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

最近的研究表明，脂筏在TCR信号转导中的功能作用。在TCR接合后，各种信号传导分子显示出在筏中富集。尽管参与TCR信号转导的分子在T细胞刺激后迁移到筏的事实强烈表明筏在TCR信号转导中的功能作用，但分子迁移到筏仍有可能用于信号转导以外的目的。为了测试筏在TCR信号转导中的必要性，已经进行了实验，其中筏的结构被诸如甲基-b-环糊精的试剂破坏。已知该试剂选择性地从细胞膜提取胆固醇。结果表明，用这些试剂破坏筏结构会损害早期TCR信号传导事件，表明筏充当TCR信号转导的功能区室。然而，由于胆固醇也是细胞膜的非筏区域的组分，因此用胆固醇处理细胞是不可能的。 关于我们 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于筏，并分析其对TCR信号转导的后续影响。SHP-1是一种具有两个SH 2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节因子。我们已经构建了一个嵌合分子，将激活形式的SHP-1定位到筏上。激活的SHP-1能够抑制TCR介导的T细胞活化时，它被定位到筏，而质膜靶向激活的SHP-1不诱导这种抑制。这些结果清楚地表明，筏中信号分子的调节磷酸化对于TCR信号的进展是必不可少的。我们的研究结果还表明，筏靶向活化的SHP-1损害LAT激活和随后的下游信号传导事件，而不影响近端步骤，如TCRzeta和ZAP-70的酪氨酸磷酸化，以及Lck的激酶活性。此外，我们观察到内源性SHP-1募集筏和相关的LAT后TCR接合，表明SHP-1参与筏介导的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