Quantitative phosphoproteomics reveals novel Vav1-dependent pathways in T cell receptor signaling (IRM10P.736)

Abstract

Abstract Vav1, a Rac/Rho guanine nucleotide exchange factor, is a critical component of the T cell receptor (TCR) signaling cascade and is rapidly tyrosine phosphorylated in response to T cell activation. Previous studies using Vav1 knockout models have determined its importance in TCR-dependent signaling. However, a wide-scale characterization of Vav1-dependent phosphorylation events is still lacking. Using quantitative mass spectrometry, a phosphoproteomic analysis of 668 tyrosine phosphorylation sites was performed on Vav1-deficient T cells across four time points of TCR stimulation. Compared to controls, cells lacking Vav1 expression showed statistically significant decreases in phosphorylation on actin cytoskeletal signaling proteins, supporting the role of Vav1 in cytoskeletal reorganization. Interestingly, loss of Vav1 led to perturbations in phosphorylation on signaling molecules proximal to the TCR. Furthermore, proteins implicated in negative feedback pathways, including CBL and PAG, showed altered phosphorylation profiles. Effects further downstream of the TCR were also observed in Vav1-deficient cells, notably, hyperphosphorylation of inhibitory sites on various cyclin dependent kinases, suggesting cell cycle arrest. This data demonstrates novel signaling networks regulated by Vav1 and support the pleiotropic functions of Vav1 downstream of the TCR.