Characterization of Receptor-Associated Protein Complex Assembly

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Characterization of receptor-associated protein complex assembly in Interleukin (IL)-2- and IL-15-activated T-cell lines Nerea Osinalde, Virginia Sanchez-Quiles, Vyacheslav Akimov, Kerman Aloria, Jesus M Arizmendi, Blagoy Blagoev, and Irina Kratchmarova J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00233 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Journal of Proteome Research

IL-2- and IL-15-induced receptor complex assembly in T-cells 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Characterization of receptor-associated protein complex assembly in Interleukin (IL)-2- and IL-15-activated T-cell lines Nerea Osinalde1†, Virginia Sanchez-Quiles1‡, Vyacheslav Akimov1, Kerman Aloria2, Jesus M. Arizmendi3, Blagoy Blagoev1 and Irina Kratchmarova1 1

Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense

M, Denmark. 2Proteomics Core Facility-SGIKER, University of the Basque Country, UPV/EHU, 48940 Leioa, Spain. 3Department of Biochemistry and Molecular Biology, University of the Basque Country, UPV/EHU, 48940 Leioa, Spain.

Running title: IL-2- and IL-15-induced receptor complex assembly in T-cells

Keywords: Cell signaling, interleukin, T-lymphocyte, quantitative proteomics, SILAC, phosphoproteomics, affinity enrichment, protein complexes

Current address: †Department of Biochemistry and Molecular Biology, University of the Basque Country UPV/EHU, 01006 Vitoria-Gasteiz, Spain. ‡ Oncology Group, UMR 144 CNRS, Curie Institute. 26, rue d'Ulm. 75248 Paris, France.

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ABSTRACT

It remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptorassociated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and –independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor β subunit (IL-2Rβ). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step towards a better understanding of the early signaling aspects of the two closely-related cytokines in T-lymphocytes.


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INTRODUCTION Interleukin-15 (IL-15) is a cytokine that was initially described as an IL-2 mimicking factor in vitro (12)

. Nevertheless, subsequent studies have convincingly demonstrated that, besides having overlapping

roles, each cytokine possesses unequal features to modulate the immune response. Indeed, both interleukins stimulate the growth of several subsets of T-cells and promote the formation of cytotoxic T-lymphocytes. IL-2 and IL-15 also induce the generation and activation of natural killer (NK)-cells and enhance the synthesis of immunoglobulins by B-cells (3-5). However, IL-2 is unique in favouring the immune tolerance by means of eliminating autoreactive T-cells due to its dominant role in the maintenance of regulatory T-cells that suppress effector T-lymphocytes(6-7) Consequently, low-dose IL-2 therapy holds great promise for treating autoimmune and inflammatory diseases by supporting the development and homeostasis of Treg cells (8-9). IL-15 in turn inhibits antigen-induced cell death by promoting the survival of CD8+ memory T-cells (10). These functional differences have pivotal implications in the use of both cytokines to enhance T-cell responses in cancer immunotherapy, which aims to target and eradicate the tumour by harnessing the immune system (11-12). At present, IL-2 is the major cytokine administered to augment T-cell responses in anti-cancer therapies (13). However, due to the severe side effects associated with the IL-2 treatment, major efforts are directed to improve anticancer therapies and IL-15 has lately emerged as a promising candidate (14-15).

IL-2 and IL-15 are closely-related cytokines mainly due to the utilization of the same receptor subunits to initiate signal transduction. Besides the common gamma chain receptor subunit (IL-2Rγ or γc) that is also shared by IL-4, IL-7, IL-9 and IL-21, IL-2 and IL-15 use the same receptor beta subunit, referred to here as IL-2Rβ, to initiate a complex cascade of signaling networks that results in an adequate immune response (16). But the question remains: how can they produce divergent cellular outcomes by signaling through the same receptor β and γc chains? So far, numerous works have attempted to explain this functional dichotomy by proposing several possible mechanisms. The specific α-subunit of each cytokine receptor (IL-2Rα and IL-15Rα), which appears to modulate ligand affinity rather than participate in signal transduction per se, is likely to contribute to such difference. IL-2Rα and IL-15Rα subunits display distinct spatial and temporal expression and associate with their 3 ACS Paragon Plus Environment


