The kinase Lck phosphorylates the T cell antigen receptor (TCR) complex when antigen is encountered and therefore its activity sets a critical threshold for T cell activation. Yet how Lck activity is regulated in resting or stimulated cells remains poorly understood. We have identified that a cryptic phosphosite within Lck, Y192, profoundly affects Lck activity when mutated. Notably, mutation of Y192 blocks critical TCR proximal signaling events including phosphorylation of the effector kinase Zap70 and subsequent calcium response. Accordingly, thymocyte development in retrogenic mice harboring a Y192 mutation is severely impaired. These mice are unable to produce mature T cells and accumulate double negative and double positive (CD4/CD8) thymocytes. We determined that these defects are caused by hyperphosphorylation of the canonical inhibitory phosphorylation site (Y505) within Lck's C-terminus. Lck's C-terminus is phosphorylated by the inhibitory kinase Csk and we have employed a regulatable reconstituted system to acutely inhibit Csk activity. Upon Csk inhibition, the phosphatase CD45 dephosphorylates Lck's C-terminus (Y505). Interestingly, mutation of Y192 disrupts CD45's ability to dephosphorylate Y505 and activate Lck. Because Y192 phosphorylation decreases in response to small molecule inhibition of Zap70, an Lck substrate, our findings support a mechanism of negative feedback. We predict that tonic or basal signaling events that occur in the absence of TCR ligand use negative feedback, including Y192 phosphorylation, to tune Lck activity. Because modulation of Lck activity impacts TCR sensitivity, feedback mechanisms are likely critical to establishing a threshold for T cell activation.
Citation Format: Adam H. Courtney, Theresa A. Kadlecek, Byron Au-Yeung, Arthur Weiss. A Zap70-dependent negative feedback loop governs Lck activation by CD45 [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A074.
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