Ser149 is another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs |
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Authors: | Xiao Jianying Liu Chao Hou Junjie Cui Cheng Wu Didi Fan Huiyu Sun Xiaohan Meng Jun Yang Fuquan Wang Enhua Yu Bingzhi |
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Institution: | Institute of Pathology and Pathopysiology, China Medical University, Shenyang, Liaoning Province 110001, China. |
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Abstract: | 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 Ser(149), Ser(229), and Ser(321) of Cdc25B, and explored the role of Ser(149) in G(2)/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr(15), 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-Ser(149) was phosphorylated at G(1) and S phases, whereas dephosphorylated at G(2) and M phases, and the phosphorylation of Cdc25B-Ser(149) was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G(1) and S phases and translocated to the nucleus at the G(2) phase. Collectively, our findings provide evidence that Ser(149) may be another potential PKA phosphorylation target of Cdc25B in G(2)/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. |
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Keywords: | Cyclin-dependent Kinase Cell Cycle MS Protein Kinase A (PKA) Protein Phosphorylation CDC25B M Phase-promoting Factor (MPF) Fertilized Mouse Eggs In Vitro Phosphorylation |
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