Ser149 Is Another Potential PKA Phosphorylation Target of Cdc25B in G2/M Transition of Fertilized Mouse Eggs*
Ser149 Is Another Potential PKA Phosphorylation Target of Cdc25B in G2/M Transition of Fertilized Mouse Eggs*
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DOI：
10.1074/jbc.m110.150524
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发表时间：
2011-01
期刊：
影响因子：
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通讯作者：
Jian-ying Xiao;Chao Liu;Junjie Hou;Cheng Cui;Di-di Wu;Huiyun Fan;Xiaohan Sun;J. Meng;Fuquan Yang;E. Wang;Bing‐zhi Yu
中科院分区：
文献类型：
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作者：
Jian-ying Xiao;Chao Liu;Junjie Hou;Cheng Cui;Di-di Wu;Huiyun Fan;Xiaohan Sun;J. Meng;Fuquan Yang;E. Wang;Bing‐zhi Yu
在哺乳动物中，蛋白激酶A（PKA）通过磷酸化细胞分裂周期25同源物B（Cdc 25 B），对M期促进因子（MPF）起负调节作用。然而，其分子机制尚不清楚。本研究通过LC-MS/MS分析，在体外鉴定了Cdc 25 B蛋白PKA的磷酸化位点，包括Ser 149、Ser 229和Ser 321，并探讨了Ser 149在小鼠受精卵G2/M转换中的作用。结果表明，过表达的Cdc 25 B-S149 A突变体通过Cdc 2-Tyr 15的直接去磷酸化启动有效的MPF激活，导致在Cdc 25 B-WT之前触发有丝分裂。相反，与对照组相比，磷酸模拟Cdc 25 B-S149 D突变体的过表达没有显着差异。此外，我们发现Cdc 25 B-Ser 149在G1和S期被磷酸化，而在G2和M期被去磷酸化，并且Cdc 25 B-Ser 149的磷酸化受到PKA的调节。此外，我们研究了内源性和外源性Cdc 25 B，其主要在G1和S期的细胞质中表达，并在G2期易位到细胞核。因此，Ser 149可能是Cdc 25 B在小鼠受精卵G2/M期的另一个潜在PKA磷酸化靶点，Cdc 25 B可能是哺乳动物PKA的直接下游底物，在小鼠胚胎早期发育调控中发挥重要作用。
It is well documented that protein kinase A (PKA) acts as a negative regulator of M phase promoting factor (MPF) by phosphorylating cell division cycle 25 homolog B (Cdc25B) in mammals. However, the molecular mechanism remains unclear. In this study, we identified PKA phosphorylation sites in vitro by LC-MS/MS analysis, including Ser149, Ser229, and Ser321 of Cdc25B, and explored the role of Ser149 in G2/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr15, resulting in triggering mitosis prior to Cdc25B-WT. Conversely, overexpression of the phosphomimic Cdc25B-S149D mutant showed no significant difference in comparison with the control groups. Furthermore, we found that Cdc25B-Ser149 was phosphorylated at G1 and S phases, whereas dephosphorylated at G2 and M phases, and the phosphorylation of Cdc25B-Ser149 was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G1 and S phases and translocated to the nucleus at the G2 phase. Collectively, our findings provide evidence that Ser149 may be another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs and Cdc25B as a direct downstream substrate of PKA in mammals, which plays important roles in the regulation of early development of mouse embryos.