磷酸酶通过Mps1对SAC及卵子发生的调控研究

Mps1 is a kind of kinase which is important in the regulation of mitotic and meiotic spindle assembly checkpoint(SAC). The function of Mps1 is related to it's phosphorylation. Two phosphates, PPM1A and PPM1B, are identified to be the specific phosphatases of Mps1. Futher experiments demonstrated that both PPM1A and PPM1B could dephosphorylated the S821 site which is the autophosphorylation and substrate recruitment site of Mps1. We hope to address if PPM1A and PPM1B could regulate the function of Mps1 by dephosphorylation of Mps1. Based on our previous works, we will further study the regulation of Mps1 function by PPM1A/1B in vitro,in cell culture and in vivo respectively: Study of biochemical regulation of PPM1A/1B to Mps1 and explore the subcelluar localization and migration of PPM1A and PPM1B in cell during cell cycle progression in vitro; In cell culture, study of the effect and mechanism of mitotic SAC, and related cell cycle progression, genome stability regulated by PPM1A/1B through Mps1 by using PPM1A and PPM1B overexpressed or knockdown cells; By making PPM1A/1B conditional gene knockout (KO) mouse separately, we will study the effect of PPM1A and PPM1B on the meiotic SAC and oogenesis in vivo. Results of this project will provide important information to the prevention and treatment of cancer and reproductive diseases.

Mps1是调节有丝分裂和减数分裂纺锤体组装检验点(SAC)的重要激酶，功能与其磷酸化状态有关。申请人在前期工作中鉴定了两种Mps1特异性磷酸酶，PPM1A和PPM1B，发现二者可使Mps1自身磷酸化及募集底物必需的S821位点去磷酸化。为了验证PPM1A和PPM1B是否通过对Mps1的去磷酸化作用对其功能进行调控，本项目拟在原有基础上分别在体外、细胞和个体水平上开展PPM1A/1B对Mps1相关功能调控的机制研究：体外将研究PPM1A/1B对Mps1的生化调控并探明二者的亚细胞定位及运动过程；在细胞水平上采取过表达及调低表达的方法，研究PPM1A/1B对有丝分裂SAC及其相关的细胞周期进程和染色体稳定性的影响及机制；分别制备PPM1A和PPM1B条件基因敲除小鼠，在个体水平上初步研究二者通过调控Mps1活性对减数分裂SAC以及卵子发生的影响。研究将为恶性肿瘤和生殖相关疾病防治提供理论参考。

Mps1是调节细胞分裂纺锤体组装检验点(SAC)的重要激酶。在前期工作中鉴定了两种Mps1特异性磷酸酶PPM1A和PPM1B。为验证二者是否通过对Mps1的去磷酸化作用对其进行调控，本项目开展了PPM1A/1B和Mps1体外相互作用研究。在细胞水平采取调低磷酸酶表达的方法，研究了PPM1A/1B对Mps1在细胞中定位的影响。研究结果显示，PPM1A和PPM1B可以在体内和体外分别与Mps1产生结合，从而初步证明PPM1A/1B是通过对Mps1的去磷酸化作用对其功能进行调控。细胞水平上研究显示，在敲低表达PPM1A/1B的细胞中，Mps1在有丝分裂前期发生磷酸化结合到着丝粒并形成SAC以后，因为没有受到PPM1A/1B的去磷酸化作用，在有丝分裂中期依然保持磷酸化状态，因而在有丝分裂中期依然定位在着丝粒上。这一结果说明，PPM1A/1B具有通过控制Mps1的磷酸化状态来达到控制有丝分裂SAC活性的作用。为了进一步验证磷酸酶对Mps1功能的调节作用，还进行了相关基因敲除小鼠的制备。目前已经得到Mps1基因敲除小鼠，为后续的Mps1在不同组织中的功能研究，尤其为卵子发生的减数分裂细胞周期研究提供了重要的动物模型。