REGULATION OF CA PUMP BY THE PROTEIN KINASE PATHWAY

描述：这是一项关于蛋白激酶途径的研究。 细胞外激动剂调节质膜钙泵。《钙离子》 泵存在于心脏、平滑肌、肾脏、脑的质膜中， 肠上皮、红细胞等多种细胞类型。它是 参与控制各种细胞的细胞内钙水平和 在钙跨上皮细胞层的移动中，如肠道和 肾小管上皮细胞。质膜磷酸化的研究 将结合蛋白激酶对CA2泵的作用进行研究 这种磷酸化对质膜钙泵的活性有影响。 有关蛋白激酶对这个泵的影响的信息将提供一个 确定激动剂如何影响细胞内功能的基本环节 通过钙信号。 该泵由一个分子量约为1的多肽链组成。 135,000(确切的分子量取决于异构体)。我们有 成功地在COS细胞中表达了该泵，并开发了一种分析方法 测定所表达的酶的活性的系统。我们建议进行研究 旨在确定泵控制的确切轨迹和性质 S的活性受蛋白激酶的影响。我们将使用截断突变体和点 突变的泵，以确定磷酸化的确切位置和意志 在几乎与以下条件相同的条件下进行活性分析 磷酸化以确定给定时间点的磷酸化如何 激活这种酶。我们将提高针对磷酸酶的抗体， 这应该是非常有用的，以确定磷酸化后的作用 刺激特定种类的细胞。

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.