CDC25B蛋白亚细胞定位调控小鼠1-细胞期受精卵发育

CDC25B Subcellular Localization Influences One-cell Stage Mouse Embryos

为探讨小鼠细胞分裂周期25B(CDC25B)蛋白149位丝氨酸磷酸化状态对小鼠1-细胞期受精卵中CDC25B的亚细胞定位和发育的影响,应用定制的CDC25B-pS149位的磷酸化和非磷酸化抗体检测小鼠1-细胞期受精卵各细胞时期的磷酸化和非磷酸化状态;应用免疫荧光观察各期受精卵中CDC25B蛋白的定位情况;将质粒pEGFP-CDC25B-WT、pEGFP-CDC25B-S149A和pEGFP-CDC25B-S149D融合质粒及空载体质粒显微注射入G1期受精卵中,观察不同显微注射组小鼠1-细胞期受精卵中外源性CDC25B蛋白亚细胞定位.结果显示,CDC25B-S149位丝氨酸在G1和S期被磷酸化,在G2和M期去磷酸化.1-细胞期受精卵从G2向M期的转换过程中,发生了CDC25B向细胞核区的移位,到2-细胞初期,部分CDC25B蛋白又从细胞核回到细胞浆.实验结果提示,小鼠1-细胞期受精卵G2/M期转换过程中,CDC25B的S149位点磷酸化修饰可能是对CDC25B细胞内定位及其活性的精确调节方式.

To investigate the effect of S149 phosphorylation of cell division cycle 25（CDC25）B on its subcellular localization as well as the subsequent influence in mouse embryos development, one-cell stage mouse embryos was stained with a specific antibody to detect the CDC25B with phosphorylated and nonphosphorylated S149. The subcellular localization of endogenous and exogenous CDC25B were also compared, using immunofluorescence and microinjection of the plasmids of pEGFP-CDC25B-WT, pEGFP-CDC25B-S149A and pEGFP-CDC25B-S149D. The results showed that CDC25B-Ser149 was phosphorylated at G1 and S phases,whereas dephosphorylated at G2 and M phases. The localization of endogenous and exogenous fluorescent signals suggested that CDC25B was transferred from cytoplasm to the nucleus at the G2/M transition, and then exported from the nucleus to the cytoplasm at two-cell stage. We postulated that the phosphorylation of S149 might be involved in the regulation of CDC25B subcellular localization and play an important role in the G2/M transition in the early development of one-cell stage mouse embryos.