SFB 854, TP19: Regulation of the Src-family kinase Lck by posttranslational modification and TCR/Lck interactions

The Src family kinase (SFK) Lck is crucial for T cell receptor (TCR)-mediated signaling. Lck’s activity is regulated via phosphorylation of tyrosine residues Y394 and Y505, which also regulate the conformation of Lck. Taking advantage of sophisticated FLIM/FRET measurements and biochemical analyses we have shown that de novo phosphorylation of Lck-Y394 upon TCR engagement is mandatory to induce T cell activation. Moreover, constitutively active/open Lck (a Y505F mutant) only activates T cells if the TCR is simultaneously engaged by antigen. A major goal of this proposal is to understand how the TCR and Lck together orchestrate the activation of membrane proximal T cell signaling employing novel biochemical, cellular and mouse models.

Beyond Y505 and Y394, Lck possesses additional amino acids which are involved in the regulation of its activity. However, the function of these sites for TCR-mediated signaling and T cell activation is not understood. Recently we obtained knock-in mice expressing Y192F and Y192E mutants of Lck. We show that the Y192E mutation severely alters thymic development of T cells. The in depth analysis of the Y192E mouse and the functional/biochemical characterization of Lck-Y195E is an additional goal of our proposal. We have also shown that conserved cysteines (in particular C476) play a role in the regulation of Lck. A further goal is thus to investigate the functional role of these residues in T cells. We recently obtained a knock-in mouse expressing a C476A mutant Lck, which we will phenotypically and functionally characterize during the 3^rd funding period of CRC854. Altogether we expect that our project will shed new light into the long lasting question how the function of Lck is regulated by posttranslational modifications. We believe that a deeper molecular understanding of the TCR-Lck interplay leading to ITAM phosphorylation might open new perspectives to modulate T cell activation in auto-immune diseases and/or to construct better chimeric antigen receptors (CARs) for cancer immunotherapy.