Co‐recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD 5 as a key regulator of TCR ‐induced ubiquitylation

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 combined 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 most 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. By combining co‐recruitment analysis with biochemical analysis, we demonstrated that the CD 5 transmembrane receptor constitutes a key scaffold for CBL ‐ and CBLB ‐mediated ubiquitylation following TCR engagement. Our results offer an integrated view of the CBL and CBLB signaling complexes induced by TCR stimulation and provide a molecular basis for their negative regulatory function in normal T cells.