A functional role of lipid rafts in TCR-mediated signal transduction

最近的研究表明，脂筏在TCR信号转导中起着重要作用。当TCR结合时，各种信号分子被显示在筏子中被富含。尽管参与TCR信号转导的分子在T细胞刺激后迁移到RAFT，这一事实有力地表明了RAFT在TCR信号转导中的功能作用，但仍有可能分子迁移到RAFT而不是信号转导。为了验证木筏在TCR信号转导中的必要性，进行了用甲基-b-环糊精等试剂破坏木筏结构的实验。众所周知，这种试剂可以选择性地从细胞膜中提取胆固醇。结果表明，这些试剂对RAFT结构的破坏损害了早期的TCR信号事件，表明RAFT作为TCR信号转导的功能区段。然而，由于胆固醇也是细胞膜的非RAFT区域的一种成分，因此用…治疗细胞更重要的是，这些试剂可能引起了与RAFT结构变化无关的淋巴细胞功能的破坏。在本研究中，我们开发了一种将激活的磷酸酶靶向RAFT的实验方法，并分析了随后对TCR信号转导的影响。SHP-1是一种具有两个SH2结构域的磷酸酶，主要在造血细胞中表达，通常被认为是细胞信号的负调控因子。我们已经构建了一个嵌合分子，它将SHP-1的激活形式定位到筏子上。当活化的SHP-1定位于RAFT时，它能抑制TCR介导的T细胞活化，而活化的SHP-1的质膜靶向不能诱导这种抑制。这些结果清楚地表明，RAFT中受调控的信号分子的磷酸化对于TCR信号的进展是不可或缺的。我们的结果还表明，针对激活的SHP-1的RAFT靶向损害了LAT的激活和随后的下游信号事件，而不影响TCRzeta和ZAP-70的酪氨酸磷酸化以及Lck的激酶活性等近端步骤。此外，我们观察到内源性SHP-1在TCR参与后募集到RAFT并与LAT相关，提示SHP-1参与RAFT介导的T细胞激活。较少

Recent studies suggest a functional role for lipid rafts in TCR signaling. Upon TCR engagement, various signaling molecules are shown to be enriched in rafts. Although the fact that molecules involved in TCR signal transduction migrate to rafts following T cell stimulation strongly suggests a functional role of rafts in TCR signaling, it is still possible that the molecules migrate to rafts for purposes other than signal transduction. To test the necessity of rafts in TCR signal transduction, experiments in which the structure of rafts is destroyed by reagents such as methyl-b-cyclodextrin have been performed. This reagent is known to selectively extract cholesterol from cell membranes. It was shown that disruption of raft structure with these reagents impairs early TCR signaling events, indicating that rafts act as functional compartments for the transduction of the TCR signal. However, since cholesterol is also a component of non-raft areas of cell membranes, treatment of cells with … More these reagents may have caused disruptions in lymphocyte function that were unrelated to the changes rendered to raft structure.In this study, we have developed an experimental method where activated phosphatase is targeted to rafts and the subsequent effect on TCR signal transduction is analyzed. SHP-1, a phosphatase with two SH2 domains, is expressed mainly in haematopoietic cells and is generally regarded as a negative regulator of cell signaling. We have constructed a chimeric molecule that localizes the activated form of SHP-1 to rafts. Activated SHP-1 was able to inhibit TCR-mediated T cell activation when it was localized to rafts, whereas the plasma membrane targeting of activated SHP-1 did not induce this inhibition. These results clearly demonstrate that regulated phosphorylation of signaling molecules in rafts is indispensable for progression of the TCR signal. Our results also indicate that raft targeting of activated SHP-1 impairs LAT activation and subsequent downstream signaling events without affecting proximal steps such as tyrosine phosphorylation of TCRzeta and ZAP-70, and the kinase activity of Lck. Moreover, we observed that endogenous SHP-1 recruited to rafts and associated with LAT after TCR engagement, suggesting that SHP-1 is involved in raft-mediated T cell activation. Less