Article pubs.acs.org/jpr
Wide-Scale Quantitative Phosphoproteomic Analysis Reveals That Cold Treatment of T Cells Closely Mimics Soluble Antibody Stimulation Qinqin Ji† and Arthur R. Salomon*,†,‡ †
Department of Chemistry, Brown University, Providence, Rhode Island 02912, United States Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02903, United States
‡
S Supporting Information *
ABSTRACT: The activation of T lymphocytes through antigen-mediated T cell receptor (TCR) clustering is vital in regulating the adaptive immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperatures have initiated some of the hallmarks of TCR signaling, such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum, as well as coalesced the T cell membrane microdomains. The precise mechanism of the TCR signaling initiation due to temperature change remains obscure. One critical question is whether the signaling initiated by the cold treatment of T cells differs from the signaling initiated by the cross-linking of the T cell receptor. To address this uncertainty, we performed a wide-scale, quantitative massspectrometry-based phosphoproteomic analysis on T cells stimulated either by temperature shifts or through the cross-linking of the TCR. Careful statistical comparisons between the two stimulations revealed a striking level of identity among the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, in unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns that were nearly identical to those of soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells. KEYWORDS: cold stimulation, Jurkat, T cell signaling, immunology, mass spectrometry, phosphoproteome
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INTRODUCTION Signals from the recognition of peptide−MHC complexes by T cell receptors (TCR) are important for T cell development, survival, and death, as well as for the subset lineage specification and differentiation into effector or memory T cells in response to foreign antigens.1,2 TCR signaling is initiated through the engagement of the TCR by a cognate peptide−MHC molecule, resulting in the sequential activation of the Src kinases Lck and Fyn that then phosphorylate the ζ chain immunoreceptor tyrosine-based activation motifs (ITAMs).3 Phosphorylated ITAMs recruit and activate the Syk-family protein kinase ZAP70, which then phosphorylates the adaptor proteins LAT and SLP-76, thus forming a signalosome complex essential for the assembly of downstream signaling proteins.4,5 Although the molecular events and the protein components that are involved in TCR signaling have been extensively © 2015 American Chemical Society
studied, fundamental questions like the identification of the mechanisms for signaling initiation and early signaling transduction are still disputed. In vivo, T cell signaling is initiated through the binding of the T cell receptor to the peptide− MHC complex presented on the surface of the antigenpresenting cells, which leads to the productive clustering of T cell signaling proteins.6 The strength of the stimulation of the T cell receptor is an important part of the context used to understand the physiologically relevant responses of these cells, and the use of antibodies leads to levels of stimulation stronger than those observed in vivo.7 Soluble antibodies, such as the anti-CD3/CD4 antibody, engage with the receptor and are used for receptor cross-linking to initiate the TCR signaling Received: November 11, 2014 Published: April 3, 2015 2082
DOI: 10.1021/pr501172u J. Proteome Res. 2015, 14, 2082−2089
Journal of Proteome Research
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pathways that lead to the activation of many of the same downstream effectors as the physiological stimulation with peptide−MHC complexes.7,8 The aggregation of lipid rafts through the aggregation of the ganglioside GM1 using the cholera toxin b subunit and the anticholera toxin also leads to many of the hallmarks of T cell activation.9 Interestingly, without the ligation of the receptor by a soluble antibody, low temperature alone is able to induce the coalescence of membrane microdomains and the activation of signaling pathways, although the precise mechanism of activation was not fully resolved in these studies.10,11 An important downstream event in the TCR signaling pathway, the tyrosine phosphorylation of ERK, was observed both in Jurkat cells and in human primary T cells after the reduced temperature treatment.10,11 Immune blotting with the pan-phosphotyrosinespecific antibody 4G10 revealed that other T cell signaling proteins, including LAT, Lck, Fyn, Src-family PTKs, and ZAP70, may show increased tyrosine phosphorylation in Jurkat cells when treated with low temperature alone, although information about the individual sites of phosphorylation was not revealed in this data.10 The increased phosphorylation of T cell signaling proteins was reversible when the cells were switched from a low temperature back to physiological temperature (37 °C). Furthermore, low-temperature-induced stimulation proved sufficient to evoke an increase in intracellular free Ca2+ concentrations, one of the obligatory events during T cell activation.11 As T cell signaling is controlled by the balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs),12 cold-induced activation could, at least hypothetically, be due to a differential impact of the temperature shift on the kinetics of individual PTKs and PTPs, leaving the former relatively more active and the latter relatively less active at low temperatures. The impact of temperature on the specific kinetics of the individual kinases and phosphatases in T cells has not been specifically studied. Other researchers have proposed that the cold-induced coalescence of T cell plasma membrane microdomains activates the signaling pathways. In these studies, Lck and CD3 were evenly distributed at 37 °C, and cold treatment induced a patchy plasma membrane distribution in which CD3 and Lck were considerably colocalized.11,12 These studies support the important role of membrane microdomains in TCR signaling. Although some progress has been made in investigating a limited number of the signaling events induced by the low temperature treatment of T cells, a comprehensive comparison between the signaling initiated by temperature shifts and the signaling initiated through cross-linking of the TCR could shed new light on the precise mechanisms of signaling initiation. A central question is whether the signaling events initiated by specific cross-linking of the T cell receptor with antibodies are in any way divergent from the pathways initiated by the coldstimulation-induced rearrangement of lipid microdomains. Here we report a wide-scale quantitative analysis of 1344 unique sites of tyrosine phosphorylation observed in T cells stimulated by a cold shift or through a receptor cross-link. This analysis revealed that the phosphorylation signaling networks induced by receptor cross-linking using soluble anti-CD3/CD4 antibody are nearly identical to those of the cold-induced coalescence of T cell plasma membrane microdomains.
