Kobayashi Michimoto, Katagiri Takuya, Kosako Hidetaka, Iida Naoyuki, Hattori Seisuke
Division of Cellular Proteomics (BML), Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.
Electrophoresis. 2007 Jun;28(12):2035-43. doi: 10.1002/elps.200600675.
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.
脂筏被认为是质膜内具有独特脂质组成的特殊微区，其脂质组成不同于周围的膜。在T细胞受体（TCR）刺激后，脂筏在T细胞/抗原呈递细胞（APC）接触部位组装，该部位被称为免疫突触，其内膜作为下游信号成分的激活或停靠位点。为了了解脂筏中发生的信号事件，我们使用二维荧光差异凝胶电泳全面分析了TCR/CD28共刺激过程中脂筏蛋白的动态变化。我们检测到多个在共刺激后强度增强的斑点，并通过肽质量指纹图谱（PMF）鉴定了这些斑点中的蛋白质。鉴定出的蛋白质包括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。特别值得注意的是，鉴定出了许多含有普列克底物蛋白同源（PH）结构域的蛋白质。生化和组织化学分析证实了这些蛋白质从细胞质转移到脂筏中。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系统分析细胞外刺激过程中脂筏蛋白动态方面的有效性。