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respective cytokines with distinct affinities (17-19). Moreover, both cytokines differ in their major mode of presentation. Whereas IL-15 is prone to be presented in trans by an IL-15Rα-expressing cell to a Tlymphocyte bearing the remaining two receptor subunits (20-21), IL-2 usually binds to heterotrimeric receptors located in the same surface of the same T-cell. Nevertheless, dendritic cells lacking IL-2Rβ but expressing IL-2Rα have been shown to use the later IL-2R subunit to trans-present IL-2 to IL-2RαT-lymphocytes which is crucial for subsequent T-cell expansion (22). In addition, cleaved and soluble IL-2 and IL-15 receptor alpha chains have distinct effects on IL-2 and IL-15 signaling pathways, respectively (23-25). The distinct kinetics reported in the signaling cascades initiated by IL-2 and IL-15 are also likely to contribute to the functional dichotomy between the two closely-related cytokines (2627)

. In spite of the numerous efforts towards the elucidation of the molecular mechanisms underlying

the divergent functions of IL-2 and IL-15, those remain elusive and require further investigation.

It is well established that IL-2/IL-2R as well as IL-15/IL-15R engagement results in the activation of JAK tyrosine kinases which are responsible for phosphorylating different substrates. The resulting modified residues serve as anchoring sites for effector proteins that are assembled into fine tune modulated complexes that transmit signals along multiple pathways until the corresponding cellular response is promoted (28). The signaling proteins that bind to the interleukin-activated receptor have been painstakingly described in the past following traditional biochemical approaches in which only a discrete number of proteins was analyzed at a time (29-33). Currently, sophisticated quantitative mass spectrometry (Q-MS)-based strategies allow the unbiased analysis of thousands of proteins simultaneously, enabling the global study of complex systems such as protein networks triggering signal transduction (34-37). Indeed, some recent studies have relied on Q-MS approaches to characterize and compare the phosphotyrosine (pY)-dependent signal transduction initiated by IL-2 and IL-15 (3839)

.

Combining Q-MS strategies with interaction proteomics and phosphoproteomics analyses, the present study characterizes the early assembly of cytokine triggered IL-2R and IL-15R protein complexes in CD4+ T-lymphocytes. This study constitutes, to our knowledge, the first attempt to decipher, using


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unbiased proteomics strategies, the formation of the active cytokine/receptor complex in interleukintreated T-cells. The investigation reveals that early assembled IL-2/IL-2R and IL-15/IL-15R protein complexes are practically indistinguishable except for their specific receptor alpha subunit. We detect several site-specific phosphorylation events within IL-2R β and γ subunits, including previously not reported ones, and unveiled that some of the cytokine-dependent phosphosites detected have the capacity to interact with distinct effector proteins involved in signal transduction. Together with the IL-2Rβ and IL-2Rγ interactome dataset generated, we define the prominent components of the active IL-2R and IL-15R protein complex and distinguish between effector proteins that are constitutively associated with the signaling receptor subunits and the molecules that are recruited via the cytoplasm. Overall, this work represents the first global analysis of receptor complex assembly following IL-2 and IL-15 stimulation that provides novel insights into the early activation events following cytokine/receptor engagement in CD4+ T-lymphocytes.

EXPERIMENTAL PROCEDURES Reagents, antibodies and synthetic peptides

Human recombinant IL-2 was kindly provided by “AIDS Research and Reference Reagent Program”, Division of AIDS (NIH, National Institute of Health) and IL-15 was purchased from PeproTech. The following antibodies were used in the study: IL-2Rβ (sc-671) and IL-2Rγ (sc-667) were obtained from SantaCruz Biotechnology, JAK1 (J24320) from Transduction Labs, JAK3 (3775) from Cell Signaling and SHC (610082) and GRB2 (610111) from BD Transduction. The HPR-conjugated secondary antibodies anti-mouse (NA931) and anti-rabbit (NA934) were purchased from GE Healthcare. Protein sepharose A-beads used for coupling primary antibodies for immunoprecipitation (IP) was obtained from GE Healthcare and streptavidin magnetic beads for coupling biotinylated peptides from Pierce. AKT inhibitor MK-2206 was purchased from Selleckchem and MEK inhibitor U0126 from Promega. Biotinylated peptide/phosphopeptide pairs used for peptide pull down assays and stable isotopelabeled phosphopeptides (SIL phosphopeptides) used for parallel reaction monitoring (PRM) analysis



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were synthesized by Peptide&Elephants and Thermo Scientific, respectively. Peptide sequences are shown in Table S1.

