Liu Yanchun, Deng Xin, Wu Didi, Jin Minglin, Yu Bingzhi
Department of Biochemical and Molecular Biology, China Medical University, Shenyang, Liaoning 110122, P.R. China.
Liaoning Blood Center, Shenyang, Liaoning 110044, P.R. China.
Exp Ther Med. 2019 Nov;18(5):3281-3290. doi: 10.3892/etm.2019.7959. Epub 2019 Aug 29.
Protein kinase C type δ (PKCδ) is involved in B-cell signaling and the regulation of growth, apoptosis and differentiation of a variety of cell types. Cell division cycle 25 (Cdc25) is a key mediator of cell cycle progression that activates cyclin-dependent kinase complexes that drive the cell cycle and participates in the regulation of DNA damage checkpoints. Cdc25B is a member of the Cdc25 family of phosphatases. The present study investigated the role and mechanism of PKCδ in regulating the fertilization of mouse embryos in early development. The expression and subcellular localization of PKCδ and Cdc25B were detected using reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence in one-cell stage mouse embryos. Specific small interfering RNAs targeting PKCδ were used to knockdown the expression of PKCδ. Subsequently, Scansite software was used to predict the target of phosphorylated Cdc25B. Western blotting was used to measure the effects of phosphorylation and dephosphorylation in one-cell stage mouse embryos at different cell cycle phases. PKCδ was expressed during M phase and served a positive role in one-cell stage mouse embryos. Immunofluorescence data revealed that PKCδ and Cdc25B were expressed during G, S, G and M phases of the cell cycle. Furthermore, phosphorylated levels of Cdc25B-Ser96 were observed during G and M phases. Microinjection with mimics of phosphorylated Cdc25B-Ser96 mRNA promoted the development of one-cell stage mouse embryos. When PKCδ was suppressed, microinjection with mimics of phosphorylated Cdc25B-Ser96 mRNA reversed the inhibition of PKCδ. To conclude, PKCδ serves a positive role in the first cell cycle of mouse embryos by phosphorylating Cdc25B-Ser96, and provides novel insights for the regulation of early embryonic development.
蛋白激酶Cδ型（PKCδ）参与B细胞信号传导以及多种细胞类型的生长、凋亡和分化调节。细胞分裂周期25（Cdc25）是细胞周期进程的关键调节因子，可激活驱动细胞周期的细胞周期蛋白依赖性激酶复合物，并参与DNA损伤检查点的调节。Cdc25B是Cdc25磷酸酶家族的成员。本研究探讨了PKCδ在调节小鼠胚胎早期发育受精过程中的作用及机制。采用逆转录定量聚合酶链反应、蛋白质免疫印迹法和免疫荧光法检测单细胞期小鼠胚胎中PKCδ和Cdc25B的表达及亚细胞定位。使用靶向PKCδ的特异性小干扰RNA来敲低PKCδ的表达。随后，使用Scansite软件预测磷酸化Cdc25B的靶点。采用蛋白质免疫印迹法检测不同细胞周期阶段单细胞期小鼠胚胎中磷酸化和去磷酸化的作用。PKCδ在M期表达，并在单细胞期小鼠胚胎中发挥积极作用。免疫荧光数据显示，PKCδ和Cdc25B在细胞周期的G1、S、G2和M期均有表达。此外，在G2和M期观察到Cdc25B-Ser96的磷酸化水平。显微注射磷酸化Cdc25B-Ser96 mRNA模拟物可促进单细胞期小鼠胚胎的发育。当PKCδ被抑制时，显微注射磷酸化Cdc25B-Ser96 mRNA模拟物可逆转PKCδ的抑制作用。总之，PKCδ通过磷酸化Cdc25B-Ser96在小鼠胚胎的第一个细胞周期中发挥积极作用，并为早期胚胎发育的调节提供了新的见解。
