Abstract B042: Regulation of Lck activity by a cryptic phosphosite

Abstract

Abstract Src family kinases (SFKs) are initiators of receptor-mediated signaling events, enabling cells to perceive and interact with their environment. For immune cells SFKs are especially crucial because they couple antigen recognition to intracellular signaling pathways. In T cells, the SFK Lck phosphorylates the T cell antigen receptor (TCR) complex upon antigen encounter. Because Lck phosphorylates the TCR complex, its activity sets a critical threshold for T cell activation; yet how Lck activity is regulated in resting or stimulated cells is poorly understood. Through quantitative mass spectrometry (SILAC), we recently identified a cryptic Lck phosphosite (Y192) as sensitive to inhibition of the effector kinase Zap70. Because Zap70 is an Lck substrate, and dramatic reduction in phosphorylation of Y192 occurred upon Zap70 inhibition, we reasoned this site might function to regulate Lck in response to changes in Zap70 activity. To test this hypothesis we reconstituted Lck-deficient Jurkat T cells with Lck Y192 variants. Mutation of Y192 reveals that this site has a profound impact on Lck activity. Cells reconstituted with Y192E or Y192A show a marked reduction in Lck activity, characterized by hyperphosphorylation on Lck's inhibitory C-terminus (Y505) and a reduction in Lck autophosphorylation within the activation loop (Y394). Consistent with a defect in Lck activity, cells that express Lck Y192E/A variants, when assessed for hallmarks of T cell activation, reveal striking defects in TCR signaling. Interestingly, these defects are reminiscent of CD45-deficient T cells. Preliminary findings suggest that mutation of Y192 disrupts the ability of the phosphatase CD45 to dephosphorylate Lck's inhibitory C-terminus (Y505). Because of the profound effect that Y192 modification exerts on Lck activity, and therefore TCR signaling, we believe that phosphorylation of Y192 comprises a Zap70-dependent negative feedback mechanism that tunes Lck activity. Such a mechanism predicts that increases in Zap70 activity cause phosphorylation of Y192 within Lck's SH2 domain, favouring Lck inactivation by preventing CD45-mediated dephosphorylation of the inhibitory C-terminus. Citation Format: Adam H. Courtney, Hanna E. Sjolin-Goodfellow, Qinqin Ji, Theresa A. Kadlecek, Arthur R. Salomon, Arthur Weiss. Regulation of Lck activity by a cryptic phosphosite. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B042.