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NETWORK OF CALCIUM-BINDING PROTEINS IN THE SIGNAL CASCADE

信号级联中的钙结合蛋白网络

基本信息

批准号：
07670103
负责人：
NIKI Ichiro
金额：
$ 1.6万
依托单位：
NAGOYA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670103/
关键词：
CALCIUM-BINDING PROTEINS CALCIUM-SIGNALLING HORMONE SECRETION PANCREATIC B-CELL INSULIN DIABETES MELLITUS CALMODULIN CALCYCLIN

项目摘要

We have obtained new findings on the roles of calcium-binding proteins in the secretory events as described below.Secretion studies from permeabilized pancreatic B-cells suggested that calmodulin and calmodulin-dependent protein phosphorylation modulate Ca^<2+>-dependent insulin secretion via acting on a proximal step in the secretory cascade. Furthermore, observation of the intracellular movement of secretory granules in the living pancreatic B-cell under a phase-contrast microscope revealed that myosin light chain kinase and CaM kinase II,two calmodulin-dependent protein kinases, are involved in the control of the insulin granules via phosphorylating their common substrate, myosin regulatory light chain. Visualization of the granules in the living B-cell enabled us to obtain direct evidence that activation of the granule movement results in recruitment of the granules to a ready-release zone in the B-cell. We demonstrated that these two kinases are critical members which transduce ca … More lcium-signalling in the secretory events and that they play distinct roles in the control of the movement dependently on the concomitant Ca^<2+> concentrations. Calcyclin, was another calcium-binding proteins found to participate in the control of insulin secretion by ourselves. We obtained purified materials of rodent calcyclin by the expression system in E.Coli. We are now attempting to identify new calcyclin-binding proteins which react with calcyclin in the pancreatic B-cell line in a Ca^<2+>-dependent manner. We also selective antibodies against the expressed calcyclin and demonstrated that this protein is dominantly distributed in the cytoplasm by immunohistochemical and immunoblotting approaches. Since the roles of calcyclin will be better understood if we can visualize the localization of this calcyclin-binding protein in a living cell, we are now attempting to let insulinoma cells express the fusion protein between calcyclin and green fluorescent protein as a fluorescent marker. Less

我们在钙结合蛋白在分泌事件中的作用方面取得了新的发现，如下所述。来自渗透性胰腺B细胞的秘密研究表明，钙调蛋白和钙调蛋白依赖的蛋白磷酸化通过作用于分泌级联中的近端步骤来调节钙依赖的胰岛素分泌。此外，在相差显微镜下观察活的胰腺B细胞分泌颗粒在细胞内的运动情况表明，肌球蛋白轻链激酶和CaM激酶II这两种钙调蛋白依赖的蛋白激酶通过磷酸化它们共同的底物肌球蛋白调节轻链参与胰岛素颗粒的调控。活的B细胞中颗粒的可视化使我们能够获得直接的证据，即颗粒运动的激活导致颗粒重新聚集到B细胞中的准备释放区。我们证明这两个激酶是转导ca-…的关键成员。在分泌活动中有更多的钙信号，它们在运动控制中发挥着明显的作用，这取决于伴随的钙离子浓度。Calcyin是我们自己发现的另一种参与胰岛素分泌调控的钙结合蛋白。通过在E.Coli中的表达系统获得了啮齿动物钙周期蛋白的纯化材料。我们现在正试图鉴定新的钙周期蛋白结合蛋白，它与胰腺B细胞系中的钙周期蛋白以钙依赖的方式发生反应。我们还选择了针对表达的钙周期蛋白的抗体，并通过免疫组织化学和免疫印迹方法证明该蛋白主要分布在细胞质中。由于如果我们能够可视化这种钙周期蛋白结合蛋白在活细胞中的定位，将更好地了解钙周期蛋白的作用，我们现在正尝试让胰岛素瘤细胞表达钙周期蛋白和绿色荧光蛋白之间的融合蛋白作为荧光标记物。较少