APEX2-based quantitative proteomics of LAT and CD3σ interactomes in living human Jurkat T cells unveils new interactors.

TCR stimulation induces a signaling cascade starting by the phosphorylation of immunoreceptor tyrosine-based activation motif (ITAMs) present in the TCR-CD3 complex. This is followed by the phosphorylation of proteins including LAT, which once phosphorylated interacts with multiple proteins allowing signal diversification and amplification. We take advantage of APEX2-based peroxidase- catalyzed proximity labeling combined with quantitative mass spectrometry to track the formation of CD3σ and LAT interactome dynamics in TCR activated Jurkat cells. We find more than 1 000 high confidence proteins for each bait and provide a quantitative molecular map of proteins enriched or reduced from the vicinity of CD3σ and LAT, after stimulation. We detail and compare the recruitment kinetics of signaling proteins to CD3σ and LAT and identify uncharacterized mediators of T cell activation. We show that the kinase MARK2, which is in the proximity of LAT and CD3σ at resting state and lost upon activation, is a negative regulator of cytokine production by T cells. This study provides a resource for uncovering the complex signaling networks that regulate TCR activation and highlights new players of this signaling cascade.