The purpose of this study is to reveal the role of Girdin in regulating the aggregation of actin filaments by studying the relationship between PKB/Akt and Girdin. First we used Scansite software (http://scansite.mit.edu) to predict relevant target sites of PKB/Akt on mouse Girdin. To gain insight into the role of phosphorylation of Girdin by PKB/Akt, we assessed the location of phosphorylated Girdin in fertilized eggs by staining with anti-P-Girdin 1 417 Ab. We detected a distinct increase in the fluorescence signal of F-actin and P-Girdin 1 417 after microinjection of Akt WT and myr-Akt. The addition of myr-Akt induced phosphorylation of Girdin in mouse fertilized eggs. In addition, siRNA-mediated Akt-knockdown blocked phosphorylation of Girdin. The distribution of actin filaments was obviously scattered. These results strongly suggest that PKB/Akt could directly phosphorylate Girdin on Ser1 417 and promote its function in mouse fertilized eggs.
通过研究PKB/Akt 与Girdin 蛋白之间的关系，目的在于揭示Girdin 蛋白在PKB/Akt 调控小鼠受精卵微丝聚集中的作用。研究中首先结合软件 (http：//scansite.mit.edu//) 预测了PKB/Akt 对Girdin 蛋白的磷酸化位点，并制定特定位点磷酸化抗体，通过蛋白免疫印迹检测经PKB/Akt mRNA 或PKB/Akt siRNA 处理后的小鼠受精卵中Girdin 蛋白磷酸化改变情况。同时检测了磷酸化的Girdin 蛋白亚细胞定位及同微丝骨架的定位关系。进一步通过显微注射PKB/Akt mRNA 再注射Girdin siRNA 检测小鼠受精卵微丝骨架的聚集。结果显示经持续激活型PKB/Akt mRNA、野生型PKB/Akt mRNA 处理后的小鼠受精卵中p-Girdin-1 417 分布位置更加集中在2-细胞分裂沟处。经野生型和持续激活型PKB/AktmRNA 注射组p-Girdin-1 417 表达增强，但是并不影响Girdin 蛋白的总的表达。说明siRNA 介导的PKB/Akt 表达敲低非常明显地降低了Girdin 蛋白的磷酸化。激光共聚焦研究显示在Akt mRNA 和Girdin siRNA 共注射组微丝骨架分布明显散乱。本研究结果充分说明Girdin蛋白在小鼠受精卵中受到PKB/Akt 的调节，PKB/Akt 通过磷酸化Girdin 蛋白改变微丝骨架的聚集。.
Keywords: F-actin; Girdin; PKB/Akt; mouse fertilized eggs.