蛋白激酶A对CDC25B蛋白S149与S321位点磷酸化的修饰抑制小鼠1-细胞期受精卵有丝分裂  

PKA-regulated phosphorylation status of S149 and S321 sites of CDC25B inhibits mitosis of fertilized mouse eggs

作　　者：肖建英[1,2] 刘超[1,2] 孙小涵[1,2] 于秉治[1] 

机构地区：[1]中国医科大学生物化学与分子生物学教研室,沈阳l10001 [2]辽宁医学院生物化学与分子生物学教研室,锦州121001

出　　处：《生理学报》2012年第1期33-40,共8页Acta Physiologica Sinica

基　　金：supported by the National Natural Science Foundation of China(No.81070489)

摘　　要：在小鼠1-细胞期受精卵,蛋白激酶A（protein kinase A,PKA）可通过磷酸化细胞分裂周期25B（cell division cycle25B,CDC25B）的321位Ser引起受精卵分裂阻滞。本文旨在研究PKA对CDC25B149位点Ser的磷酸化对小鼠受精卵发育的影响,及其与321位点的关系。质粒pBSK-CDC25B-WT（野生型）、pBSK-CDC25B-S149A（149位Ser突变成Ala）、pBSK-CDC25B-S321A（321位Ser突变成Ala）、pBSK-CDC25B-S149A/S321A（149位和321位Ser联合突变成Ala）体外转录成mRNAs;显微注射入小鼠S期受精卵中,在PKA抑制剂H-89预处理的M16培养基中培养,观察其对受精卵发育、有丝分裂成熟促进因子（matu-ration promoting factor,MPF）活性及CDC2-Tyr15磷酸化状态的影响。结果显示,H-89呈剂量（0~50μmol/L）依赖性的方式加速小鼠受精卵卵裂。然后选择卵裂率接近100%、H-89（40μmol/L）培养的小鼠受精卵,与对照组相比,CDC25B各种mRNAs注射组受精卵卵裂率明显增高,MPF活性提前达到高峰;CDC2-Tyr15磷酸化状态和MPF活性变化相一致,以上结果说明,CDC25B蛋白的149位Ser也是PKA在体内调控CDC25B的重要靶点。因此在小鼠1-细胞期受精卵有丝分裂过程中,PKA对小鼠CDC25B蛋白S149位点与S321位点的磷酸化修饰可能是控制受精卵G2/M转换的重要方式。To further test whether protein kinase A （PKA） can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase （22 h after hCG injection） were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 （0–50 μmol/L）, the G2 phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 μmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B （CDC25B） on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor （MPF） activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 μmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development ofmouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G2/M transition in the mitotic cell cycle of fertilized mouse eggs.

关 键 词：细胞分裂周期25B 蛋白激酶A 有丝分裂成熟促进因子 H-89 小鼠受精卵 

分 类 号：Q954.4[生物学—动物学]