APEX2-based quantitative proteomics of LAT and CD3ζ interactomes in living human Jurkat T cells unveils new interactors.

T cell receptor (TCR) stimulation induces a signaling cascade that starts with the phosphorylation of immunoreceptor tyrosine-based activation motifs (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 and dynamics of CD3ζ (also known as CD247) and LAT interactomes in TCR-activated Jurkat T cells. We identify, with high confidence, more than 1000 proteins for each bait, and we provide a quantitative molecular map of proteins that are enriched or depleted in the vicinity of CD3ζ and LAT after TCR stimulation. We detail and compare the recruitment kinetics of signaling proteins to CD3ζ and LAT, and identify uncharacterized mediators of T cell activation. We show that the kinase MARK2, which is in the proximity of LAT and CD3ζ 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 involved in this signaling cascade.