Cell culture and stimulation

The human leukemic T-cell line Kit225 was maintained in RPMI 1640 media (Gibco) supplemented with 10 % FBS, 1 % penicillin/streptomycin, 1 % sodium pyruvate, 1 % glutamine and 16 U/ml of recombinant human IL-2 at a density of 1.106 cell/ml at 37 ºC and 5 % CO2. For the SILAC-based immunoprecipitation experiments, Kit225 cells were grown in media containing either Lys/Arg (0/0), Lys/Arg (4/6) or Lys/Arg (8/10) for two weeks to allow complete labeling of their proteome. Prior stimulation, cells were synchronized at G1 phase of the cell cycle by IL-2 starvation for 48 hours aiming to mimic resting T-cells. Cells were stimulated by incubation with 200 U/ml of IL-2 (Lys4/Arg6) or IL-15 (Lys8/Arg10) for 5 minutes at 37 ºC and subsequently cytokine treatment was interrupted by placing cells on ice for 5 min. As a control, Kit225 cells grown in light media (Lys0/Arg0) were kept unstimulated. Cells were lysed with ice-cold co-immunoprecipitation buffer (25 mM TrisHCl pH 7.5, 100 mM NaCl, 1 % NP-40, 1 mM sodium pervanadate, 5 mM betaglycerophosphate, 5 mM NaF, complete protease inhibitor cocktail (complete tablets, Roche)) and protein concentrations were estimated using BCA protein assay kit (Pierce).

Protein immunoprecipitation and peptide processing

Protein lysates corresponding to non-stimulated and cytokine-stimulated Kit225 cells were combined in protein concentration ratios 1:1:1 and pre-cleared with sepharose A-beads for 1h at 4 ºC. For immunoprecipitating IL-2Rβ and IL-2Rγ together with their corresponding binding partners, precleared lysates were incubated with antibodies against IL-2Rβ (sc-671) and IL-2Rγ (sc-667) respectively, for 4 h at 4 ºC. Subsequent procedure was followed as previously described (40). In brief, immunoprecipitated proteins were washed, eluted and run in two parallel lanes of a precast 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 (41).


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Whereas peptides derived from one of the lanes were directly concentrated and desalted using C18 stage tips to further analyze by LC-MS/MS, peptides derived from the other lane were subjected to TiO2 enrichment (42-43) prior to MS analysis. We performed two independent biological replicates for the immunoprecipitation of each receptor subunit.

Peptide pull-down assay and on-bead protein digestion

SILAC-labeled cells (Arg0/Lys0 and Arg10/Lys8) were lysed in co-immunoprecipitation buffer and pre-cleared for 2 h at 4 ºC while immobilized streptavidin beads were loaded with biotinylated peptides. Then, equal amounts of pre-cleared lysate proteins were incubated with the respective immobilized peptide for 2.5 h at 4 ºC. After extensive washing with lysis buffer, the beads loaded with the phosphorylated and non-phosphorylated version of the same peptide were combined (1:1) and attached proteins were on-bead digested. Briefly, protein were resuspended in urea buffer (8 M urea, 10 mM TrisHCl pH 7.5) and subjected to reduction and alkylation prior to LysC digestion. Finally, urea concentration was decreased up to 2 M so that proteins could be digested with trypsin. Resulting peptides were desalted and fractionated with C18 stage tips by eluting with increasing acetonitrile concentration (Figure S1). We performed two independent biological replicates for each of the four different pull down assays, swapping the SILAC labels for replicate experiments.

LC-MS/MS and data analysis

LC-MS/MS analysis of peptides and phosphopeptides enriched by IP and TiO2 was carried out using a reverse phase liquid chromatography system (EASY-nLC 1000 ultra-high pressure, Thermo Fisher Scientific) interfaced with a Q Exactive mass spectrometer (Thermo Fischer Scientific) via a nanoelectrospray source (Thermo Fisher Scientific) whereas peptide pull down experiments were analyzed on Velos Orbitrap MS system (Thermo Fischer Scientific). Acidified peptides were loaded on an analytical in-house packed column (20 cm x 75 µm, ReproSil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH)) in solvent A (0.5 % acetic acid) and eluted by a nonlinear 120 min solvent B gradient (0.5 % acetic acid, 80 % ACN) at a flow rate of 250 nl/min. Q Exactive was operated in a top 10 data



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dependent mode. Survey scans were acquired at a resolution of 70,000 (m/z 400) and fragmentation spectra at 35,000 (m/z 400). Precursors were fragmented by higher energy C-trap dissociation (HCD) with normalized collision energy of 25 eV. The maximum injection time was 120 ms for survey and 124 ms for MS/MS scan whereas the AGC target values of 1e6 and 1e4 were used for survey scans and for MS/MS scans, respectively. In the Velos Orbitrap MS system survey full-scan MS spectra (m/z range, 300-1750; resolution 30,000 at m/z 400) were acquired and the 8 most intense multiply charged ions were fragmented by HCD (resolution 15,000 at m/z 400). Repeat sequencing of peptide was minimized by excluding the selected peptide candidates for 45 s.

