Phosphoproteomic Analysis Reveals New SLP76 and PLCg1 Feedback Pathways That Regulate Lck in T Cells

Abstract

Abstract T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. Lck tyrosine kinase is the central regulator of T cell activation regulated through its phosphorylation state. The exact mechanisms that regulate the spatial equilibrium of Lck between the TCR and downstream regulators are not well defined. While some of the important regulatory proteins involved in Lck feedback loops in T cells are also beginning to be defined, the pathways leading to the activation and recruitment of these inhibitory molecules are much more obscure. Modern proteomic methods using quantitative mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways. Our recent data reveal a previously uncharacterized regulatory network where N-terminal tyrosine residues on SLP76 are involved in both positive and negative feedback loops, which regulate the phosphorylation of the activation loop on Lck. We also collected phosphoproteomic data that suggests that Lck targeting but not activity is regulated by PLCγ1. We examined the phosphoproteome of PLCγ1 deficient and reconstituted T cells through a timecourse of TCR stimulation. These data indicate that Lck activity was shifted from TCR to the SLP76 complex in the absence of PLCγ1. Altogether, wide-scale modern methods in quantitative mass spectrometry drive the molecular characterization of T cell signaling circuits and facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation and protein-protein interactions through a variety of activation states.