Mechanism Analysis Of Itk Molecule On The Activation Of T Lymphocyte

Protein tyrosine kinases (PTKs) of the Src, Syk/ZAP-70 and Tec are crucial for antigen-receptor-induced lymphocyte activation. Itk is one of the Tec family non-receptor tyrosine kinases, its tyrosine phosphorylation and interaction between Itk and other signal molecules play a crucial role in regulating the T-cell receptor signaling transduction pathways. However, the current study is not enough to clarify the mechanisms. So our work focuses on the influence of Itk in the T-cell receptor signal pathway.In the first chapter, to explore the possibility influence of Itk mutants on the signaling pathway, seven mutants (W208K, T279D, I282A, C288A, Y292A, V330A, R332K) were generated. Co-immunoprecipitation assay verified that those mutants could interact with SLP76, three mutants (W208K, T279D, Y292A) significantly disrupted the interaction between Itk and SLP76. Interestingly, we observed that Itk induced PLC-yl phosphorylation was independent on SLP76 in 293T cells, four Itk mutants (W208K, T279D, C288A, Y292A) drastically diminished the tyrosine phosphorylation of PLC-γ1 at Y775 and Y783 by western blot assay.Influenza A virus can cause a highly contagious acute respiratory illness and is still a major cause of human infectious diseases among the viruses. Influenza virus-induced signaling involves a complex network of different overlapping molecules and signal cascade. Whether this signal pathway in Jurkat cells is affected by influenza virus infection was investigated here. In the second chapter, we observed that Jurkat cells can be infected directly by influenza virus and Itk translocated from the cytoplasma to membrane in influenza virus-infected Jurkat cells. Strikingly, cellular localization of PLC-yl was not changed, but phosphorylation levels of PLC-γ1 at Y775 and Y783 increased clearly during influenza A virus infection. Moreover, a stable Itk-deficient Jurkat cell line was generated. We observed that in this cell line the phosphorylation of PLC-yl at Y783 was down-regulated during the infection with influenza virus, indicating that phosphorylation of PLC-yl at Y783 is Itk dependent in influenza virus-infected Jurkat cells.Taken all together, these results suggest that influenza virus infection may affect the activation of T cell through the Itk/PLC-yl signaling pathway. Furthermore, the research of association among Itk,SLP76 and PLC-yl will help us to understand the molecular mechanism for activation of T-cell.