All raw data files acquired were searched against the UniProt human database version 2014.01 (with 88,479 sequence entries) with MaxQuant proteomics computational platform version 1.3.0.5 and using Andromeda search engine (44). In triple SILAC experiments light, medium and heavy labels were set as Arg0/Lys0, Arg6/Lys4 and Arg10/Lys8 whereas for double SILAC Arg0/Lys0 and Arg10/Lys8 were selected. Precursor and fragment mass tolerances were set to 7 and 20 ppm, respectively. Enzyme specificity was set to trypsin, allowing for cleavage N-terminal to proline and between aspartic acid and proline (with a maximum of 2 missed cleavages). Carbamidomethylation of C was set as fixed modification whereas oxidation of M, protein N-terminal acetylation, NQ deamidation and STY phosphorylation were selected as variable modifications for database searching. For peptide and protein identification a minimal peptide length of 7 amino acids was required and the false discovery rate was set at 0.01. Additionally, for protein identification we demanded at least two peptides, of which at least one was unique to the protein group. Both razor and unique peptides, except STY phosphorylated peptides were considered for protein quantification. For the analysis of phosphopeptides, 1% FDR, a minimum localization probability of 0.75 and a score difference of at least 5 was used (43). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (45) via PRIDE partner repository with the dataset identifier PXD002386.

Sample preparation for targeted phosphoproteomics analysis 8 ACS Paragon Plus Environment

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Prior stimulation with IL-2, Kit225 T-cells deprived with the cytokine for 48 h were treated with 5 µM MK-2204, 15 µM U0126 or vehicle for 30 min whereas starved cells were used as control. Cells were lysed using urea buffer (Urea 8 M, TrisHCl 10 mM pH 7.5) followed by sonication. Proteins were insolution reduced with 1 mM DTT for 45 min, alkylated with 5.5 mM CAA (2-Chloroacetamide) for 1 h and digested with LysC for 4 h followed by overnight trypsin digestion. Resulting peptides were acidified, purified over a Sep-Pak cartridge C18 column (Waters) and lyophilized for 48 h. Then, samples were resuspended in 60% ACN/ 1% TFA, phosphopeptides were enriched using TiO2 beads (42-43)

and purified using C18 Stage Tips.

Parallel Reaction Monitoring (PRM) and data analysis

PRM analyses were performed using a Q Exactive mass spectrometer (ThermoFisher Scientific) interfaced with an Easy-nLC 1000 nanoUPLC System (ThermoFisher Scientific). Phosphopeptides were air dried in a Speedvac and resuspended with the SIL phosphopeptide mixture. Samples were loaded onto an Acclaim PepMap100 precolumn (75 µm x 2 cm, ThermoFisher Scientific) connected to an Acclaim PepMap RSLC (50 µm x 15 cm, Thermo Scientific) analytical column. Peptides were eluted with a 90 min linear gradient from 3% to 30% of acetonitrile in 0.1% of formic acid at a flow rate of 300 nl/min directly onto the nanoES Emitter (ThermoFisher Scientific). The Q Exactive was operated in Targeted-MS2 mode and method optimization was achieved by analysis of SIL phosphopeptide by Full MS, Full MS/dd-MS2 (Top10) and Targeted-MS2. Then, selected m/z values were incorporated in an inclusion list and specific retention time windows were applied based on method optimization results. Spectra were acquired at a resolution of 17,500 (m/z 200). Peptide selection was done with an isolation window of 2.0 Th and normalized collision energy of 28 was applied for peptide fragmentation. The maximum injection time was 500 ms and an AGC target value of 5e5 was used. Raw files were processed and searched with Proteome Discoverer 1.4 (ThermoFisher Scientific) in order to identify the selected peptides. Precursor and fragment mass tolerances were set to 10 ppm and 0.05 Da respectively, up to 1 missed cleavage was allowed and a Human SwissProt database (version 2013_09) was used. Carbamidomethylation of C was set as fixed 9 ACS Paragon Plus Environment


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modification and M oxidation, STY phosphorylation, and heavy isotope labels as variable modifications (13C6-R, 13C615N4-R, 13C4-K and 13C615N2-K). For peptide relative quantification Skyline v2.6.0.6851 software (46) was used. All integrated peaks were manually inspected to ensure correct peak detection and total peak area values were exported into Microsoft Office Excel (Microsoft) for further data analysis.

