Regulation of phospholemman by phosphorylation

描述(申请人提供)：磷脂酶(PLM)是一种72个氨基酸。 与其他蛋白相互作用参与的膜磷蛋白 离子和渗透分子的跨膜通量。它是一种主要的底物 CAMP依赖的蛋白激酶(PKA)和蛋白激酶C的磷酸化 心脏和骨骼肌，可能是细胞中的中央调节元件 通过蛋白激酶传递信号。然而，人们对此知之甚少。 磷酸化调节PLM。拟议工作的长期目标 是为了了解磷酸化对PLM的影响。我们的初步研究 显示PLM与其他细胞信号分子相互作用，并且 PKA的磷酸化增加卵母细胞和小鼠卵母细胞中PLM的数量和活性 转基因哺乳动物细胞。要了解更多关于PLM监管的信息，请 为了实现磷酸化，我们提出了两个特定的目标。 第一个目标是确定磷酸化在调节中的作用。 PLM和二聚体伙伴如MLP之间的蛋白质相互作用， 一种肌肉LIM蛋白和一种来自卵母细胞的新的激酶锚定蛋白，该蛋白 已在酵母双杂交试验中得到鉴定。为了实现这一目标，我们将 共沉淀法在转基因哺乳动物中共沉淀PLM和伴侣蛋白的研究 细胞和注射RNA的非洲爪哇卵母细胞中，并测量离子电流和体积 对细胞的调节。假设是不同的磷酸化不同 PLM中的位点将改变蛋白质之间的相互作用。第二个目标是 确定PKA激活增加PLM数量和数量的机制 注射RNA的蛙卵母细胞和转基因哺乳动物细胞中的活性。至 为了实现这一目标，我们将研究表达野生型和 使用生化、电生理和容量方法的突变的PLM。这个 假说是PLM丝氨酸68位的磷酸化导致增加 蛋白质表达和/或稳定性。 这些目标的成功实现将对我们的 了解磷酸化如何通过PLM调节细胞功能， 尤其是在心脏和骨骼肌中， 肾上腺素能系统是深奥的。此外，关于细胞信号的新知识 通过蛋白激酶会被产生。

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.