Ding Xia, Deng Hui, Wang Dongmei, Zhou Jiajia, Huang Yuejia, Zhao Xuannv, Yu Xue, Wang Ming, Wang Fengsong, Ward Tarsha, Aikhionbare Felix, Yao Xuebiao
Anhui Key Laboratory of Cellular Dynamics and Chemical Biology, University of Science and Technology of China, Hefei 230027, China.
J Biol Chem. 2010 Jun 11;285(24):18769-80. doi: 10.1074/jbc.M110.129007. Epub 2010 Apr 1.
The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437-1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin(S66D). Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.
埃兹蛋白-根蛋白-膜突蛋白家族可在膜蛋白与皮质细胞骨架之间建立起调控连接，还参与信号转导通路。埃兹蛋白定位于壁细胞的顶端膜，将蛋白激酶A激活级联反应与受调控的盐酸分泌相偶联。我们最近的蛋白质组学研究揭示了一种对挥发性膜重塑至关重要的埃兹蛋白-ACAP4-ARF6蛋白复合物（方Z、苗Y、丁X、邓H、刘S、王F、周R、沃森C、傅C、胡Q、利拉德JW、小鲍威尔M、陈Y、福特JG和姚X（2006年）《分子细胞蛋白质组学》5，1437 - 1449）。然而，ACAP4是否与埃兹蛋白发生物理相互作用以及它们的相互作用如何整合到膜-细胞骨架重塑过程中，目前仍不清楚。在此，我们首次提供证据表明，埃兹蛋白通过ACAP4的N端400个氨基酸以蛋白激酶A介导的磷酸化依赖性方式与ACAP4相互作用。在静息壁细胞中，ACAP4定位于细胞质膜，但在组胺刺激后会转位至顶端质膜。在分泌型而非静息型壁细胞裂解物中，ACAP4与埃兹蛋白共沉淀，提示存在磷酸化调控的相互作用。事实上，这种相互作用在磷酸酶处理后消失，并通过使用模拟磷酸化的埃兹蛋白（S66D）的体外重组试验得到验证。重要的是，埃兹蛋白决定了分泌型壁细胞中ACAP4的顶端分布，因为抑制埃兹蛋白或过表达不可磷酸化的埃兹蛋白都会阻止ACAP4的顶端定位。此外，过表达缺乏GTP酶激活蛋白的ACAP4会导致顶端膜-细胞骨架重塑和胃酸分泌受到抑制。综上所述，这些结果定义了一种将ACAP4-埃兹蛋白相互作用与极化上皮分泌相联系的新分子机制。
