Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation.

Guillaume Voisinne,Laura Girard,Frédéric Fiore,Romain Roncagalli,Elise Bergot,Odile Burlet‐Schiltz,Antonio García‐Blesa,Bernard Malissen,Anne Gonzalez De Peredo,Karima Chaoui,Marie Malissen

doi:10.15252/msb.20166837

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.

Highlights

Affinity purification followed by mass spectrometry (AP-MS) enables analysis of protein–protein interaction (PPI) networks and of signaling complexes in their physiological cellular context (Gingras et al, 2007; Hein et al, 2015)
By combining co-recruitment analysis of the time-resolved and quantitative CBL and CBLB signalosomes with biochemical analysis, we demonstrated that CD5, a transmembrane protein expressed at the surface of T cells, constitutes a scaffold that has a central role in the CBL- and CBLB-mediated ubiquitylation events that follow T-cell receptor (TCR) engagement
To identify by AP-MS the proteins interacting with CBL and CBLB prior to or after TCR-mediated activation of primary mouse T cells, we generated two lines of gene-targeted mice expressing OneSTrEP-tag (OST) (Junttila et al, 2005) at the carboxyl-terminus of endogenous CBL and CBLB proteins (Fig EV1A and B)

Summary

Introduction

Affinity purification followed by mass spectrometry (AP-MS) enables analysis of protein–protein interaction (PPI) networks (interactomes) and of signaling complexes (signalosomes) in their physiological cellular context (Gingras et al, 2007; Hein et al, 2015). Such large-scale studies have resulted in comprehensive views of several signalosomes, they provided limited information on their internal organization and dynamics of assembly. Following TCR engagement, the protein tyrosine kinases (PTK) LCK and ZAP-70 are activated and phosphorylate several transmembrane and cytosolic proteins, leading in turn to the recruitment of CBL and CBLB via their tyrosine kinase binding (TKB) domains or indirectly via the GRB2 adaptor. Modification of the TCR and of its signaling partners with ubiquitin promotes their sorting to multivesicular

Methods

Results

Conclusion