Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing, 100069, China.
Mol Cells. 2013 Jul;36(1):47-54. doi: 10.1007/s10059-013-0014-0. Epub 2013 Jun 14.
The actin cytoskeleton plays an important role in cell shape determination, adhesion and cell cycle progression. Ezrinradixin-moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na(+)-H(+) exchanger regulatory factor 1 (NHERF1), associates with actin cytoskeleton and is related to cell cycle progression. Its Ser279 and Ser301 residues are phosphorylated by cyclin-dependent kinase 2 (cdc2)/cyclin B during the mitosis phase. However, the biological significance of EBP50 phosphorylation mediated by cdc2/cyclin B is not clear. In the present study, MDA-MB-231 cells with low levels of endogenous EBP50 protein were stably transfected with constructs of EBP50 wild type (WT), phosphodeficient (serine 279 and serine 301 mutated to alanine-S279A/S301A) or phospho-mimetic (serine 279 and serine 301 mutated to aspartic acid-S279D/S301D) mutants. Subsequently, multiple phenotypes of these cells were characterized. Failure of cdc2/cyclin B-mediated EBP50 phosphorylation in cells expressing S279A/S301A (AA cells) significantly increased F-actin content, enhanced the adherence of cells to the extracellular matrix, altered cell morphology and caused defects in cytokinesis, as reflected in the formation of giant cells with heteroploid DNA and multinucleation or giant nuclei. Furthermore, knockdown of EBP50 expression in AA cells rescued cell defects such as the cytokinesis failure and abnormal cell morphology. EBP50 S279A/ S301A had a weaker binding affinity with actin than EBP50 S279D/S301D, which might explain the increase of F-actin content in the AA cells. The present results suggest that cdc2/cyclin B-mediated EBP50 phosphorylation may play a role in the regulation of various cell functions by affecting actin cytoskeleton reorganization.
肌动蛋白细胞骨架在细胞形态决定、黏附和细胞周期进程中发挥重要作用。Ezrinradixin-moesin（ERM）结合磷蛋白 50（EBP50），也称为 Na(+)-H(+)交换体调节因子 1（NHERF1），与肌动蛋白细胞骨架相关，并与细胞周期进程有关。其丝氨酸 279 和丝氨酸 301 残基在有丝分裂期由细胞周期蛋白依赖性激酶 2（cdc2）/细胞周期蛋白 B 磷酸化。然而，cdc2/细胞周期蛋白 B 介导的 EBP50 磷酸化的生物学意义尚不清楚。在本研究中，用 EBP50 野生型（WT）、磷酸化缺陷型（丝氨酸 279 和丝氨酸 301 突变为丙氨酸-S279A/S301A）或磷酸模拟型（丝氨酸 279 和丝氨酸 301 突变为天冬氨酸-S279D/S301D）突变体的稳定转染 MDA-MB-231 细胞，使其内源性 EBP50 蛋白水平降低。随后，对这些细胞的多种表型进行了表征。在表达 S279A/S301A（AA 细胞）的细胞中，cdc2/细胞周期蛋白 B 介导的 EBP50 磷酸化失败会显著增加 F-肌动蛋白含量，增强细胞与细胞外基质的黏附，改变细胞形态，并导致胞质分裂缺陷，表现为异倍体 DNA 形成的巨细胞和多核或巨核。此外，在 AA 细胞中敲低 EBP50 表达可挽救细胞缺陷，如胞质分裂失败和异常细胞形态。EBP50 S279A/S301A 与肌动蛋白的结合亲和力弱于 EBP50 S279D/S301D，这可能解释了 AA 细胞中 F-肌动蛋白含量的增加。本研究结果表明，cdc2/细胞周期蛋白 B 介导的 EBP50 磷酸化可能通过影响肌动蛋白细胞骨架重组来调节各种细胞功能。
