Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation

SUMMARY The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi‐omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10‐mediated oxidative phosphorylation in T cell quiescence exit. Our multi‐layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence. HIGHLIGHTSProteome and phosphoproteome profiling reveals temporal dynamics of T cell activationTCR activates interconnected functional modules, kinases, and transcription factorsmTORC1 links TCR to mitoribosome biogenesis and complex IV activityCOX10 is crucial for T cell activation in vitro and in vivo &NA; Tan et al. apply multi‐layer proteomic profiling and systems biology approaches to define T cell proteome and phosphoproteome landscapes, and they identify signaling networks and bioenergetics pathways that mediate T cell quiescence exit. These data establish the function and mechanisms of oxidative phosphorylation and mitochondrial activation in antigen‐induced T cell responses.