Statistical analysis

Perseus statistical software was employed for the calculation of the statistical significance B, a pvalue that depends on protein intensities and ratios. It was also used to perform the hierarchical clustering analysis in which protein groups were clustered according to their ratios and following next settings: row and column distance calculated using the Euclidean algorithm; row and column linkage – average (47). The relative abundance of the proteins enriched in IL-2R β and γ immunoprecipitations was calculated averaging the intensity of their three most intense peptides and normalizing the value to the IL-2Rβ/ IL-2Rγ complex level. The normalized values were then converting into percentage, using the highest value of each experiment as 100. Similarly, relative protein abundance in the peptide pull down assays was calculated using the peptide intensity information and converting then into percentages. MS/MS spectra of the phosphorylated peptides corresponding to IL-2Rβ and IL-2Rγ were validated and annotated using MaxQuant viewer expert system (47). KinasePhos was used to search for putative kinase motifs (48).Viewer Graphs were generated using Microsoft Office Excel (Microsoft) and Venn diagram Plotter.

RESULTS In order to depict the interleukin receptor-associated protein complex in IL-2- and IL-15-activated Tcells, we followed the experimental design represented in Figure 1A in duplicate. The approach consisted of two complementary SILAC-based interaction proteomic analyses that aimed to identify cytokine-dependent and independent interacting proteins of IL-2Rβ and IL-2Rγ as well as site-specific phosphorylation events occurring in both receptor subunits.


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Figure 1 could be placed around here

According to the current model of interleukin signaling (Figure 1B), the tyrosine kinase JAK1 is constitutively associated with IL-2Rβ, whereas its family member JAK3 is pre-bound to γc. Upon ligand engagement, both receptor subunits are juxtaposed and consequently JAK kinases become active, phosphorylating the receptors and thus, initiating a complex network of signaling cascades that result in T-cell proliferation. Based on this model, we should expect that in the immune complexes obtained from IL-2Rβ immunoprecipitation (IP), both IL-2Rβ and JAK1 would display a ratio of 1 between resting and cytokine-treated T-lymphocytes (Figure 1C). However, as a consequence of receptor oligomerization, IL-2Rγ and its binding partner JAK3 should be more abundant in stimulated conditions. On the contrary, as IL-2Rγ and JAK3 are believed to be constitutively associated, both should show a ratio of 1 in the immune complexes obtained from precipitating IL-2Rγ. Nevertheless, in the same immune complexes IL-2Rβ and JAK1 should be more abundant upon interleukin stimulation, which would indicate that they interact with the receptor gamma subunit only in cytokineactivated T-lymphocytes.

As evident from this illustrative example, the combined analysis of cytokine-driven IL-2Rβ and IL2Rγ interactome allows distinguishing between: 1) proteins that are already associated with each receptor subunit in resting T-cells and 2) proteins that are recruited to the oligomerized receptor complex in a cytokine-dependent manner. Therefore, in order to unveil and compare the receptor complex assembly triggered by IL-2 and IL-15 in T-lymphocytes, it is imperative to dissect the interactome of the two signaling receptors shared by both cytokines. Accordingly, the study presented here focuses on investigating the binding partners of IL-2Rβ and IL-2Rγ in resting as well as in IL-2and IL-15-treated T-cells. Moreover, bearing in mind the pivotal role of protein phosphorylation in orchestrating the interleukin receptor complex assembly, site-specific phosphorylation events occurring in IL-2Rβ and γc were also examined in detail.

Defining the interactome of IL-2Rβ and IL-2Rγ in IL-2- and IL-15-treated T-lymphocytes



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To identify the proteins recruited to IL-2Rβ in response to IL-2 and IL-15 stimulation, we performed two independent immunoprecipitations of the IL-2R beta subunit and subsequently analyzed the precipitated complexes by Q-MS. In addition, a portion of the immune complex was enriched in phosphopeptides using TiO2 beads prior to MS analysis.

In total 1368 proteins were confidently quantified in the two replicas showing a Pearson coefficient of 0.65 and 0.58 for IL-2- and IL-15-induced protein recruitment, respectively. It is worth mentioning that the majority of the proteins quantified (>97 %) are within a one-fold range difference in quantitation between the replicas (Figures 2A-B and Table S2).


