Characterization of the T Cell Receptor (TCR) pathway in primary T lymphocytes
One of our research areas is to decipher, at the cellular and molecular levels, the functional mechanisms of T lymphocytes using proteomic approaches. In collaboration with teams bringing complementary expertise in immunopathology and T cell signaling (B. Malissen, CIML, Marseille; R. Lesourne, CPTP, Toulouse), we are involved in the detailed characterization of the T Cell Receptor (TCR) pathway, by analyzing the signalosome of several key components of this complex system using AP-MS strategies.
In collaboration with R. Roncagalli and B. Malissen (CIML, Marseille), we use KI mouse model expressing different bait proteins of the TCR pathway with a C-terminal One-Strep-tag to enrich endogenous signaling complexes at different time points following TCR activation. Time resolved proteomics allows to obtain dynamics information and to gain insight into the mechanisms of action of several proteins and the internal organization of the interactomes.
AP-MS workflow for analysis of the TCR signalosome
Using this methodology, we characterized the interactome of PAG (Phosphoprotein associated with glycosphingolipid-enriched microdomains), a ubiquitously expressed transmembrane adaptor protein present in lipid rafts and involved in the regulation of TCR signaling.
Conversely to the previously proposed model, we showed that PAG is quickly phosphorylated and binds CSK and other inhibitory molecules upon T cell activation.
Analysis of PAG interactome
|Identification of new interactors
||Binding kinetics of proteins interacting with PAG
Fine characterization of phosphorylation kinetics of PAG tyrosines
We also applied a similar approach to analyze the differences between the molecular networks formed around the E3 ubiquitin–protein ligases CBL and CBLB, two negative regulators of the TCR.
We monitored the dynamics of the CBL and CBLB signaling complexes that assemble in T cells following TCR stimulation, and exploit correlations in protein association with CBL and CBLB as a function of time 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 CBLBmediated ubiquitylation following TCR engagement.
Analysis of Cbl and Cbl interactomes
Analysis of Themis1 interactome
MS-based analysis of Themis1 interactome in thymocytes
|Evaluation of absolute protein partners abundance|
Reginald et al (2015) Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells. Journal of immunology 195, 5472-5481.
Voisinne et al (2016) Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation. Mol Syst Biol 12, 876.
Zvezdova et al (2016) Themis1 enhances T cell receptor signaling during thymocyte development by promoting Vav1 activity and Grb2 stability. Sci Signal 9, ra51.