Functional Analysis of Tyrosinekinase in B Cells with Fluorescent Molecular Rotars

大鼠嗜碱性白血病细胞（RBL-2H3）的IgE受体（Fc epsilon RI）在β亚基和γ亚基中包含一个被称为免疫受体酪氨酸激活基序（ITAM）的一致序列。在ITAM中存在两种磷酸酪氨酸，它们在信号转导级联中起重要作用。在本研究中，我们合成了大鼠IgE受体β亚基和γ亚基的酪氨酸磷酸化ITAM肽，分别为（DRLYEELNHVYSPIYSELC）和（DAVYTGLNTRNQETYETLC），其中Y代表磷酸化的酪氨酸。这些磷酸化肽以Fmoc- tyr -(PO_3Me_2)为原料，采用固相Fmoc化学合成。[rbr - 2h3细胞ITAM图像分析]在获得的针对含磷酸化酪氨酸肽的单克隆抗体中，J-59和J-69单克隆抗体在细胞抗原刺激后识别存在于细胞膜和细胞质附近的蛋白质，特别是后者与存在于细胞膜附近的蛋白质结合更强。受此结果的鼓舞，通过分析共聚焦荧光显微镜获得的图像来追踪刺激后J-69对膜蛋白识别的时间过程。在刺激后5分钟和15分钟，J-69更优先识别靠近细胞膜的ITAM。然而，在1小时和2小时后，荧光强度明显下降，表明抗体从膜蛋白中去除。这种现象可以通过考虑到磷酸酶对γ亚基的去磷酸化和由此产生的J-69去除来解释。

The IgE receptors (Fc epsilon RI) of rat basophilic leukemia cells (RBL-2H3) contain a consensus sequence termed immunoreceptor tyrosine-based activation motif (ITAM) in beta and gamma subunits. In ITAM there exist two phosphotyrosines, which play an important role in signal transduction cascade.In this study, we have synthesized tyrosine phosphorylated ITAM peptides for beta and gamma subunits in rat IgE receptors, that is, (DRLYEELNHVYSPIYSELC) and (DAVYTGLNTRNQETYETLC) for beta and gamma subunits, respectively, where Y represents phosphorylated tyrosine. These phosphorylated peptides were synthesized by solid-phase Fmoc chemistry by using Fmoc-Tyr-(PO_3Me_2).[Image Analysis of ITAM in RBL-2H3 Cells]Among the obtained monoclonal antibodies against the peptides containing phosphorylated tyrosines, J-59 and J-69 monoclonal antibodies recognized proteins existing near cell membranes and cytoplasm after the antigen stimulation of the cells, especially the later one binds more strongly to proteins existing near cell membranes than the former. Encouraged by this result, time course of J-69 recognition for the membrane proteins after the stimulation was pursued by analyzing images obtained by a confocal fluorescence microscopy. In 5 and 15minutes after the stimulation J-69 recognized more preferentially ITAM near cell membranes. However, the fluorescence intensity decreased remarkably in 1 hr and 2 hr later, indicating the removal of antibodies from the membrane protein. This phenomenon can be explained by taking account of dephosphorylation of the gamma subunits by phosphatase with the resultant J-69 removal.