CX43 IN MITOSIS

有丝分裂中的 CX43

• 批准号: 8169604
• 负责人: PAUL D. LAMPE
• 金额: $ 1.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169604
• 关键词: Biochemical; C-terminal; CDC2 Protein Kinase; Cell Cycle; Cells; Computer Retrieval of Information on Scientific Projects Database; Connexin 43; Connexins; Consensus; Cyclin B; Cytokinesis; Funding; Gap Junctions; Grant; Homeostasis; Immunofluorescence Immunologic; Institution; Ions; Life; Mitosis; Mitotic; Morphology; Nature; Nocodazole; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Protein Biosynthesis; Protein Kinase; Protein Synthesis Inhibitors; Proteins; Recycling; Research; Research Personnel; Resolution; Resources; Role; Serine; Signal Transduction; Site; Source; Spottings; Structure; Technology; United States National Institutes of Health; Western Blotting; cell growth; cellular imaging; intercellular communication; light microscopy; lysosomal proteins; protein degradation; 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. 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. The cell cycle helps to regulate cell growth by maintaining constant volumes and homeostasis. Gap junctions (GJs) are an integral part of this process because it is partially through intercellular communication that cells help to maintain uniform cytosolic metabolite and ion concentrations. 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 typically found in the C-terminal region, although Cx56 has an additional phosphorylation site located in the cytoplasmic loop. 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 sequence. Nonetheless, the carboxyl terminus can be deleted and GJs will still assemble so the significance of phosphorylation sites in assembly is not yet understood. Treatment of cells released from nocodazole block with proteosomal, lysosomal and protein synthesis inhibitors demonstrated that protein degradation plays a role in the disappearance of the P3-Cx43 after mitosis. While substantial protein synthesis occurs during reformation of GJs after mitosis, there is intriguing evidence from live cell imaging that some connexin recycling may also be occurring. At the light microscopy resolution level, there is no morphology information on the nature of connexin bearing structures since the intracellular localization appears as small bright spots and old versus new protein cannot be distinguished from each other. This project is focused on determining whether recycling occurs during mitosis and uses the tetracysteine technology to do so.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细胞周期通过维持恒定的体积和稳态来帮助调节细胞生长。 间隙连接（GJ）是这一过程的一个组成部分，因为它部分是通过细胞间的通信，细胞有助于保持均匀的胞质代谢物和离子浓度。 有丝分裂是细胞周期的一个组成部分，在整个细胞中引起显著的形态学和生物化学变化。 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内化其GJ时，因为它们进入有丝分裂并经历细胞变圆和胞质分裂。 在未受刺激的（正常运输细胞）NRK细胞中，从免疫沉淀的细胞裂解物中分离的Cx43在Western印迹上显示三条带。 这些包括非磷酸化形式（NP）和两种磷酸化形式（P1和P2），主要在多个未识别的丝氨酸位点上磷酸化。 GJ斑主要含有P1和P2型，而NP型位于细胞内。使用免疫荧光，诺考达唑同步的有丝分裂细胞显示Cx43的完全细胞内定位。 在这些有丝分裂细胞中发现的Cx43是唯一磷酸化的，并以p34 cdc 2/细胞周期蛋白B激酶依赖的方式作为不同的P3物种迁移。大多数连接蛋白含有多个磷酸化位点。 这些磷酸化位点通常在C-末端区域发现，尽管Cx 56具有位于细胞质环中的额外磷酸化位点。 这些磷酸化位点对于适当的蛋白质运输、组装和降解是重要的，并且可能在信号转导中起作用，因为这些连接蛋白含有几个蛋白激酶共有磷酸化序列。 尽管如此，羧基末端可以被删除，GJ仍然会组装，因此磷酸化位点在组装中的意义还不清楚。用蛋白体、溶酶体和蛋白质合成抑制剂处理从诺考达唑阻断释放的细胞表明，蛋白质降解在有丝分裂后P3-Cx43的消失中起作用。 虽然大量的蛋白质合成发生在有丝分裂后GJ的重组过程中，但活细胞成像的有趣证据表明，也可能发生一些连接蛋白再循环。在光学显微镜分辨率水平上，没有关于连接蛋白承载结构的性质的形态学信息，因为细胞内定位表现为小亮点，并且新旧蛋白不能彼此区分。该项目的重点是确定有丝分裂过程中是否发生再循环，并使用四半胱氨酸技术来实现。