REGULATION OF CA PUMP BY THE PROTEIN KINASE PATHWAY

蛋白激酶途径对 CA 泵的调节

• 批准号: 2750139
• 负责人: JOHN T PENNISTON
• 金额: $ 6.37万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2750139
• 关键词: active sites; antibody formation; calcium transporting ATPase; calmodulin dependent protein kinase; cell line; chimeric proteins; enzyme activity; laboratory rabbit; mutant; phosphorylation; point mutation; protein isoforms; protein kinase A; protein kinase C

DESCRIPTION: This is a study of the protein kinase pathway by which extracellular agonists regulate the plasma membrane Ca2+ pump. The Ca2+ pump exists in the plasma membrane of heart, smooth muscle, kidney, brain, intestinal epithelium, erythrocyte and many other cell types. It is involved in control of intracellular Ca2+ levels in cells of all kinds and in movement of Ca2+ across epithelial cell layers such as the intestinal and kidney tubule epithelia. Studies on the phosphorylation of plasma membrane Ca2+ pump by protein kinases will be combined with studies on the effect of such phosphorylation on the activity of the plasma membrane Ca2+ pump. Information about the effect of protein kinases on this pump will provide an essential link in determining how agonists influence intracellular function by means of the Ca2+ signal. The pump consists of a single polypeptide chain of molecular weight about 135,000 (the exact molecular weight depends on the isoform). We have succeeded in expressing this pump in COS cells and have developed an assay system for determining activity of the expressed enzyme. We propose studies designed to determine the exact locus and nature of the control of the pump s activities by protein kinases. We will use truncated mutants and point mutants of the pump to determine the exact site of phosphorylation and will carry out activity assays under conditions almost identical to those of phosphorylation to determine how the phosphorylation at a given point activates the enzyme. We will raise antibodies against the phosphoenzymes, which should be very useful in determining the role of phosphorylation after stimulation of specific kinds of cells.

描述：这是一项对蛋白激酶途径的研究，通过该途径 细胞外激动剂调节质膜 Ca2+ 泵。 钙2+ 泵存在于心脏、平滑肌、肾、脑、 肠上皮、红细胞和许多其他细胞类型。 这是 参与控制各种细胞的细胞内 Ca2+ 水平 Ca2+ 穿过上皮细胞层（例如肠道和细胞）的运动 肾小管上皮。 质膜磷酸化的研究 蛋白激酶的 Ca2+ 泵将与以下因素的影响研究相结合： 这种磷酸化对质膜Ca2+泵的活性的影响。 有关蛋白激酶对该泵的影响的信息将提供 确定激动剂如何影响细胞内功能的重要环节 通过 Ca2+ 信号。 该泵由分子量约为 135,000（确切的分子量取决于异构体）。 我们有 成功在 COS 细胞中表达该泵并开发了一种检测方法 用于测定表达酶活性的系统。 我们提出研究 旨在确定泵控制的精确轨迹和性质 蛋白激酶的活性。 我们将使用截短的突变体和点 泵的突变体以确定磷酸化的确切位点并将 在几乎相同的条件下进行活性测定 磷酸化以确定给定点的磷酸化程度 激活酶。 我们将产生针对磷酸酶的抗体， 这对于确定磷酸化后的作用非常有用 刺激特定种类的细胞。