From those, a stringent list of potential cytokine induced IL-2Rβ interactors was generated containing the proteins fulfilling the following conditions: Consistent SILAC ratio between the two replicas, the ratio between interleukin treatment and control (IL-2/Ctr and/or IL-15/Ctr) ≥ 2, Significance B p-value < 0.001 and Ratio Count ≥ 2. Following these criteria that accounts for generation of high confidence data, 15 proteins were found with higher abundance in the immune complexes upon IL-2 and IL-15 treatment and therefore represent the set of effectors that are recruited to the beta chain of the receptor after cytokine stimulation of Kit225 T-cells. Table 1 summarizes the relevant information about those proteins.

Table 1 could be placed around here

As expected, based on the receptor complex formation model presented above (Figure 1B), IL-2Rγ and its constitutive interactor JAK3 were highly enriched in the IL-2Rβ-containing immune complexes under both cytokine treatments, whereas the enrichment of IL-2Rβ and JAK1 was unaffected (Figure 2C, indicated with diamond shape and Table 1, upper panel).

Besides γc and JAK3, several proteins were consistently enriched in the IL-2Rβ IP constituting the set of effectors that associate, either directly or through IL-2Rγ, with the IL-2R β chain in cytokine-


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treated T-cells. Most of those proteins were also found enriched in our previous study where the phosphotyrosine immune complexes of IL-2- and IL-15-treated T-lymphocytes were analyzed, thus demonstrating that they are bone fide effectors of the IL-2 and IL-15 signaling pathways (39). This group of proteins includes the adaptors SHC1, FAM59A, GAB2, GAB3, members of the GRB2/SOS complex, as well as numerous members of the PI3K family (Table 1). Moreover, for the first time, the present work shows that the ubiquitin ligase SOCS2, the adaptor protein SH2B1 and the serine/threonine kinase TNK2 also associate with IL-2Rβ in cytokine-treated T-cells, suggesting they may play a role in the signaling cascades initiated by both interleukins. Our study also reveals that protein recruitment to IL-2Rβ induced by IL-2 and IL-15 is virtually identical, showing a Pearson correlation coefficient of 0.98 (Figure 2C). This result is in line with recently published data in which quantitatively comparing the phosphotyrosine signaling networks of IL-2 and IL-15 pinpointed that signaling properties of both cytokines are highly similar in T-cell lines (38-39).

Regarding the analysis of IL-2Rγ interacting partners, our Q-MS procedure provided confident quantification of 1260 proteins in the two biological replicates performed. The overall correlation coefficient between the two replicas of IL-2- and IL-15-induced recruitment was 0.43 and 0.37. Once again, the quantification value obtained for most of the proteins (>96 %) differed less than one-fold between the replicas (Figures 3A-B and Table S3).


We found that 7 proteins were enriched (Interleukin/Ctr>2, significance B p-value 2, Significance B p-value < 0.001), including several well-known IL-2 effector proteins (Figure 7A).


Our study revealed that IL-2Rβ pS431-containing peptide is able to recruit the three clathrin subunits (CLTA, CLTB and CTLC) composing the clathrin triskelion, whereas IL-2Rβ pT476 seemed not to recruit any IL-2 effector at all (Figure 7B). On the contrary, various mediators of the IL-2 signaling pathways were found to be specific interactors of the phosphorylated peptide bearing IL-2Rγ Y325 and/or Y357. Among the 7 proteins that were found to specifically associate with IL-2Rγ pY325containing peptide, protein binding level analysis suggests that SOCS2 is the most prevalent interactor (Figure 7C). The same proteins were also enriched using IL-2Rγ Y357-bearing phosphopeptide. But unlike observed for pY325, not only SOCS2 but also the phosphatase PTPN11 and adaptor GRB2 appear to be among the principal interactors of IL-2Rγ pY357 (Figure 7B-C). Additionally, PLCG1, members of the PI3K family (PIK3R2 and PIK3CD) and the ubiquitin ligases CBL and CBLB were specifically and exclusively found to associate with IL-2Rγ pY357-containing peptide. Among them CBLB showed the highest binding level (Figure 7C).

Altogether, these data discloses the biological relevance of those site-specific phosphorylation events occurring on the cytokine-activated IL-2R signaling subunits by demonstrating their capacity to recruit effector molecules and contribute to the adequate assembly of the IL-2R complex that is required to transduce the signal initiated at the cell membrane inside the cell.



