蛋白激酶Pkmyt1在小鼠受精卵早期发育过程中活性调控及机制研究

The early development of mouse embryos was regulated by a variety of protein kinases and Pkmyt1 negatively regulates the mitosis of one-cell stage mouse embryos, however, the underlying mechanism of Pkmyt1 activity remains unsolved. Our primary data demonstrated Pkmyt1 is expressed in a dynamic spatiotemporal pattern during the first cell cycle of mouse embryos, suggesting Pkmyt1 plays key roles during earlier embryogenesis of mouse embryos. Therefore, Yeast two-hybrid were applied to screen out target proteins RASAL2 and IQGAP1 which interacted with Pkmyt1. In this study, the protein and protein interactions between Pkmyt1 and RASAL2/IQGAP1 were veritified in vitro and in vivo and further confirmed the interaction domain. Then, overexpressing and downregulating RASAL2/IQGAP1 in one-cell stage mouse embryos, we will observe the changes of protein activity, distribution, expression and stability of Pkmyt1 and the effects of RASAL2 /IQGAP1 on the cleavage of mouse embryos and investigate how RASAL2/IQGAP1 regulates M-phase promoting factor(MPF) activity and the mechanisms underlying the regulations to further decipher the mechanism of G2/M transmit of one-cell-stage mouse embryos. Therefore, our study will shed new light on the molecular mechanism of earlier embryogenesis and open new avenue to the optimization of human assisted reproduction under in vitro condition.

小鼠受精卵早期发育受严格调控，我们前期研究发现蛋白激酶Pkmyt1通过MPF负调控小鼠1-细胞期受精卵有丝分裂进程，但Pkmyt1活性调控机制尚未阐明。因此，我们利用酵母双杂交系统筛选出与Pkmyt1互作蛋白RASAL2和IQGAP1，并进一步证明与Pkmyt1存在直接相互作用并确定互作结构域；其次，以小鼠 1-细胞期受精卵为研究对象，干预RASAL2和IQGAP1，①探讨对Pkmyt1的活力、定位、蛋白表达水平及稳定性的影响；②观察小鼠1-细胞期受精卵卵裂率、MPF活性、Cdc2- Thr14/Tyr15磷酸化状态和Cyclin B蛋白水平，以探讨RASAL2和IQGAP1通过Pkmyt1调控小鼠受精卵的发育。本研究对揭示小鼠受精卵早期发育的分子机制和生殖机理具有十分重要的意义，为优化人类辅助生殖提供理论和实验依据。

双特异性蛋白激酶Pkmyt1通过磷酸化细胞周期蛋白依赖激酶使其失活，进而负调控细胞周期进程。Pkmyt1属于蛋白激酶Wee1家族成员之一，为了深入的探讨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，并通过蛋白互作验证RASAL2与Wee1家族成员（Wee1,Wee2,Pkmyt1）存在相互作用。鉴于有关RASAL2在小鼠卵细胞和早期胚胎中的功能研究很少，本课题组首先研究了RASAL2在小鼠1-细胞期受精卵细胞中的表达定位，研究其生物学功能及作用机制，通过生物信息学、细胞生物学和分子生物学等实验手段深入探讨RASAL2在小鼠卵细胞激活过程中对原核形成的作用及对早期胚胎发育的影响，有利于更好地探究哺乳动物早期胚胎的发育，并为在临床上进行胚胎流产畸形方面研究打下一定的基础。