Functional Analysis of Tyrosinekinase in B Cells with Fluorescent Molecular Rotars

荧光分子旋转体 B 细胞酪氨酸激酶的功能分析

• 批准号: 08672483
• 负责人: NOJI Masahide
• 金额: $ 1.02万
• 依托单位: Suzuka University of Medical Science and Technology (1997)Nagoya City University (1996)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672483/
• 关键词: tyrosinekinase; phosphorylated peptides; monoclonal antibody; ITAM; Image analysis; リン酸化チロシン; IgEレセプター; チロシンキナーゼ; 可視化解析; RBL-2H3

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.

大鼠嗜碱性白血病细胞(RBL-2H3)的IgE受体(Fc Epsilon RI)在β和γ亚基中含有一个共同的序列，称为基于免疫受体酪氨酸的激活基序(ITAM)。ITAM中存在两种在信号转导通路中起重要作用的磷酸化酪氨酸，在本研究中，我们合成了大鼠IgE受体β和γ亚基的酪氨酸磷酸化ITAM多肽，即β亚基的(DRLYEELNHVYSPIYSELC)和伽马亚基的(DAVYTGLNTRNQETYETLC)，其中Y代表磷酸化酪氨酸。这些磷酸化多肽是用Fmoc-Tyr-(PO_3Me_2)固相Fmoc化学方法合成的。[RBL-2H3细胞中ITAM的图像分析]在获得的抗磷酸化酪氨酸多肽的单抗中，J-59和J-69单抗识别细胞抗原刺激后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得更强。在这一结果的鼓舞下，通过分析共聚焦荧光显微镜获得的图像，追踪了刺激后J-69识别膜蛋白的时间过程。刺激后5分钟和15分钟，J-69更多地识别细胞膜附近的ITAM。然而，在1小时和2小时后，荧光强度显著下降，表明抗体从膜蛋白中去除。这一现象可以通过考虑磷酸酶对伽马亚基的去磷酸化以及由此产生的J-69去除来解释。