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Analysis of the Vav1 interactome in primary T cells

ABSTRACT: In this study, we combined mouse genetics and quantitative proteomics to characterize in a comprehensive manner the VAV1 interactome. A line of knock-in mouse expressing endogenous VAV1 molecules with a genetic tag permitting affinity purification was generated and combined with affinity purification and mass spectrometry (AP-MS) to analyze the VAV1 signaling complex that assembles in primary CD4+ T cells over 300 s of activation. This allowed us to describe in a time-resolved manner the interactome of VAV1 in primary CD4+ T cells and to identify 44 previously unknown bindings partners of VAV1. The dataset contains mass spectrometry results from the analysis 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 pervanadate for different time lengths. Control samples for each time point were prepared from wild-type mice. 8 different conditions were thus analyzed: - VAV1-OST transgenic mice, CD4+ T cells non stimulated (noted Vav_0) - VAV1-OST transgenic mice, CD4+ T cells stimulated 30s (noted Vav_30) - VAV1-OST transgenic mice, CD4+ T cells stimulated 120s (noted Vav_120) - VAV1-OST transgenic mice, CD4+ T cells stimulated 300s (noted Vav_300) - 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) Four biological replicates were prepared for these 8 different conditions (noted S1, S2, S3, S4), yielding 32 analyzed samples. Three technical nanoLC-MS runs were acquired for each sample (noted R1, R2, R3), except for some samples of the biological series S4 for which 2 technical nanoLC-MS runs were performed. This led to 91 nanoLC-MS raw files composing the dataset.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Primary Cell, T Cell

SUBMITTER: Karima Chaoui

LAB HEAD: Anne Gonzalez de Peredo

PROVIDER: PXD004217 | Pride | 2018-06-26

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.

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Analysis of the Vav1 interactome in primary T cells

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