Experimental procedure.

<p>Two cell populations of human Jurkat T cell clones (J14-76-11 and J14) were incubated with RPMI 1640 medium containing normal or heavy isotope labeled arginine and lysine amino acids, physically differentiating the two proteomes by a shift in molecular weights. Each cell population was then pre-incubated with OKT3 and OKT4 antibodies for 10 minutes at 4°C and then crosslinked with anti-IgG at 37°C for the times indicated. After cell lysis, light and heavy cell lysates were combined at an equal protein concentration ratio for each timepoint. Proteins were then reduced, alkylated, and trypsin-digested into peptides. Peptides were desalted by Sep-Pak cartridges, enriched by phosphotyrosine peptide immunoprecipitation and Fe³⁺ IMAC, and then subjected to reversed-phase LC-MS/MS analysis. MS shifts introduced by heavy isotope labeling allow for differentiation between light and heavy peptide counterparts in MS spectra. Selected ion chromatogram (SIC) peak areas of light and heavy isotope labeled phosphopeptides were calculated for relative quantification of peptide abundance. Individual SIC peak areas were normalized to the SIC peak area of the copurified synthetic peptide LIEDAEpYTAK in the same timepoint. A label-free heatmap was generated based on peptide abundance for a certain peptide in SLP-76 reconstituted Jurkat cells through a time course of receptor stimulation and SILAC ratio heatmaps were generated based on the ratio of abundance between light (SLP-76 reconstituted) and heavy (SLP-76 deficient) peptide counterparts for each timepoint (SLP-76 deficient in relative to SLP-76 reconstituted).</p

Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

<div><p>The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.</p> </div

Phosphorylation kinetics of Itk, PLCγ1, and PLCγ2 in SLP-76 reconstituted and deficient cells.

<p>Phosphorylation kinetics of A) Itk Y512, B) PLCγ1 Y771, and C) PLCγ2 Y753 in the presence (WT) and absence of SLP-76 (ΔSLP76) are represented for 8 timepoints. Results represent the means of three replicate experiments (error bars indicate SD). “*” represents timepoints with significant changes (less than 2% false discovery rate) in phosphorylation abundance between WT and ΔSLP76 cells.</p

Quantitative phosphoproteomic analysis of proteins not previously known to be associated with TCR signaling (con't).

<p>Listed above is a portion of the data collected, representing proteins not previously established to be involved in TCR signaling. Temporal changes in phosphorylation abundance are represented as heatmaps from 3 replicate experiments as described in methods. The label free heatmap represents the change of abundance of phosphopeptides in SLP-76 reconstituted cells across 10 minutes of TCR stimulation while the SILAC heatmap represents the ratios of abundance of phosphopeptides in SLP-76 deficient cells relative to SLP-76 reconstituted cells at each of the TCR stimulation timepoints. The label free and SILAC heatmaps are described in detail as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046725#pone-0046725-g003" target="_blank">Figure 3</a>.</p

Phosphorylation kinetics of PAG, PI3K, WASP in SLP-76 reconstituted and deficient cells.

<p>Phosphorylation kinetics of A) PAG, B) PI3K, and C) WASP in the presence (WT) and absence of SLP-76 (ΔSLP76) are represented for 8 timepoints. Results represent the means of three replicate experiments (error bars indicate SD). “*” represents timepoints with significant changes (less than 2% false discovery rate) in phosphorylation abundance between WT and ΔSLP76 cells.</p

Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

<div><p>Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate <i>NLRP1</i> gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to <i>NLRP1</i> promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for <i>NLRP1</i> induction. This study provides further evidence that links ER stress with innate</p></div

Erk positive feedback on Lck, ZAP70, and CD3 ITAMs.

<p>Phosphorylation kinetics of Lck, ZAP70, and CD3 ITAMs from SLP-76 reconstituted (WT) and deficient (ΔSLP76) cells are presented for 8 timepoints. The differences of phosphorylations between WT and ΔSLP76 cells (ΔSLP76–WT) are also presented to show the trend of phosphorylation changes. Results represent the means of three replicate experiments (error bars indicate SD). “*” represents timepoints with significant changes (less than 2% false discovery rate) in phosphorylation abundance between WT and ΔSLP76 cells.</p

Quantitative phosphoproteomic analysis of proteins not previously known to be associated with TCR signaling.

<p>Listed above is a portion of the data collected, representing proteins not previously established to be involved in TCR signaling. Temporal changes in phosphorylation abundance are represented as heatmaps from 3 replicate experiments as described in methods. The label free heatmap represents the change of abundance of phosphopeptides in SLP-76 reconstituted cells across 10 minutes of TCR stimulation while the SILAC heatmap represents the ratios of abundance of phosphopeptides in SLP-76 deficient cells relative to SLP-76 reconstituted cells at each of the TCR stimulation timepoints. The label free and SILAC heatmaps are described in detail as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046725#pone-0046725-g003" target="_blank">Figure 3</a>.</p

Canonical TCR signaling pathway.

<p>Established signaling cascades in activated T cells. Proteins identified in our quantitative phosphoproteomic analysis are highlighted in red and the detailed data regarding these proteins, their phosphorylation kinetics in both SLP-76 reconstituted and deficient Jurkat T cells upon TCR stimulation, were presented later in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046725#pone-0046725-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046725#pone.0046725.s006" target="_blank">Figure S3</a>.</p

NLRP1 mRNA and protein are up-regulated upon ER stress.

<p>(A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or <i>NLRP1</i>^{<i>−/−</i>} HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)</p