The Role of Tyrosine Kinase Emt/itk in T-cell Signaling
Description:... Expressed in Mast and T-cells/IL-2 Inducible Tyrosine Kinase (Emt/Itk) is a protein tyrosine kinase required for T-cell antigen receptor (TCR)-induced activation and development. A physical interaction between Emt/Itk and the TCR has not been previously described and the mechanism through which Emt/Itk mediates its effector functions is poorly understood. Here, I have studied the role of Emt/Itk in T-cell antigen receptor (TCR) signal transduction by tracking its localization in response to stimulation through the TCR. Furthermore, I have investigated the mechanism of Emt/Itk localization and activation via interactions with the adapter protein LAT. I have utilized laser-scanning confocal microscopy to demonstrate that antibody-mediated engagement of the CD3[epislon]; chain induces the co-localization of Emt/Itk with TCR/CD3 at the plasma membrane. Removal of the Emt/Itk Pleckstrin Homology domain ([delta]PH-Emt/Itk) abrogates the association of the kinase with the cell membrane, as well as its activation-induced co-localization with the TCR complex and subsequent tyrosine phosphorylation. The addition of a membrane localization sequence from Lck to [delta]PH-Emt/Itk restores its co-localization with the TCR but not its activation-induced tyrosine phosphorylation. The data suggest that the PH domain of Emt/Itk can be replaced with another membrane-localization signal without affecting the membrane targeting and activation-induced colocalization of the kinase with the TCR. However, the PH domain is indispensable for the activation-induced tyrosine phosphorylation of the kinase. I also found that the Emt/Itk SH2 domain is critical for transphosphorylation and activation of Emt/Itk catalytic activity. I show that the Emt/Itk SH2 domain is essential for Emt/Itk to form TCR/CD3 inducible Emt/Itk-LAT complexes whereas the SH3 domain and catalytic activity are not required. I also demonstrate that Emt/Itk-LAT complexes are biologically important because Jurkat T-cells with deficient LAT expression (JCaM2) fail to increase Emt/Itk tyrosine phosphorylation upon TCR/CD3 stimulation. Furthermore, I confirm the stimulation dependent clustering of Emt/Itk and LAT with the TCR complex using confocal microscopy. The present data indicate that the Ernt/Itk SH2 domain mediates the formation of a complex with LAT at the TCR which is essential for Emt/Itk activation and function.
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