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

Abstract

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.

The multiple faces of CD5

“…Sequence analysis of the 429–441 region showed that this region resembles ITAM domains present in other receptors; however, different functional studies suggest it acts more like an ITIM domain than an ITAM domain (this will be discussed later). Recently, it has been demonstrated that CD5 is an important scaffold for Cbl and Cbl‐b ubiquitinylation following TCR engagement . In this context, Roa et al.…”

“…C-Cbl activation and degradation of Vav and CD5 itself has also been demonstrated to be regulated by the C-terminal region of this receptor. Finally, other signaling pathways have been proposed to be modulated through CD5, including phosphorylation of Vav and PI3K by Lck as well as RasGA-mediated downmodulation of TCR signaling demonstrated that CD5 is an important scaffold for Cbl and Cbl-b ubiquitinylation following TCR engagement 43. In this context, Roa et al have shown the importance of the carboxy-terminal region of human CD5 in c-Cbl-mediated degradation of Vav, thus reinforcing the role of this receptor as a negative regulator of TCR signaling.…”

Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry

“…Co-IP of CD5 and ARAP1 from cell lysates of WT CD4 + T cells stimulated with anti-CD3 plus anti-CD4 antibodies for 2 min independently validated the interaction between CD5 and ARAP-1 with GRB2 identified by AP-SWATH (Figure S3D). CD5 is an important organizer of ubiquitylation following TCR stimulation (Voisinne et al., 2016), and the identification of GRB2 within the CD5 signalosome further unveils its complexity. Taken together, these results and the ones provided by the AP-SWATH analysis of the GRB2 OST molecule delineate comprehensive maps of the GRB2 interactome in both resting and activated primary mouse CD4 + T cells.…”

“…Interestingly, this particular kinetic pattern was also observed with SATB1 and ARAP1 (Figure S6). This observation might suggest the existence of a complex that involves GRB2, PTPRA, SATB1, and ARAP1, since correlations in protein association with a given bait as a function of time of TCR stimulation support the occurrence of physical association between them (Voisinne et al., 2016). Although further experiments would be needed to test this hypothesis, our results led us to conclude that the workflow presented in this study provided a sufficient level of quantitative accuracy to distinguish similar and differential protein interaction dynamics between two functionally distinct primary T cell populations.…”

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells

“…More specifically, the function of T cells is negatively regulated by the E3 ubiquitin-protein ligases CBL and CBLB. Voisinne et al utilized AP-MS to analyze the dynamics of the CBL and CBLB signaling complexes formed after TCR stimulation [99]. They identified several proteins that had not yet been implicated in those signaling complexes and demonstrated that the CD5 transmembrane receptor constitutes a key scaffold for ubiquitin-ligase mediated ubiquitylation following TCR engagement [99].…”