COBRE: UNR: SIGNAL TRANSDUCTION PATHWAYS REGULATED BY CELL VOLUME

COBRE：UNR：由细胞体积调节的信号转导途径

• 批准号: 7720383
• 负责人: Ilia A Yamboliev
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720383
• 关键词: Actin-Binding Protein; Actins; Address; Anions; Binding; Biochemical; Blood Vessels; Canis familiaris; Cell Proliferation; Cell Volumes; Cells; Chloride Ion; Chlorides; ClC-3 channel; Computer Retrieval of Information on Scientific Projects Database; Environment; Funding; Glucocorticoids; Goals; Grant; In Vitro; Institution; Maintenance; Mediating; Membrane; Microfilaments; Molecular; Muscle Tonus; Myocardium; NIH 3T3 Cells; Phosphorylation; Phosphotransferases; Physiological; Protein Kinase; Pulmonary artery structure; Regulation; Research; Research Personnel; Resources; Serum; Signal Transduction Pathway; Smooth Muscle Myocytes; Source; Swelling; Testing; Ubiquitination; United States National Institutes of Health; in vivo; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Maintenance of physiological cell volume is crucial for cell proliferation, differentiation and survival. Anion channels, and particularly chloride (Cl-) channels, participate in other important physiological functions including maintenance of the rhythmic cycle of the heart and the muscle tone of blood vessels. The Cl- channel ClC-3 is a major volume regulator in pulmonary artery smooth muscle cells (PASMCs), however its regulation is undefined. The major goal of the present proposal is to define the fundamental intracellular mechanisms regulating ClC-3 in anisotonic cell environment, by addressing the following specific aims: (1) Test the hypothesis that protein kinases regulate the volume-sensitive outwardly rectifying anion channel (VSOAC)/ClC-3 by phosphorylation. Biochemical and molecular approaches will be utilized in experiments with PASMCs and NIH 3T3 cells to establish that ClC-3 is regulated by phosphorylation of its N-terminus (NT) and/or C-terminus (CT), and to identify protein kinases that phosphorylate ClC-3 in vitro and in vivo. (2) Test the hypothesis that the serum and glucocorticoid-dependent kinase (SGK) regulates VSOAC/ClC-3 by indirect phosphorylation and/or ubiquitination. This aim will involve experiments to test the possibility that hypotonic swelling-mediated activation of SGK leads to activation of a currently unknown intermediate kinase, or by decreased ubiquitination and increased membrane expression of ClC-3 in NIH 3T3 cells and canine PASMCs. (3) Test the hypothesis that subcortical actin and actin-binding proteins interact with VSOAC/ClC-3 channel and contribute to its activation. This aim will include experiments to assess the binding capacity of the cytosolic ClC-3 termini with actin filaments and actin-binding proteins, to identify ClC-3 domains involved in the association, and to test the hypothesis that inhibition of this interaction interferes with the activation of ClC-3.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 维持生理细胞体积对于细胞增殖、分化和存活至关重要。阴离子通道，特别是氯离子 (Cl-) 通道，参与其他重要的生理功能，包括维持心脏的节律周期和血管的肌张力。 Cl 通道 ClC-3 是肺动脉平滑肌细胞 (PASMC) 中的主要容量调节剂，但其调节尚不明确。本提案的主要目标是通过解决以下具体目标来定义在不等渗细胞环境中调节 ClC-3 的基本细胞内机制：（1）测试蛋白激酶通过磷酸化调节体积敏感的外向整流阴离子通道（VSOAC）/ClC-3 的假设。将在 PASMC 和 NIH 3T3 细胞实验中采用生化和分子方法，以确定 ClC-3 通过其 N 末端 (NT) 和/或 C 末端 (CT) 磷酸化进行调节，并鉴定在体外和体内磷酸化 ClC-3 的蛋白激酶。 (2) 检验血清和糖皮质激素依赖性激酶 (SGK) 通过间接磷酸化和/或泛素化调节 VSOAC/ClC-3 的假设。这一目标将涉及实验来测试低渗肿胀介导的 SGK 激活是否会导致目前未知的中间激酶激活，或者通过减少 NIH 3T3 细胞和犬 PASMC 中 ClC-3 的泛素化和膜表达增加来激活。 (3) 检验皮质下肌动蛋白和肌动蛋白结合蛋白与 VSOAC/ClC-3 通道相互作用并有助于其激活的假设。该目标将包括评估胞质 ClC-3 末端与肌动蛋白丝和肌动蛋白结合蛋白的结合能力的实验，以确定参与关联的 ClC-3 结构域，并测试抑制这种相互作用会干扰 ClC-3 激活的假设。