Regulation of phospholemman by phosphorylation

描述（由申请人提供）：Phospholemman（PLM）是一种72个氨基酸 一种膜磷蛋白，与其他蛋白质相互作用，参与 离子和渗透物的跨膜通量。它是一种主要的基质， cAMP依赖性蛋白激酶（PKA）和蛋白激酶C的磷酸化， 且可能是细胞中中心调节元件 通过蛋白激酶传递信号。然而，人们对如何做到这一点知之甚少。 磷酸化调节PLM。拟议工作的长期目标 是了解磷酸化对PLM的影响。我们的初步研究 表明PLM与其他细胞信号分子相互作用， PKA的磷酸化增加了卵母细胞中PLM的数量和活性， 转染的哺乳动物细胞。要了解有关PLM监管的更多信息， 磷酸化，我们提出了两个具体的目标。 第一个目的是确定磷酸化在调节细胞凋亡中的作用。 PLM和二聚化配偶体如MLP之间的蛋白质-蛋白质相互作用， 一种肌肉LIM蛋白和一种来自卵母细胞的新激酶锚定蛋白， 已经在酵母双杂交测定中鉴定。为了实现这一目标，我们将 在转染哺乳动物中研究PLM和伴侣蛋白的共沉淀 细胞和RNA注射的非洲爪蟾卵母细胞，并测量离子电流和体积 调节细胞。假设是不同的磷酸化 PLM中的位点将改变蛋白质-蛋白质相互作用。第二个目标是 确定PKA激活增加PLM数量的机制， 在RNA注射的青蛙卵母细胞和转染的哺乳动物细胞中的活性。到 为了完成这一目标，我们将研究表达野生型和 突变PLM使用生物化学，电生理和体积手段。的 假设是PLM的丝氨酸68处的磷酸化导致增加的 蛋白质表达和/或稳定性。 这些目标的成功实现将大大有助于我们的 了解磷酸化如何通过PLM调节细胞功能， 特别是在心脏和骨骼肌中， 肾上腺素能系统是深刻的。此外，关于细胞信号传导的新知识 通过蛋白激酶将被产生。

DESCRIPTION (provided by applicant): Phospholemman (PLM) is a 72 amino acid membrane phosphoprotein that interacts with other proteins to participate in transmembrane flux of ions and osmolytes. It is a major substrate for phosphorylation by cAMP-dependent protein kinase (PKA) and protein kinase C in heart and skeletal muscle and may be a central regulatory element in cell signaling through protein kinases. Very little, however, is known about how phosphorylation regulates PLM. The long-term objectives of the proposed work are to understand the effect of phosphorylation on PLM. Our preliminary studies show that PLM interacts with other cell signaling molecules, and that phosphorylation by PKA increases PLM quantity and activity in oocytes and in transfected mammalian cells. To learn more about regulation of PLM by phosphorylation, we propose two specific aims. The first aim is to determine the role of phosphorylation in modulating protein-protein interactions between PLM and dimerization partners such as MLP, a muscle lim protein, and a novel kinase anchoring protein from oocyte that have been identified in yeast two-hybrid assays. To complete this aim, we will study co-precipitation of PLM and partner proteins in transfected mammalian cells and in RNA-injected Xenopus oocytes, and measure ion currents and volume regulation of the cells. The hypothesis is that phosphorylation of different sites in PLM will alter the protein-protein interactions. The second aim is to determine the mechanism by which activation of PKA increases PLM quantity and activity in RNA-injected frog oocytes and in transfected mammalian cells. To complete this aim, we will study transfected cells expressing wild-type and mutant PLMs using biochemical, electrophysiological and volumetric means. The hypothesis is that phosphorylation at serine 68 of PLM leads to increased protein expression and/or stability. Successful completion of these aims will contribute greatly to our understanding of how phosphorylation regulates cell function through PLM, particularly in heart and skeletal muscle where the functional impact of the adrenergic system is profound. In addition, new knowledge on cell signaling through protein kinases will be generated.