NETWORK OF CALCIUM-BINDING PROTEINS IN THE SIGNAL CASCADE

我们对钙结合蛋白在分泌过程中的作用有了新的发现，如下所述。通透性胰腺b细胞的分泌研究表明，钙调蛋白和钙调蛋白依赖蛋白磷酸化通过作用于分泌级联的近端步骤来调节Ca^<2+>依赖胰岛素的分泌。此外，在相差显微镜下观察活胰腺b细胞分泌颗粒的胞内运动，发现肌球蛋白轻链激酶和CaM激酶II，两种钙调素依赖性蛋白激酶，通过磷酸化它们的共同底物肌球蛋白调节轻链参与胰岛素颗粒的控制。对活b细胞中颗粒的可视化使我们能够获得直接证据，证明颗粒运动的激活导致颗粒被募集到b细胞的预释放区。我们证明了这两种激酶是在分泌过程中传导钙信号的关键成员，并且它们在依赖于伴随的ca ^<2+>浓度的运动控制中发挥着不同的作用。钙调素是另一种钙结合蛋白，被发现参与控制胰岛素分泌。我们通过大肠杆菌表达系统获得了鼠钙调素的纯化材料。我们现在正试图鉴定新的钙调素结合蛋白，这些蛋白在胰腺b细胞系中以Ca^<2+>依赖的方式与钙调素发生反应。我们还通过免疫组织化学和免疫印迹方法选择了针对表达的钙调素的抗体，并证明该蛋白主要分布在细胞质中。由于钙调素的作用将更好地理解，如果我们可以可视化钙调素结合蛋白在活细胞中的定位，我们现在正试图让胰岛素瘤细胞表达钙调素和绿色荧光蛋白之间的融合蛋白作为荧光标记。少

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