Article
EXPERIMENTAL SECTION
Cell Culture, Treatment, and Lysis
Jurkat E6-1 cells were obtained from the American Tissue Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 medium (Hyclone, Logan, UT) containing 10% heat-inactivated undialyzed FBS (Hyclone, Logan, UT), 2 mM L -glutamine, 100 U/mL penicillin G, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator with 5% CO2 at 37 °C. Cells were then washed and resuspended in PBS for further treatment. Cells were incubated for 20 min at 4 °C for cold stimulation. As a control, cells were incubated at 37 °C for 20 min. For soluble antibody stimulation, cells were treated with anti-CD3 and anti-CD4 antibodies in PBS (clones OKT3 and OKT4; eBioscience, San Diego, CA) for 5 min following incubation at 37 °C for 20 min as described.13 Once treatment was completed, cells were lysed in lysis buffer (9 M urea, 1 mM sodium orthovanadate, 20 mM HEPES, 2.5 mM sodium pyrophosphate, and 1 mM βglycerophosphate, pH 8.0). Protein Reduction, Alkylation, Digestion, and Peptide Immunoprecipitation
Protein concentration was measured by the DC Protein Assay (Bio-Rad, Hercules, CA). Reduction with DTT, alkylation with iodoacetamide, and digestion with trypsin were performed as previously described.13 Tryptic peptides were desalted using C18 Sep-Pak Plus cartridges (Waters, Milford, MA) and lyophilized for 48 h to dryness.14 Peptide immunoprecipitation was performed using preconjugated P-Tyr-100 phosphotyrosine antibody beads (Cell Signaling Technology) as previously described.13 A 5 pmol fraction of the synthetic phosphopeptide LIEDAEpYTAK was added to each sample as an exogenous quantitation standard prior to the peptide immunoprecipitation. After immunoprecipitation, the samples were desalted using C18 Zip Tip pipet tips (Millipore Corporation, Billerica, MA) according to the manufacturer’s instructions. Automated Nano-LC/MS and Data Analysis
Tryptic peptides were analyzed by a fully automated phosphoproteomic technology platform.15,16 Phosphopeptides were eluted into a Linear Trap Quadropole (LTQ) Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA) through a PicoFrit analytical column (360 μm outer diameter, 75 μm inner diameter, fused silica packed on a pressure bomb with 15 cm of 3 μm Monitor C18 particles; New Objective, Woburn, MA) with a reversed-phase gradient (0−70% 0.1 M acetic acid in acetonitrile in 90 min). An electrospray voltage of 2.0 kV was applied using a split flow configuration as described previously.17 Spectra were collected in the positive ion mode and in cycles of one full MS scan in the Orbitrap (m/z: 400−1800), followed by data-dependent MS/MS scans in the LTQ of the sequential 10 most abundant ions in each MS scan (with charge state screening for +1, +2, and +3 ions and a dynamic exclusion time of 30 s). The automatic gain control was 1 000 000 for the Orbitrap scan and 10 000 for the LTQ scans. The maximum ion time was 100 ms for the LTQ scan and 500 ms for the Orbitrap full scan. Orbitrap resolution was set at 60 000. The MS/MS spectra were searched against the nonredundant UniProt complete proteome database (version released February 1, 2013), which contained 87 613 forward and an equal number of reversed decoy protein entries, using the Mascot algorithm version 2.2.07 from Matrix Science.18 2083
DOI: 10.1021/pr501172u J. Proteome Res. 2015, 14, 2082−2089
Article
Journal of Proteome Research
Figure 1. Flow chart of phosphoproteomics data acquisition and data analysis. (A) Experiment design of this proteomics study. Two stimulations were performed on human Jurkat T cells: cold stimulation and soluble antibody (CD3/4) stimulation. (B) Data analysis workflow. (C) Volcano plots for q values versus intensity changes for cold stimulation and for CD3/4 stimulation. Cyan points are considered to be significant, having q values 2 or 20) and precursor mass error (20 and a mass error
