Global analysis of dynamic changes in lipid raft proteins during T-cell activation
Global analysis of dynamic changes in lipid raft proteins during T-cell activation
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DOI：
10.1002/elps.200600675
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发表时间：
2007-06-01
期刊：
影响因子：
2.9
通讯作者：
Hattori, Seisuke
中科院分区：
文献类型：
--
作者：
Kobayashi, Michimoto;Katagiri, Takuya;Hattori, Seisuke
脂筏被认为是质膜内具有不同于周围膜的独特脂质组成的特殊微域。在t细胞受体（TCR）刺激后，脂筏聚集在t细胞/抗原呈递细胞（APC）接触位点，即免疫突触，其内部小叶作为下游信号成分的激活或对接位点。为了了解脂筏中发生的信号事件，我们使用二维荧光差凝胶电泳技术全面分析了TCR/CD28共刺激过程中脂筏蛋白的动态变化。我们发现了多个共刺激后强度增强的点，并通过PMF鉴定了这些点上的蛋白质。鉴定的蛋白包括Src家族酪氨酸激酶、酪氨酸磷酸酶、磷脂酰肌醇3-激酶（pi3 -激酶）、肌动蛋白结合蛋白和小gtp酶的调节因子。特别有趣的是，许多pleckstrin同源（PH）结构域的蛋白质被鉴定出来。生化和组织化学分析证实了这些蛋白质从细胞质溶胶到脂筏的易位。我们还证明了这些蛋白质在t细胞/APC界面组装。这些结果表明我们的系统在细胞外刺激下系统分析脂筏蛋白动力学的有效性。
Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.