A quantitative proteomics approach to the Lck interactome

Despite years of ongoing research in the field of T cell signaling, a satisfactory answer to the central question of how TCR ligation engages the key tyrosine kinase Lck to lead to T cell activation remains fragmentary, if not confused. The post-translational modifications to which Lck is subjected and their significance for activating and/or regulating the TCR signaling cascade are poorly understood. Thus, for instance, in contrast to previous believes, it has become increasingly clear that acute tail deposphorylation of Lck is not playing a dominant role in TCR triggered-activation. Rather, domain displacement by adapter proteins may be a potent stimulus for Lck activity physically funneling TCR-engagement to Lck activation. These findings may allow a paradigm shift in the field of Lck research and highlight the need for a global and unbiased reexamination of the Lck regulatory module. The ambition of the proposed project is to provide an exhaustive and quantitative description of the dynamic Lck phosphoproteome and interactome during TCR-engagement and thus set the basis for a novel view of regulatory events that govern TCR signaling thresholds. Such analyses have not been possible until relatively recently, due to the lack of sensitive instrumentation. We will use advanced mass spectrometry-based proteomics to generate a time-resolved fine map of Lck phosphosites. Relevant, regulated phoshosites will be characterized in depth by biochemical and functional approaches. In the second phase of the project, we will define the dynamic Lck interactome. This goal will be achieved using a high-affinity epitope tag that should allow minimizing handling times and thus preserving also unstable complexes. We will use experimental conditions of physiological TCR- engagement that allow modulating signal strength and quality by altered peptide ligands. Individual interaction partners will be assigned to the different Lck subdomains and alternative entry points will be epitope-tagged to refine and enlarge the Lck regulatory grid. We hope to provide the first quantitative description of the Lck- signaling network in T cells amenable to modeling, which could represent a valuable contribution for our understanding of T cell biology and Src kinase mechanisms of action. Newly identified regulatory mechanisms could represent valuable targets also for therapeutic intervention.