Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics

Abstract

Interleukin-2 (IL-2) is major cytokine involved in T cell proliferation, differentiation and apoptosis. Association between IL-2 and its receptor (IL-2R), triggers activation of complex signaling cascade governed by tyrosine phosphorylation that culminates in transcription of genes involved in modulation of the immune response. The complete characterization of the IL-2 pathway is essential to understand how aberrant IL-2 signaling results in several diseases such as cancer or autoimmunity and also how IL-2 treatments affect cancer patients. To gain insights into the downstream machinery activated by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5 min of IL-2 stimulation resulted in identification of 172 proteins among which 79 were found with increased abundance in the tyrosine-phosphorylated complexes, including several previously not reported IL-2 downstream effectors. Combinatorial site-specific phosphoproteomic analysis resulted in identification of 99 phosphorylated sites mapping to the identified proteins with increased abundance in the tyrosine-phosphorylated complexes, of which 34 were not previously described. In addition, chemical inhibition of the identified IL-2-mediated JAK, PI3K and MAPK signaling pathways, resulted in distinct alteration on the IL-2 dependent proliferation.

Introduction

Protein phosphorylation is one of the most important post-translational modifications involved in the regulation of essential cellular processes such as signal transduction, cell cycle control, proliferation, differentiation and apoptosis [1], [2], [3]. Protein kinases and phosphatases are responsible of phosphorylating and dephosphorylating the corresponding substrates, thereby altering their modification state. The combinatorial action and the specific expression of the over 500 predicted kinases and over 100 predicted phospahtases in the human genome determines response of the cells to different stimuli. At least one third of all human proteins are believed to be phosphorylated [2], [3], [4], [5], [6], [7], and it is estimated that 0.05–1.8% of protein phosphorylations occurs on a tyrosine residue [6], [8]. However, reversible tyrosine phosphorylation is the major mechanism of intracellular signal transduction initiated by receptor tyrosine kinases (RTKs) as well as signaling systems, including the IL-2 signaling pathway, where the signal is transduced from cell surface throughout the cytoplasm and to the nucleus via tyrosine phosphorylations of the receptors and downstream effectors.

IL-2, the first interleukin discovered, was initially described as a T cell growth-promoting factor [9]. Nevertheless, three decades of research have shown that its spectrum of biological activities is much broader. Apart from the immunostimulatory role, IL-2 is also known to have a pivotal immunosuppressive and immunoregulatory function [10]. Antigen binding to the T cell receptor (TCR) promotes IL-2 secretion as well as expression of IL-2 receptors (IL-2R). IL-2R comprises a heterotrimeric complex consisting of three polypeptide subunits named α, β and γ [11], [12]. The α subunit plays a critical role in regulating IL-2- mediated responses by modulating the affinity of the IL-2R for the ligand. The latter two subunits β and γ, respectively, each contain extracellular domains that bind the ligand and cytoplasmic tails that initiate the signal transduction process. IL-2/IL-2R association induces receptor oligomerization which results in the activation of several cytosolic protein tyrosine kinases (PTK) such as members from the JAK family [13], [14]. These PTKs associate physically and functionally with discrete regions of the IL-2Rβ and γ subunits, thereby phosphorylating the receptor and initializing a complex network of downstream signaling cascades [15], [16].These include the JAK/STAT, RAS/MAPK or PI3K/AKT pathways known to be activated upon IL-2 stimulation of T lymphocytes and to control transcription of genes that are directly involved in the modulation of the immune response.

Considering the multiple fundamental processes mediated by IL-2, it is not surprising that dysregulation of IL-2 signaling is involved in several diseases, such as cancer and autoimmune disorders. Conversely, recombinant IL-2 has been successfully used for therapeutic purposes in different cancer treatments [17], [18]. However, despite its abundant clinical benefits, IL-2 often causes severe side effects [19]. In order to fully decipher the clinical potential of IL-2, complete understanding of the architecture of the downstream processes activated by this cytokine is required.

Recent developments in quantitative mass spectrometry offer attractive alternatives to perform large scale proteomic experiments that only some few years ago were unfeasible. Among the existing techniques, stable isotope labeling by amino acids in cell culture (SILAC) is one of the well consolidated applications for quantitative proteomic analysis [20]. Moreover, combination of SILAC with immunoprecipitation using anti-phosphotyrosine antibodies has been shown to be very effective to quantitatively study signaling pathways downstream of receptor tyrosine kinases (RTKs) [21]. The EGF, PDGF, FGF, insulin, EphB, HGF and Her2/neu receptor mediated signaling pathways have been successfully analysed using this quantitative proteomics platform [22], [23], [24], [25], [26], [27], [28], [29].

Although individual aspects of the signaling triggered by IL-2 have been extensively studied, the distinct complex activation of its global signaling network remains not fully elucidated. In the present work, we used double encoding SILAC [30] combined with phosphotyrosine (pTyr) immunoprecipitation to identify and quantitatively assess tyrosine phosphorylation events in response to IL-2 in human T cell line Kit225. Kit225 cells represent an optimal model system to study IL-2 mediated signaling since these cells express relatively large amount of IL-2Rs and their proliferation is entirely dependent on IL-2 [31]. Depriving Kit225 cells from IL-2 48 h prior IL-2 stimulation leads to accumulation of the cells in the G1 phase of the cell cycle where the cells acquire characteristics of true resting T-cells [32]. Upon TCR mediated antigen recognition resting T-cells activate and express IL-2 as well as the IL-2Rα required for the subsequent induction of the IL-2 signaling pathway. Therefore, the IL-2 induced activation of Kit225 cells would be representative of the activation, which T-cells undergo when stimulated with the endogenously expressed and secreted IL-2 in response to the antigen presentation. Moreover, the effects observed in Kit225 cells are reproduced in other T-cell types such as NK or the murine cytotoxic T cell line CT6 [32], [33].We identified 172 proteins that showed change in abundance in anti-phosphotyrosine immunoprecipitates following treatment with IL-2, among which 79 were found with increased abundance indicating their involvement in the IL-2 signaling cascade. These proteins were as diverse as the subunits of the IL-2R, protein and lipid kinases, phosphatases, proteins involved in cytoskeletal rearrangements, endocytosis and ubiquitination. Moreover, we identified several proteins with increased abundance in the tyrosine-phosphorylated complexes not previously reported as IL-2 downstream effectors. In addition, combinatorial phosphoproteomic analysis resulted in the mapping of 99 phosphorylated sites among the identified proteins with increased abundance in the tyrosine-phosphorylated complexes, of which 34 were not previously described in the PhosphoSite database. Finaly, we used a chemical biology strategy to block the identified specific branches of the IL-2 mediated signaling to decipher their distinct effects on the IL-2 dependent proliferation of the human Kit225 T-cells.