Tyrosine Phosphorylation of Themis Induced by TCR and Cytokine Signaling Is Regulated by Multiple Src Family and Janus Family Kinases 2253039

Abstract

Abstract Introduction TCR and cytokine signaling play critical roles in thymocyte development and peripheral T cell homeostasis. Themis is a key regulator of signaling pathways in both developing thymocytes and mature T cells. Mice deficient in Themis fail to produce mature thymocytes due to defective positive selection. Previous studies have shown that tyrosine phosphorylation of Themis at Y34, mediated by the kinase Lck and regulating interaction with the phosphatase Shp1, is essential for its role in modulating TCR signaling during CD4 SP thymocyte development. However, the mechanism by which Themis regulates cytokine signaling, particularly in CD8 SP thymocytes and peripheral CD8+ T cells, remain poorly understood. Methods To identify kinases that regulate Themis phosphorylation, we cloned potential kinases expressed in T cells and co-expressed them with Themis in 293T. To determine which tyrosine residues are targeted by these kinases, we individually mutated all 18 tyrosine sites of Themis (Y-F) and assessed the impact of each mutation on kinase-dependent phosphorylation. Furthermore, to examine the intracellular localization and trafficking of Themis following TCR and/or cytokine stimulation, we generated a series of Themis—EGFP fusion proteins. Results In addition to Lck, Themis was phosphorylated by several other Src family kinases and members of the Janus kinase family. We have successfully generated a set of Themis Y-to-F mutants and are currently mapping which tyrosine residues are regulated by these kinases. Moreover, we constructed seven Themis—EGFP fusion variants, inserting EGFP at different positions within Themis. Most of these fusion proteins retained functional activity, as evidenced by their constitutive binding to Grb2, which is a characteristic feature of Themis. Conclusion Themis is regulated by multiple kinases, including members of the Src and Janus kinase families, suggesting that Themis integrates signals from both TCR and cytokine pathways to coordinate T cell development and homeostasis. Funding Source N/A Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)