Chen Xiaoxuan, Kong Xiangyu, Zhuang Wenxin, Teng Bogang, Yu Xiuyi, Hua Suhang, Wang Su, Liang Fengchao, Ma Dan, Zhang Suhui, Zou Xuan, Dai Yue, Yang Wei, Zhang Yongxing
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, Fujian, 361102, PR China.
Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning, 116001, PR China.
Sci Rep. 2016 Feb 16;6:21224. doi: 10.1038/srep21224.
Here we show that A-kinase anchoring protein 95 (AKAP95) and connexin 43 (Cx43) dynamically interact during cell cycle progression of lung cancer A549 cells. Interaction between AKAP95 and Cx43 at different cell cycle phases was examined by tandem mass spectrometry(MS/MS), confocal immunofluorescence microscopy, Western blot, and co-immunoprecipitation(Co-IP). Over the course of a complete cell cycle, interaction between AKAP95 and Cx43 occurred in two stages: binding stage from late G1 to metaphase, and separating stage from anaphase to late G1. The binding stage was further subdivided into complex binding to DNA in interphase and complex separating from DNA in metaphase. In late G1, Cx43 translocated to the nucleus via AKAP95; in anaphase, Cx43 separated from AKAP95 and aggregated between two daughter nuclei. In telophase, Cx43 aggregated at the membrane of the cleavage furrow. After mitosis, Cx43 was absent from the furrow membrane and was located in the cytoplasm. Binding between AKAP95 and Cx43 was reduced by N-(2-[P-Bromocinnamylamino]-ethyl)-5-isoquinolinesulfonmide (H89) treatment and enhanced by Forskolin. dynamic interaction between AKAP95 and Cx43 varies with cell cycle progression to regulate multiple biological processes.
我们在此表明，A激酶锚定蛋白95（AKAP95）和连接蛋白43（Cx43）在肺癌A549细胞的细胞周期进程中动态相互作用。通过串联质谱（MS/MS）、共聚焦免疫荧光显微镜、蛋白质免疫印迹和免疫共沉淀（Co-IP）检测了不同细胞周期阶段AKAP95与Cx43之间的相互作用。在完整的细胞周期过程中，AKAP95与Cx43的相互作用发生在两个阶段：从G1晚期到中期的结合阶段，以及从后期到G1晚期的分离阶段。结合阶段进一步细分为间期与DNA的复合物结合以及中期与DNA的复合物分离。在G1晚期，Cx43通过AKAP95转运至细胞核；在后期，Cx43与AKAP95分离并聚集在两个子核之间。在末期，Cx43聚集在分裂沟膜处。有丝分裂后，Cx43不在沟膜处，而是位于细胞质中。用N-（2-[对溴肉桂酰胺基]乙基）-5-异喹啉磺酰胺（H89）处理会降低AKAP95与Cx43之间的结合，而用福斯可林处理则会增强这种结合。AKAP95与Cx43之间的动态相互作用随细胞周期进程而变化，以调节多种生物学过程。
