Sun Lei, Gao Jinmin, Dong Xin, Liu Min, Li Dengwen, Shi Xingjuan, Dong Jin-Tang, Lu Xianyu, Liu Chunyong, Zhou Jun
Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.
Proc Natl Acad Sci U S A. 2008 May 20;105(20):7153-8. doi: 10.1073/pnas.0710018105. Epub 2008 May 13.
EB1 (end-binding protein 1) is a key player in the regulation of microtubule dynamics. In concert with its binding partners, adenomatous polyposis coli and p150(glued), EB1 plays a crucial role in a variety of microtubule-based cellular processes. In this study we have identified in a yeast two-hybrid screen the mitotic kinase and chromosome passenger protein Aurora-B as a binding partner of EB1. GST pull-down and immunoprecipitation experiments reveal a specific interaction between Aurora-B and EB1 both in cells and in vitro. Immunofluorescence microscopy shows that these two proteins colocalize on the central spindle in anaphase and in the midbody during cytokinesis. Kinase assays using both immunoprecipitated and purified Aurora-B demonstrate that EB1 is not a substrate of Aurora-B. Rather, EB1 positively regulates Aurora-B kinase activity. EB1 overexpression remarkably enhances Aurora-B activity and knockdown of its expression impairs Aurora-B activity. Our data further show that EB1 is able to protect Aurora-B from dephosphorylation/inactivation by protein phosphatase 2A (PP2A) by blocking PP2A binding to Aurora-B. These findings establish Aurora-B as an EB1-interacting protein and suggest that EB1 stimulates Aurora-B activity through antagonizing its dephosphorylation/inactivation by PP2A.
EB1（末端结合蛋白1）是微管动力学调节中的关键因子。EB1与其结合伴侣腺瘤性息肉病蛋白和p150（glued）协同作用，在多种基于微管的细胞过程中发挥关键作用。在本研究中，我们通过酵母双杂交筛选鉴定出有丝分裂激酶和染色体乘客蛋白Aurora-B是EB1的结合伴侣。GST下拉实验和免疫沉淀实验表明，Aurora-B与EB1在细胞内和体外均存在特异性相互作用。免疫荧光显微镜显示，这两种蛋白在后期的中央纺锤体以及胞质分裂期间的中间体上共定位。使用免疫沉淀和纯化的Aurora-B进行的激酶分析表明，EB1不是Aurora-B的底物。相反，EB1正向调节Aurora-B激酶活性。EB1的过表达显著增强Aurora-B活性，而敲低其表达则损害Aurora-B活性。我们的数据进一步表明，EB1能够通过阻止蛋白磷酸酶2A（PP2A）与Aurora-B结合，保护Aurora-B不被去磷酸化/失活。这些发现确定了Aurora-B是一种与EB1相互作用的蛋白，并表明EB1通过拮抗PP2A介导的去磷酸化/失活来刺激Aurora-B活性。
