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STUDIES IN THE ROLE OF EZRIN IN THE CYTOSKELETAL SYSTEM IN THE PARIETAL CELL

Ezrin 在壁细胞细胞骨架系统中的作用研究

基本信息

批准号：
09672216
负责人：
URUSHIDANI Tetsuro
金额：
$ 2.3万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

The aim of the present project was to elucidate the role of cytoskeletal proteins in the secretory process using the parietal cell as a model. The actual plans were to determine the functional phosphorylation site of ezrin and to develop a model system which allows an introduction of peptides into the cell in order to make a direct analysis of the function of proteins. In the first year, human ezrin was cloned and the partial sequences were treated with PKA, and no phosphorylation was observed. However, purified rabbit ezrin was phosphorylated by PKA suggesting species difference in the sequence of ezrin. In the separate experiments, we showed that many of pharmacological probes commonly used to analyze signal transduction were useless, and suggested that the establishment of permeabilized cell system was indispensable.During the next year's research, Professor Goldenring in the University of Georgia informed us that he sequenced rabbit ezrin and found no PKA sites, namely, no species … More difference in the PKA site of ezrin. In the meantime, we obtained some data suggesting that ezrin itself is not phosphorylated by PKA, whereas gastric mucosa contains an unidentified kinase which is activated by PKA and phosphorylates C-terminal site of ezrin. This direction is different from the plan at the beginning, but it has now become much more interesting project. At present, experiments are in progress to identify this kinase. For the other project, we successfully developed a new model, the beta-escin permeabilized rabbit gastric gland, which allows the introduction of peptides into the cell. Using this system, it was shown that not only the PKA-inhibitory peptide but also the inhibitory peptide for myosin light chain kinase inhibited cAMP-stimulated acid secretory response. We could also show the possible involvement of small GTP-binding proteins in the secretory process applying functional peptides to the system. This model was thus demonstrated to be a powerful tool to analyze the interaction between cytoskeletal proteins in the parietal cell vesicular transport. Less

本项目的目的是阐明细胞骨架蛋白在分泌过程中的作用，以壁细胞为模型。实际的计划是确定ezrin的功能磷酸化位点，并开发一个模型系统，允许将肽引入细胞，以便对蛋白质的功能进行直接分析。第一年克隆了人ezrin，部分序列经PKA处理，未发现磷酸化现象。然而，纯化的兔ezrin被PKA磷酸化，表明ezrin的序列存在物种差异。在单独的实验中，我们发现了许多常用的用于分析信号转导的药理学探针是无用的，并提示了通透细胞系统的建立是必不可少的。在第二年的研究中，乔治亚大学的Goldenring教授告诉我们，他对兔ezrin进行了测序，没有发现PKA位点，即没有发现物种……ezrin的PKA位点存在更多差异。同时，我们获得的一些数据表明，ezrin本身不被PKA磷酸化，而胃黏膜中含有一种未知的激酶，该激酶被PKA激活并磷酸化ezrin的c端位点。这个方向与最初的计划不同，但现在它已经成为一个更有趣的项目。目前，鉴定这种激酶的实验正在进行中。在另一个项目中，我们成功地开发了一种新的模型，β -escin渗透兔胃腺，它可以将肽引入细胞。实验结果表明，除了pka抑制肽外，肌球蛋白轻链激酶抑制肽也能抑制camp刺激的酸分泌反应。我们还可以展示小gtp结合蛋白在分泌过程中的可能参与，将功能肽应用于系统。因此，该模型被证明是分析细胞壁细胞囊泡运输中细胞骨架蛋白之间相互作用的有力工具。少