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DISCUSSION

Since the discovery of IL-15 in 1994, several mechanisms have been proposed to explain the functional dichotomy existing between IL-2 and IL-15 that signal through the same IL-2Rβ and IL2Rγ subunits. So far, the quaternary structures of IL-2/IL-2R and IL-15/IL-15R have been compared, as well as the gene signature and the pY-driven signaling cascades triggered by both cytokines (38-39,5354)

. Nevertheless, the underlying molecular mechanisms explaining the functional differences between

IL-2 and IL-15 remain elusive. Aiming to shed light on the still existing controversy, the present study focused on characterizing the initial receptor complex assembly in CD4+ T-lymphocytes following IL2 and IL-15 activation. Combining various Q-MS-based phosphoproteomic and interaction proteomic strategies, we have defined a cohesive model of protein recruitment through IL-2- and IL-15-induced phosphorylation events occurring on IL-2R β and γ subunits, the two signaling receptor subunits shared by the cytokines. In agreement with the currently established model, we confirm that IL-2 and IL-15 stimulation results in the assembly of two different heterotrimeric receptor complexes comprised by the same β and γ subunits (IL-2Rβ and IL-2Rγ) and a specific alpha chain (IL-2Rα or IL-15Rα). This is the major difference observed between IL-2- and IL-15-activated receptor complexes and, although both receptor alpha subunits are believed to solely modulate cytokine affinity, it has largely been suggested that they could partially account for the functional discrepancy existing between the two cytokines (1720)

. Indeed, an increasing body of research is demonstrating that membrane associated IL-2 and IL-15

receptor alpha chains can be shed and resulting soluble forms of the receptors can modulate the immune response either acting as agonists or antagonists of their specific cytokine (23). It should be noted that soluble IL-2Rα and IL-15Rα have been detected in Kit225 T-cell line (55-56). Although we have no direct evidence, it is plausible that IL-2- and IL-15-treated Kit225 T-cells receptor alpha chain cleavage has occurred. In fact, this would partially explain the low SILAC ratio obtained for both receptor alpha subunits in the IL-2Rβ and IL-2Rγ IP experiments performed. In addition, it is possible that IL-2Rβ/IL-2Rγ heterodimers are formed in IL-2- and IL-15-treated Kit225 T-cells or that IL2Rβ/IL-2Rγ heterodimers result from the early dissociation of the alpha chains as means of a 20 ACS Paragon Plus Environment

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regulatory mechanism to avoid over-stimulation of the system. IL-2Rβ/IL-2Rγ heterodimers comprise the intermediate-affinity receptor binding IL-2 and IL-15 with similar affinities (Kd ~ 1 nM) (57), therefore if present in cytokine-treated T-cells, it is unlikely that they account for the distinct signaling properties displayed by the two cytokines. There are two opposing models known as the “pre-assembly” and “stepwise binding” model defending that the association between the interleukin receptor alpha and beta subunits is constitutive or cytokine-induced, respectively (58-59). Although our data cannot exclude that there is a pre-bound fraction between the alpha and the beta receptor subunits, the increase in SILAC ratios indicates that there is a recruitment of alpha receptor subunit to the beta receptor subunit in support to the stepwise model.


Overall our data support the current model in which constitutively coupled IL-2Rβ/JAK1 associate with pre-bound IL-2Rγ/JAK3 upon cytokine stimulation (Figure 8). Moreover, we provide evidence indicating that additional JAK3 is recruited to the cytokine-activated receptor complex in CD4+ Tlymphocytes, which was already demonstrated by Kirken and colleagues in IL-2-treated YT NK-cells (60)

. Unlike us, they did not detect association between IL-2Rγ and JAK3 in non-activated NK cells,

whereas they found that IL-2Rβ and JAK1 are constitutively associated and additional JAK1 is recruited to the receptor beta chain in response IL-2 stimulation. These data indicate altogether that the receptor complex assembly following cytokine stimulation is cell type dependent. Therefore, a more detailed knowledge of the underlying mechanism in each case, as we deciphered for CD4+ subset of Tcells, is imperative for a better understanding of the subsequent downstream signaling events.

Independently of the mechanism, IL-2- and IL-15-induced receptor oligomerization results in the activation of JAK tyrosine kinases, which phosphorylate multiple substrates creating anchoring sites for proteins with various functions that lead to the activation of downstream signaling networks (61-62).



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For the first time, our work has revealed that IL-2Rγ Y325 and Y357 become phosphorylated in IL-2and IL-15-treated CD4+ T-lymphocytes. More importantly, we have disclosed their biological role demonstrating that both sites, when phosphorylated, are capable of recruiting several signaling proteins. None surprisingly, mutation on IL-2Rγ Y325 has been detected in patients with X-linked severe combined immunodeficiency (XSCID), a rare genetic disorder of T- and B-cell development and function that is a consequence of impaired γc signal transduction (63-64).

