Cdc25B和Wee1B核浆转位调控小鼠受精卵G2/M期转换

In all species studied, the regulation of the cell cycle is orchestrated by maturation-promoting factor (MPF), a complex of the Cdc2 kinase and the regulatory cyclin B. Activation of the Cdc2 kinase requires the dephoshorylation of Thr 14 and Tyr15 residues. The phosphorylation state of these two critical residues on Cdc2 is dictated by a balance between the activity of the Weel kinases and the Cdc25 phosphatases.The Wee1B and Cdc25B are involved in the control of the G2/M transition of the cell cycle.Subcellular localization might represent an important.aspect of the regulation of its activity. However, compartmentalization of Wee1 kinases and Cdc25B phosphatases and nucleocytoplasmic trafficking in regulating MPF is unknown in the control of early development and reproduction in mammalian embryos.Our previous study indicate that Cdc25B is distributed throughout the cytoplasm at the G1 and S phases, which may be in connection with phosphorylation modification the S149 and S321 residues of Cdc25B by protein kinase A. When the S149 and S321 of Cdc25B were mutated to unphosphorylated Ala, parts of Cdc25B shift from the cytoplasm to the nucleus, which may trigger mitosis. Whereas Wee1B partitioned primarily in the nucleus during G1 and S phase and progressively migrated to the cytoplasm as cells transitioned from G2 phase to M phase. Nucleocytoplasmic shuttling is prevalent between Wee1B and Cdc25B controlling the G2/M transition in fertilized mouse eggs is essential for their function during mitosis.In this study, we demonstrate that the sequestration of Cdc25B and wee1B regulate MPF activity in distinct subcellular compartments is critical for the G2/M transition in fertilized mouse eggs via site-directed mutagenesis, microinjection, live cell imaging system, immunofluoresence analysis and immunoprecipitation, etc. Therefore, our study provide the basis for the optimize the human assisted reproductive in vitro conditions and elucidating the mammalian ontogenesis mechanism.

蛋白激酶Wee1B和磷酸酶Cdc25B是调控细胞周期G2/M转换的关键因子，其功能与亚细胞定位密切相关。但如何调节Wee1B和Cdc25B亚细胞定位与活性平衡进而调控MPF活性及有丝分裂进程尚不清楚。我们发现在小鼠受精卵G1和S期，Cdc25B的321和149位丝氨酸磷酸化并定位胞浆，丝氨酸突变为丙氨酸后，Cdc25B发生核转运，触发受精卵有丝分裂；相反Wee1B在G1和S期定位胞核，G2末期则向胞浆转运。提示在小鼠受精卵G2/M转换时存在Cdc25B和Wee1B反向核浆转位，这可能是调控细胞G2/M转换的重要机制。本课题以小鼠受精卵为研究对象，观察Cdc25B和Wee1B野生型和突变体在1-细胞期受精卵不同时期的亚细胞定位及对受精卵有丝分裂的影响，探讨二者核浆转位机制，阐明二者调控受精卵早期发育的分子机制。本研究在深入阐明哺乳类个体发生机理的同时，为优化人类辅助生殖体外发生条件提供依据。

在哺乳动物的受精卵细胞中，如何调节Wee1 蛋白激酶和Cdc25B 磷酸酶活性的平衡及亚细胞定位进而调控MPF活性和有丝分裂进程尚不十分清楚。我们在研究Cdc25B调控小鼠1-细胞期受精卵早期发育的基础上，继续探讨双特异性蛋白激酶Wee1家族成员（Wee2和Pkmyt1）在小鼠早期受精卵中的表达、定位，下调Wee2促进小鼠1-细胞期受精卵有丝分裂，过表达Wee2和Pkmyt1可以通过磷酸化细胞周期蛋白依赖激酶（CDK1）使其失活，进而负调控细胞周期进程；小鼠1-细胞期受精卵中Wee2蛋白可能是PKA的直接作用底物，其主要功能位点是S15，PKA/Wee2通路对小鼠受精卵早期发育具有重要的调控作用。为了深入探讨Wee1的生物学功能及作用机制，我们用酵母双杂交筛选出与Wee1相互作用的候选蛋白，并通过免疫共沉淀、GST-Pull down和免疫荧光共定位等蛋白互作和原位杂交验证筛选蛋白RNA结合蛋白Khdrbs3与Wee1家族成员（Wee1,Wee2,Pkmyt1）存在相互作用，提示Khdrbs3可能通过调控母源mRNA的稳定或翻译或者通过蛋白-蛋白相互作用参与卵细胞激活这一早期事件。进而我们进一步观察了Khdrbs3在小鼠1-细胞期受精卵中的表达和作用，过表达Khdrbs3对1-细胞期受精卵的卵裂率无影响，而下调Khdrbs3表达抑制受精卵卵裂率，说明Khdrbs3促进1-细胞期受精卵有丝分裂的进程，原位杂交也表明Khdrbs3在鸡胚早期发育过程中的重要作用。本研究对揭示哺乳动物卵母细胞激活过程中原核形成和早期胚胎发育的机理打下很好基础，并为在临床上进行胚胎流产畸形方面研究奠定基础。