Wide-Scale Quantitative Phosphoproteomic Analysis Reveals That Cold Treatment of T Cells Closely Mimics Soluble Antibody Stimulation

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 mass-spectrometry-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

Vav1 Regulates T‑Cell Activation through a Feedback Mechanism and Crosstalk between the T‑Cell Receptor and CD28

Vav1, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T-cell receptor (TCR) signaling cascade is tyrosine phosphorylated rapidly in response to T-cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca²⁺ responses, and actin cytoskeleton regulation; however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck and ZAP-70, is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation. Importantly, this study revealed a new function for Vav1 in the negative feedback regulation of the phosphorylation of immunoreceptor tyrosine-based activation motifs within the ζ chains, CD3 δ, ε, γ chains, as well as activation sites on the critical T cell tyrosine kinases Itk, Lck, and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways

A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T‑Cell Receptor and SLP-76 Complex

Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca²⁺ signaling and PKC-family kinases are well characterized, PLC-γ1’s role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr¹⁹² phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck’s targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr¹⁹² phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd

Quantitative phosphoproteomic analysis of proteins associated with the KEGG TCR signaling pathway.

<p>Depicted is a SILAC heatmap representation of temporal changes in tyrosine phosphorylation of proteins associated with the KEGG TCR signaling pathway. Heatmaps were calculated from the averages of four biological replicate experiments. SILAC ratios between U0126-treated and control DMSO-treated Jurkat T cells are represented for each phosphopeptide and time point. A white dot within the SILAC heatmaps indicate a statistically significant difference (q value <0.02) in the comparison between U0126-treated and control SILAC ratios for that time point. Abbreviations: GSK3 beta, Glycogen synthase kinase 3 beta; PI3K alpha, Phosphatidylinositol 3 kinase regulatory subunit, alpha; SHP-2, Protein tyrosine phosphatase, nonreceptor type 11.</p

Effects of U0126 on the canonical TCR signaling pathway.

<p>Depicted is a model of ERK positive feedback with quantitative U0126-treated to DMSO-treated SILAC ratio heatmaps beside individual proteins, corresponding to the changes in phosphorylation between the two conditions across the four time points of TCR stimulation. Heatmaps were calculated from the averages of four biological replicate experiments. Green represents elevated phosphorylation in response to U0126 treatment relative to DMSO-treated controls, whereas red represents a decrease in phosphorylation relative to DMSO-treated controls. Blanks in the heatmap indicate that a clearly defined SIC peak was not observed for that phosphopeptide in that time point. Black represents no change. White dots within the heatmap indicate a statistically significant difference (q value <0.02) in the comparison between U0126-treated and control DMSO-treated SILAC ratios for that time point. Below each heatmap square is a color bar representing the percent CV for that time point. Orange represents a high degree of variation, while black represents a low degree of variation amongst the replicate analyses. Blanks indicate a lack of replicate data required to accurately determine the CV.</p

Experimental protocol.

<p>Human Jurkat T cells were incubated with light or heavy stable isotope-labeled arginine and lysine amino acids, physically differentiating the two proteomes by a shift in molecular weights. Cells were treated with either U0126 (heavy-labeled cells) or DMSO (light-labeled cells) for 2.5 hours prior to stimulation. Each cell population was then pre-incubated with OKT3 and OKT4 antibodies for 30 seconds at 37°C and then cross-linked with IgG at 37°C for the times indicated.</p

Phosphorylation of Ser⁵⁹ Lck across a time course of TCR stimulation.

<p>(A) Cell lysates from a time course of TCR stimulation in the presence of 0.1% DMSO or 20 µM U0126 were separated by SDS-PAGE and immunodetected with phospho-Ser⁵⁹ Lck and Lck specific antibodies. (B) Densitometric analysis of phospho-Ser⁵⁹ Lck levels normalized to Lck levels was performed. Shown is the mean ± S.D. from 4 biological replicate experiments. Statistically significant differences in relative phospho-Ser⁵⁹ levels between U0126-treated and DMSO-treated cells for each time point are indicated with an asterisk (*-p value <0.007, **-p value <0.015).</p

Phosphorylation of the activation site of ZAP-70 across a time course of TCR stimulation.

<p>(A) Cell lysates from one replicate of a time course of TCR stimulation in the presence of 0.1% DMSO or 20 µM U0126 were separated by SDS-PAGE and immunodetected with phospho-ZAP-70 (Tyr⁴⁹³) and ZAP-70 specific antibodies. (B) Densitometric analysis of phospho-ZAP-70 levels normalized to ZAP-70 levels was performed. Shown is the mean ± S.D. from 4 biological replicate experiments. Statistically significant differences in relative phospho-ZAP-70 levels between U0126-treated and DMSO-treated cells for each time point are indicated with an asterisk (*-p value <0.003).</p

Quantitative phosphoproteomic analysis of proteins implicated in integrin signaling.

<p>Heatmaps were calculated from the averages of four biological replicate experiments. White dots within the SILAC heatmaps indicate a statistically significant difference (q value <0.02) in the comparison between U0126-treated and DMSO-treated control Jurkat T cell SILAC ratios for that time point.</p

Tyrosine phosphorylation in U0126-treated and DMSO-treated Jurkat T cells across a time course of TCR stimulation.

<p>Cell lysates from one replicate of a time course of TCR stimulation in the presence of 0.1% DMSO or 20 µM U0126 were separated by SDS-PAGE and immunodetected with a monoclonal 4G10 antibody that recognizes phosphotyrosines. The immunoblot is representative of data from 4 biological replicate experiments.</p