Kane Lawrence P, Watkins Simon C
Department of Immunology, BST E-1056, University of Pittsburgh, Pittsburgh, PA 15261, USA.
J Biol Chem. 2005 Jun 10;280(23):21949-54. doi: 10.1074/jbc.M412913200. Epub 2005 Apr 6.
Tec family tyrosine kinases are key regulators of lymphocyte activation and effector function. Several Tec family kinases (Tec, Itk, Rlk/Txk) are expressed in T cells, but it is still not clear to what degree these are redundant or have unique functions. We recently demonstrated that Tec alone, among the Tec kinase family members examined, can induce nuclear factor of activated T cell-dependent transcription. This unique functional characteristic correlated with a unique pattern of subcellular localization, as Tec (but not other family members) was found in small vesicles, the appearance of which requires signaling through the T cell receptor for antigen. Here we report on our studies of these Tec-containing structures in live T cells, using total internal reflection fluorescence microscopy. With this technique, we showed that, in live T cells, the Tec vesicles are located at the plasma membrane, the vesicles are unique to Tec (and not the related kinase Itk), and their formation and maintenance require T cell receptor signaling through Src family kinases and PI 3-kinase. Finally, we have imaged isolated T cell membranes by confocal microscopy, confirming the membrane-proximal location of Tec vesicles, as well as demonstrating overlap of these vesicles with the tyrosine kinase Lck, the Tec substrate PLC-gamma1, and the early endosomal antigen 1 marker EEA1.
Tec家族酪氨酸激酶是淋巴细胞活化和效应器功能的关键调节因子。几种Tec家族激酶（Tec、Itk、Rlk/Txk）在T细胞中表达，但这些激酶在何种程度上功能冗余或具有独特功能仍不清楚。我们最近证明，在所检测的Tec激酶家族成员中，仅Tec就能诱导活化T细胞核因子依赖性转录。这种独特的功能特性与独特的亚细胞定位模式相关，因为Tec（而非其他家族成员）存在于小囊泡中，这些小囊泡的出现需要通过T细胞抗原受体进行信号传导。在此，我们报告利用全内反射荧光显微镜对活T细胞中这些含Tec结构的研究。通过这项技术，我们表明，在活T细胞中，Tec囊泡位于质膜上，这些囊泡是Tec特有的（而非相关激酶Itk），其形成和维持需要通过Src家族激酶和PI 3激酶进行T细胞受体信号传导。最后，我们通过共聚焦显微镜对分离的T细胞膜进行成像，证实了Tec囊泡位于膜近端，同时也证明了这些囊泡与酪氨酸激酶Lck、Tec底物PLC-γ1以及早期内体抗原1标记物EEA1存在重叠。