We report here that both IL-2Rγ sites mentioned above can associate with SOCS2 which contains a SH2 domain that most probably mediates the binding. We also detected that SOCS2 is recruited to IL2Rβ upon cytokine stimulation and although it cannot be discarded a direct binding between the two proteins, it seems plausible that the association is through IL-2Rγ phosphotyrosines. To our knowledge this is the first study demonstrating that SOCS2 is a component of the active IL-2Rβ/IL2Rγ protein complex in CD4+ T-cells. However, more than a decade ago its family member SOCS3 was shown to associate with IL-2Rβ/JAK1 complex in IL-2-treated peripheral blood lymphocytes (65). Considering the high sequence homology between the two family members especially within the SH2 domain, it is likely that the recruitment of SOCS3 to IL-2Rβ/JAK1 occurs also through the cytokineinduced phosphorylation of IL-2Rγ Y325 and Y357.

We identified SOCS2 as a prominent binding protein of IL-2Rγ pY325 and pY357, nonetheless according to the IL-2Rβ interactome dataset SOCS2 is associated with a minor IL-2Rβ/IL-2Rγ complex population in T-cells. A priori these results may seem contradictory. However they only reflect the limitations of each technique and uncover the need to combine diverse Q-MS-based interaction proteomic strategies and the relevance of studying the relative abundance of the interactors in order to construct an accurate model of the protein complex of interest. It is undisputable that peptide/phosphopeptide pull down screens are very convenient for studying modification-dependent protein interactions (52,66). However they do not necessarily reflect faithfully the interactome of the protein they belong to. Affinity purification experiments using short unstructured peptides reveal the potential of a specific sequence to recruit interactors but does not necessarily imply that all the


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interactors will associate with the protein the peptides correspond to. It cannot be excluded the possibility that a specific peptide-driven interaction is hampered within the biological context of the intact protein due to for example the presence of competing docking sites that are missing in the peptide used as bait in the pull down screen. For instance, that may explain why despite having the capacity to interact with IL-2Rγ pY325 and pY357-containing peptides, the tyrosine phosphatases INPPL1 and INPP5D were not found enriched in the IL-2Rβ and IL-2Rγ immunoprecipitations. Therefore, it is evident that in addition to identifying peptide- or protein-binding partners it is essential to carefully analyze and compare their relative abundance by complementary approaches in order to avoid misleading conclusions.

Unlike SOCS2, our data demonstrate that together with JAK1 and JAK3 the adaptor proteins SHC1 and GRB2 are prominent components of the signaling IL-2Rβ/IL-2Rγ complex (Figure 8). Indeed, it is well documented that cytokine-activated JAK1 phosphorylates IL-2Rβ Y364 creating a docking site for the SHC1 which then becomes tyrosine phosphorylated on Y427 and recruits GRB2 (33,67-68). We also found that the two adaptor proteins can associate with IL-2Rγ Y325 and Y357-bearing phosphopeptides independently of IL-2Rβ, which implies the existence of an additional recruitment mechanism of SHC1 and GRB2 to the functional IL-2Rβ/IL-2Rγ complex that to our knowledge has not been described before. However, considering the relative low abundance of SHC1 within the protein complexes isolated in the pull down assays, we believe that the recruitment of SHC1 is occurring through cytokine-induced phosphotyrosines present in IL-2Rβ. On the contrary, the presence of GRB2 in IL-2Rγ pY357 pull down was noticeable suggesting that the adaptor protein could also be recruited to the active interleukin receptor complex by directly binding to the IL-2Rγ pY357. Like it was demonstrated for the recruitment of GRB2 to EGFR (69), our data is pioneer in implying that GRB2 can bind the tyrosine phosphorylated IL-2Rβ/IL-2Rγ complex directly, as well as indirectly via SHC1. This could explain why GRB2 seems to be more abundant than SHC1 in the interleukin triggered receptor complex characterized.



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It has been recently shown that the rapid EGF-induced association of SHC1 and proteins involved in the activation of pro-mitogenic pathways depends on the previous recruitment of GRB2 to tyrosine phosphorylated SHC1 (70). The Ras/Rho GEFs SOS1 and SOS2 are among the rapid SHC1-binding proteins that we found to be recruited from the cytoplasm to the activated IL-2Rβ/γc complex in IL-2and IL-15-treated T-lymphocytes. Due to the stringent criteria selected to consider a protein as cytokine-dependent interactor of IL-2Rβ or IL-2Rγ (Interleukin/Ctr ratio >2; Significance B pvalue

Characterization of Receptor-Associated Protein Complex Assembly

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