【作者】 邓辉；

【导师】 姚雪彪；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2010， 博士

【摘要】 在正常的生物机体中,生物学活动是通过各种蛋白的复杂的功能作用网络实现,而蛋白功能的行使依赖于其活性的动态调控。一般而言,由蛋白分子本身可塑性所介导的基于蛋白翻译后修饰引起的构象变化,可以动态的调控蛋白分子的生物活性,进而调控其功能的执行。同一个蛋白的不同翻译后修饰途径可以引起其分子可塑性的不同改变,从而介导不同生物学功能的行使。Ezrin是一类典型的膜-骨架连结蛋白,在膜与骨架相关的生物学动态调控中具有重要作用,早期研究显示其功能受多个磷酸化位点的磷酸化修饰的调控。在胃壁细胞中,Ezrin定位于顶膜,并受到PKA对其66位丝氨酸的磷酸化调控,影响胃酸的分泌过程。在研究细胞膜-骨架动力学调控的过程中,我们鉴定了一个新的Arf6特异的GAP蛋白ACAP4,并且证实了Ezrin-ACAP4-Arf6蛋白功能复合物在调控膜-骨架重构功能中具有重要作用。基于这些结论,我们以胃壁细胞泌酸过程为模式体系,探讨了Ezrin在PKA介导的66位丝氨酸磷酸化修饰下,通过其自身构象的变化,调整与ACAP4的相互作用。在泌酸的胃壁细胞中,这种可控的相互作用使得ACAP4被Ezrin特异性的转位到顶膜,从而引起随后的一系列与膜-骨架动力学相关的动态调控功能,促进了胃壁细胞的泌酸活性。在这个过程中Ezrin-ACAP4-Arf6为中心的相互作用网络发挥了重要的作用。这其中,Ezrin的分子可塑性无疑为极化上皮细胞分泌的调控机制提供了一个新的解释。另外,还有研究显示,Ezrin的功能行使同其567位苏氨酸的磷酸化也密不可分。早期研究显示,567位的磷酸化可以打开静息期闭合的Ezrin构象,是其活化的必要步骤。而在肝癌组织中的研究显示,Ezrin的567位苏氨酸的磷酸化同肿瘤细胞的侵染活性紧密相关。利用567位磷酸化特异性的抗体对不同肝癌细胞样品进行免疫印迹可以证明在具有高侵染活性的肝癌细胞中,Ezrin的567位苏氨酸处于高度磷酸化水平。而在低侵染活性的HepG2细胞中转入567位模拟磷酸化的Ezrin突变体,可以促进其侵染能力的提高,也可以证明Ezrin的567位磷酸化对肿瘤细胞的侵染活性有重要的促进作用。同时,我们相关的原子力显微镜数据显示,在567位磷酸化过程中Ezrin经历了由N-C相互结合的闭合状态到打开状态的构象变化,从而暴露出与相关蛋白的作用位点,激活了其多样化的生物学功能。而66位的磷酸化过程则是在567位磷酸化使其N-C结合的构象打开后,进一步使其N端原本闭合的构象进一步打开。结合其构象变化同功能调控的关系,不难看出,Ezrin分子可塑性的存在,直接促成了其高度动态性的功能调控作用。综上所述,本研究阐述了Arf6-ACAp4-ezrin蛋白质作用网络在壁细胞酸分泌过程中的重要作用及调控分子机制,揭示了Ezrin在肝癌转移中的重要调控作用。并且初步阐明了Ezrin通过自身的分子可塑性,在磷酸化的调控作用下,动态行使功能的分子机理。更多还原

【Abstract】 In regular organisms, different biological functions are based on functionally organized network comprised of diversity of proteins. The protein function depends on the dynamic regulation of its activity. Generally, due to the molecular plasticity of the protein, the activity will be dynamically regulated by the protein structural change induced by specific post translational modification.Different specific site modification of a same protein may result in different plastically change of the molecule, consequently regulate different cellular function.Ezrin is a typical plasma membrane-cytoskeleton linker protein. It plays important role in membrane-cytoskeleton dynamic regulation. Our previous study revealed that its function was regulated by different site phosphorylation. In gastric parietal cells, Ezrin was localized to the apical membrane. It controls gastric acid secretion by phosphrylation of PKA on its 66 serine. In research of membrane-cytoskeleton dynamic regulation, we idetified a novel Arf6 specific GAP--ACAP4, and proved that a typical function complex comprized of Ezrin-ACAP4-Arf6 play an important role in membrane-cytoskeleton remodeling. Thus, we use gastric parietal cells as our model system, to study that upon the phosphorylation of PKA on 66 serine of Ezrin, the structural change of Ezrin rearranged the interaction between Ezrin and ACAP4.This rearrangement translocated ACAP4 to the specific area on apical membrane,and induced a subsequently membrane-cytoskeleton remoduling. As a result, the gastric acid secretion was avitvated. Undoubtedly, Ezrin-ACAP4-Arf6 complex function importantly on this secretion process. And the molecular plasticity may play the most important role.Also, we have revealed that the function of Ezrin was activated by its 567 threonine phosphorylation. Our previous study showed that this site phosphorylation will open the close form of Ezrin which is in dormant stage, and is the most important step on its activation. And our heptocellular carcinoma research inferred that 567 threonine phosphorylation by Rho kinase of Ezrin is critical for tumor cell metastasis. Using a phospho-amino acid specific antibody, the western blotting analysis showed that Thr567 hyperphosphorylation is correlated with the invasive phenotype of hepatocellular carcinoma and poorclinical outcome. Using phospho-mimicking mutants, it was demonstrated that the phosphorylation of ezrin at Thr567 promotes HepG2 cell invasion.Meanwhile, our correlated AFM data showed that the Thr567 phosphorylation made Ezrin change from the close form of N-C interaction to an open form, and expose the multiple functional sites in order to activate different cellular function. And Ser66 phosphorylation function after Thr567 phosphorylation and can open the close form of the N-terminal head of Ezrin. In all, the molecular plasticity insures the highly dynamic regulation of Ezrin’s function.更多还原