Co‐recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 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 CD5 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. Synopsis The composition and dynamics of the signalosomes operated by the E3 ubiquitin–protein ligases CBL and CBLB were determined in primary T cells after TCR stimulation. Analysis of correlations in protein association as a function of time reveals the importance of the CD5 transmembrane receptor in the regulation of ubiquitylation. The signalosomes of the E3‐ubiquitin ligases CBL and CBLB exhibit both redundant and distinct features in mature CD4 + T cells. Analysis of correlations in protein association with CBL and CBLB accurately predicts interactions between recruited proteins. CD5 is identified as a key regulator of CBL‐ and CBLB‐mediated ubiquitylation following TCR engagement. Graphical Abstract The composition and dynamics of the signalosomes operated by the E3 ubiquitin–protein ligases CBL and CBLB were determined in primary T cells after TCR stimulation. Analysis of correlations in protein association as a function of time reveals the importance of the CD5 transmembrane receptor in the regulation of ubiquitylation.