支架蛋白RACK1调控小鼠1-细胞期受精卵G2/M期转换机制的研究

The decision to enter mitosis is mediated by a network of proteins that regulate the activation of the Cdc2-Cyclin B complex and accumulating evidence demonstrates that scaffolding protein RACK1 (receptor of activated protein kinase C) plays a key role in the network during embryogenesis and tumorigenesis. However, the roles of RACK1 in one-cell-stage mouse embryos remain unsolved. Our primary data demonstrated RACK1 is expressed in a dynamic spatiotemporal pattern during the first cell cycle of mouse embryos and,once disturbed,the first mitosis of mouse embryos is abnormal suggesting RACK1 plays key roles during earlier embryogenesis of mouse embryos. In this study, by over expressing and down regulating RACK1 in one-cell-stage mouse embryos, we will observe the effects of RACK1 on the cleavage of mouse embryos and investigate how RACK1 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.

研究表明多功能支架蛋白RACK1是调控真核细胞有丝分裂的关键因子，但RACK1在小鼠1-细胞期受精卵的作用及其机制尚未阐明。我们前期研究发现RACK1在小鼠1-细胞期受精卵各个细胞时期动态表达，M期表达明显增多；下调RACK1抑制小鼠受精卵卵裂，提示RACK1在受精卵早期发育中有重要作用。为此，本课题以小鼠1-细胞期受精卵为研究对象，干预RACK1，观察小鼠受精卵卵裂、MPF活性及Cdc2磷酸化状态和Cyclin B蛋白水平的变化；探讨RACK1对MPF的直接调节者Cdc25B和Wee1B/Pkmyt1的活力、翻译调控、蛋白表达水平及其稳定性影响，并发现RACK1与Cdc25B和Wee1B/Pkmyt1是否存在直接相互作用，阐明RACK1调控小鼠1-细胞期受精卵G2/M期转换的机制。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。

RACK1 是多功能的支架蛋白, 参与体内各种生物过程的调节，包括信号传导、免疫应答、细胞生长、迁移和分化以及 MAPK 活化，血管生成，肿瘤生长，神经元反应，细胞凋亡，染色质重塑和生物钟的正常功能。RACK1表达沉默抑制小鼠1-细胞期受精卵的卵裂率，进一步说明RACK1促进小鼠1-细胞期受精卵有丝分裂的进程。双特异性蛋白激酶Wee1家族（Wee1、Wee2、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。RACK1 调控细胞周期，是否也参与Wee1的活性调控呢？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RACK1，并通过蛋白互作验证RACK1与Wee1家族成员（Wee1,Wee2,Pkmyt1）存在相互作用。提示RACK1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，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。同时，我们也通过进一步利用卵巢癌和胃癌细胞为实验材料继续探讨RACK1与WEE1相互作用调控细胞生长增殖的可能机制，为肿瘤增殖和转移等临床疾病研究开辟新的思路。