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Analysis of the TCR signalosome in primary T cells

ABSTRACT: T-cell receptor (TCR) signaling is essential for the function of T cells. Here we combine mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the composition and dynamics of the signaling complexes that assemble around 15 nodes of the TCR signaling cascade of primary CD4+ T cells. This dataset contains the experiments performed with 14 different bait proteins • Cbl • Cblb • Fyb • Inpp5d • Itk • Lck • Lcp2 • Nck1 • Nfatc2 • Plcg1 • Ptpn22 • Ptpn6 • Themis • Vav1 Each of them contains mass spectrometry results from the analysis of AP-MS experiments, based on the endogenous expression of One-Strep-tagged (OST) bait proteins in engineered mice models, and affinity purification of these proteins from primary CD4+ T cells, using Streptactin beads. Purification of OST proteins was performed at 5 different time points of stimulation of CD4+ T cells with anti-CD3 and anti-CD4 antibodies (0s; 30s; 120s; 300s; 600s). Each AP-MS purification of an OST- protein is associated with a corresponding control (purification from WT CD4+ T cells) at the same time point of stimulation. Several biological replicate experiments (time course OST series + associated WT controls) were performed for each bait. Several MS replicates were acquired for each sample. In addition, we analyzed the total proteome of CD4+ T cells isolated from each engineered mice model (14 different mice expressing an OST bait) and from WT mice, in order to calculate copy numbers of the baits and their associated proteins.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Primary Cell, T Cell

SUBMITTER: Karima Chaoui

LAB HEAD: Odile Schiltz

PROVIDER: PXD007660 | Pride | 2019-09-26

REPOSITORIES: Pride

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Project description:T-cell receptor (TCR) signaling is essential for the function of T cells and negatively regulated by the E3 ubiquitin-protein ligases CBL and CBLB. Here we combine mouse genetics and affinity purification coupled to quantitative mass spectrometry to monitor the dynamics of the CBL and CBLB signaling complexes that assemble in normal T cells over 600 seconds of TCR stimulation. We identify many previously known CBL and CBLB interacting partners, as well as a majority of proteins that have not yet been implicated in those signaling complexes. We exploit correlations in protein association with CBL and CBLB as a function of time of TCR stimulation for predicting the occurrence of direct physical association between them. The dataset is divided in two distinct subsets corresponding to the Cbl and to the Cblb interactomes. Each of them contains mass spectrometry results from the analysis of 10 different conditions of AP-MS purifications (based on affinity purification on Streptactin beads of One-Strep-tagged proteins) starting from CD4+ T cells which were either non stimulated or stimulated with anti-CD3 and anti-CD4 antibodies as follows: CBL interactome: - CBL-OST transgenic mice, CD4+ T cells non stimulated (noted Cbl_0) - CBL-OST transgenic mice, CD4+ T cells stimulated 30s (noted Cbl_30) - CBL-OST transgenic mice, CD4+ T cells stimulated 120s (noted Cbl_120) - CBL-OST transgenic mice, CD4+ T cells stimulated 300s (noted Cbl_300) - CBL-OST transgenic mice, CD4+ T cells stimulated 600s (noted Cbl_600) - WT mice, CD4+ T cells non stimulated (noted WT_0) - WT mice, CD4+ T cells stimulated 30s (noted WT_30) - WT mice, CD4+ T cells stimulated 120s (noted WT_120) - WT mice, CD4+ T cells stimulated 300s (noted WT_300) - WT mice, CD4+ T cells stimulated 600s (noted WT_600) For the CBL interactome, 3 biological replicates were prepared for these 10 different conditions (noted Ech1, Ech2, Ech3), yielding 30 analyzed samples. Three technical nanoLC-MS runs were acquired for each sample (noted R1, R2, R3), leading to 90 nanoLC-MS raw files. CBL interactome: - CBLB-OST transgenic mice, CD4+ T cells non stimulated (noted Cblb_0) - CBLB-OST transgenic mice, CD4+ T cells stimulated 30s (noted Cblb_30) - CBLB-OST transgenic mice, CD4+ T cells stimulated 120s (noted Cblb_120) - CBLB-OST transgenic mice, CD4+ T cells stimulated 300s (noted Cblb_300) - CBL-OST transgenic mice, CD4+ T cells stimulated 600s (noted Cblb_600) - WT mice, CD4+ T cells non stimulated (noted WT_0) - WT mice, CD4+ T cells stimulated 30s (noted WT_30) - WT mice, CD4+ T cells stimulated 120s (noted WT_120) - WT mice, CD4+ T cells stimulated 300s (noted WT_300) - WT mice, CD4+ T cells stimulated 600s (noted WT_600) For the CBLB interactome, 4 biological replicates were prepared for these 10 different conditions (noted S1, S2, S3, S4), yielding 40 analyzed samples. For series S1 to S3, 2 technical nanoLC-MS runs were acquired for each sample (noted R1, R2) and for serie S4, 3 technical nanoLC-MS runs were acquired for each sample (noted R1, R2, R3), leading in total to 90 nanoLC-MS raw files.

			Action	DRS
	Cbl.zip	Other
	Cblb.zip	Other
	Fyb.zip	Other
	Inpp5d.zip	Other
	Itk.zip	Other

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Analysis of the TCR signalosome in primary T cells

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