC fluorophore and further analyzed using FACS analyses. Figure 5B shows that both IL-2 and IL-15 stimulation resulted in a decrease in APC-labeled Kit225 population, indicating that IL-2R␤ was internalized upon stimulation with both cytokines. However, as evident from the graph in Fig. 5B, IL-2R␤ internalization was significantly more dramatic following incubation with IL-2. Thus, for the first time here we demonstrate that the subtle differences among the www.proteomics-journal.com

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proteins involved in receptor internalization result in a different T-cell response as evident from the increased IL-2R␤ endocytosis rate of IL-2-treated cells.

4

Concluding remarks

Despite sharing the ␥c of the receptor for signal transduction, the IL-2 family of cytokines do not exert identical responses in immune cells. This is clearly evidenced in our study showing that whereas IL-15 mimics the proliferative effect of IL2 in the lymphocytic T cell line Kit225, their family partner IL-7 does not. The intriguing paradigm of how IL-2 and IL-15 can act differently despite sharing both the ␤ and the ␥ receptor subunits prompted us to compare the global signaling networks triggered by both cytokines aiming to identify potential molecular differences between both systems. Combining our large-scale phosphoproteomic data with already published reports, we managed to reconstruct the molecular snapshot at 5-min stimulation with IL-2 and IL-15 covering the principal signaling branches involved in T-cell proliferation. Although the signaling mechanisms triggered by IL-2 and IL-15 were found to be highly similar, we demonstrate that several endocytosis-related proteins display higher enrichment in the pY immunocomplexes of IL-2-treated cells. Accordingly, further analysis confirmed that receptor internalization was accelerated in cells stimulated with IL-2. These results demonstrate that despite their high similarity, the IL-2 and IL-15 signaling networks are not identical and suggest that the functional dichotomy between the two closely related cytokines may be in part explained by the different half-life of their surface-bound receptor.  C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 5. Activation of the endocytic machinery in response to IL-2 and IL15. (A) Schematic representation of clathrin-mediated endocytosis of IL-2R. (B) Cell surface IL-2R␤ was detected by incubating starved cells and cells stimulated with IL-2 and IL-15 with APC-IL2R␤ antibody using nonpermeabilized conditions and analyzing by FACS. The histogram represents the mean fluorescence intensities normalized to untreated control cells from each experiment. Results are mean values of three independent experiments (*t-test, p < 0.05).

The MS proteomics data in this paper have been deposited in the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository [57]: dataset identifier PXD000XXX PXD001129. We are grateful to Oriane Cedile for help with the FACS analysis. This work was supported by a grant from the Novo Nordisk Foundation, the Lundbeck Foundation, and the Augustinus Foundation. N.O. is supported by the Lundbeck Foundation. I.K. is supported by grants from the Danish Natural Science Research Council and the Danish Medical Research Council. The authors have declared no conflict of interest.

5

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