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

最近的研究表明脂筏在TCR信号传导中的功能作用。在TCR参与后，各种信号分子被证明在筏中富集。尽管参与TCR信号转导的分子在T细胞刺激后迁移到筏上这一事实强烈表明筏在TCR信号转导中的功能作用，但仍有可能分子迁移到筏上的目的并非信号转导。为了验证筏在TCR信号转导中的必要性，我们进行了用甲基-b-环糊精等试剂破坏筏结构的实验。这种试剂可以选择性地从细胞膜中提取胆固醇。研究表明，这些试剂破坏筏结构会损害早期TCR信号转导事件，表明筏作为TCR信号转导的功能区室。然而，由于胆固醇也是细胞膜非筏区的一个组成部分，用这些试剂处理细胞可能会导致淋巴细胞功能的破坏，而这些破坏与筏结构的变化无关。在本研究中，我们开发了一种实验方法，将活化的磷酸酶靶向筏，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传导的负调控因子。我们已经构建了一个嵌合分子，将活化形式的SHP-1定位到筏上。激活的SHP-1能够抑制tcr介导的T细胞激活，当它定位在筏上时，而激活的SHP-1的质膜靶向并没有诱导这种抑制。这些结果清楚地表明，筏中信号分子的磷酸化调控对于TCR信号的进展是必不可少的。我们的研究结果还表明，raft靶向活化的SHP-1会损害LAT的激活和随后的下游信号事件，而不会影响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参与后被募集到筏上并与LAT相关，这表明SHP-1参与了筏介导的T细胞活化。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