CX43 IN MITOSIS

有丝分裂中的 CX43

• 批准号: 8361911
• 负责人: PAUL D. LAMPE
• 金额: $ 2.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361911
• 关键词: Biochemical; CDC2 Protein Kinase; Cell Communication; Cell Cycle; Cell Proliferation; Cells; Connexin 43; Connexins; Consensus; Cyclin B; Cytokinesis; Developmental Process; Disease; Event; Funding; Gap Junctions; Grant; Homeostasis; Image Analysis; Immunofluorescence Immunologic; Mediating; Mitosis; Mitotic; Morphology; National Center for Research Resources; Nocodazole; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Principal Investigator; Protein Kinase; Proteins; Recycling; Research; Research Infrastructure; Resources; Role; Serine; Signal Transduction; Site; Source; Structure; Technology; United States National Institutes of Health; Western Blotting; carcinogenesis; cost; intercellular communication; loss of function; protein transport; tissue/cell culture; trafficking

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Direct cell-cell communication as mediated by gap junctions has been shown repeatedly to be a necessary component of homeostasis and is highly regulated during the cell cycle, developmental processes and cell proliferation. Connexin diseases result when gap junction proteins mis-traffic or mis-function and loss of gap junction intercellular communication is concomitant with carcinogenesis. Mitosis, an integral part of the cell cycle, causes significant morphological and biochemical changes throughout the entire cell. Certainly, some of the biggest changes in GJ structure morphology and distribution occur as tissue culture cells internalize their GJs as they go into mitosis and undergo cell rounding and cytokinesis. In unstimulated (normally trafficking cells) NRK cells, Cx43 isolated from immunoprecipitated cell lysates show three bands on Western blots. These include a nonphosphorylated form (NP) and two phosphorylated forms (P1 and P2) that are predominately phosphorylated on multiple, unidentified serine sites. GJ plaques contain predominantly the P1 and P2 forms while the NP form is localized intracellularly. Using immunofluorescence, nocodazole synchronized mitotic cells display an entirely intracellular localization of Cx43. The Cx43 found in these mitotic cells is uniquely phosphorylated and migrates as a distinct P3 species in a p34cdc2/cyclin B kinase-dependent manner. Most connexins contain sites for multiple sites for phosphorylation. These phosphorylation sites are important for proper protein trafficking, assembly and degradation and likely play a role in signal transduction as these connexins contain several protein kinase consensus phosphorylation sequences. This project is focused on determining whether recycling occurs during mitosis, correlation with serine phosphorylation events and where old versus new protein is found in the cell during mitosis, using the NCMIR tetracysteine technology to do so.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 由间隙连接介导的直接细胞间通讯已被多次证明是体内平衡的必要组成部分，并且在细胞周期、发育过程和细胞增殖过程中受到高度调节。当间隙连接蛋白错误运输或功能异常以及间隙连接细胞间通讯丧失与癌发生同时发生时，就会导致连接蛋白疾病。有丝分裂是细胞周期的一个组成部分，会导致整个细胞发生显着的形态和生化变化。 当然，GJ 结构形态和分布的一些最大变化发生在组织培养细胞进入有丝分裂、细胞变圆和胞质分裂时内化其 GJ 时。 在未刺激（通常是运输细胞）的 NRK 细胞中，从免疫沉淀的细胞裂解物中分离出的 Cx43 在蛋白质印迹上显示出三个条带。 其中包括一种非磷酸化形式 (NP) 和两种磷酸化形式（P1 和 P2），它们主要在多个未识别的丝氨酸位点上被磷酸化。 GJ 斑块主要包含 P1 和 P2 形式，而 NP 形式位于细胞内。使用免疫荧光，诺考达唑同步有丝分裂细胞显示 Cx43 完全细胞内定位。 在这些有丝分裂细胞中发现的 Cx43 被独特地磷酸化，并以 p34cdc2/cyclin B 激酶依赖性方式作为独特的 P3 物种迁移。大多数连接蛋白含有多个磷酸化位点。这些磷酸化位点对于正确的蛋白质运输、组装和降解非常重要，并且可能在信号转导中发挥作用，因为这些连接蛋白含有多个蛋白激酶共有磷酸化序列。该项目的重点是使用 NCMIR 四半胱氨酸技术确定有丝分裂期间是否发生回收、与丝氨酸磷酸化事件的相关性以及有丝分裂期间细胞中新旧蛋白质的位置。