Nuclear Import of β-Dystroglycan Is Facilitated by Ezrin-Mediated Cytoskeleton Reorganization

The β-dystroglycan (β-DG) protein has the ability to target to multiple sites in eukaryotic cells, being a member of diverse protein assemblies including the transmembranal dystrophin-associated complex, and a nuclear envelope-localised complex that contains emerin and lamins A/C and B1. We noted that the importin α2/β1-recognised nuclear localization signal (NLS) of β-DG is also a binding site for the cytoskeletal-interacting protein ezrin, and set out to determine whether ezrin binding might modulate β-DG nuclear translocation for the first time. Unexpectedly, we found that ezrin enhances rather than inhibits β-DG nuclear translocation in C2C12 myoblasts. Both overexpression of a phosphomimetic activated ezrin variant (Ez-T567D) and activation of endogenous ezrin through stimulation of the Rho pathway resulted in both formation of actin-rich surface protrusions and significantly increased nuclear translocation of β-DG as shown by quantitative microscopy and subcellular fractionation/Western analysis. In contrast, overexpression of a nonphosphorylatable inactive ezrin variant (Ez-T567A) or inhibition of Rho signaling, decreased nuclear translocation of β-DG concomitant with a lack of cell surface protrusions. Further, a role for the actin cytoskeleton in ezrin enhancement of β-DG nuclear translocation was implicated by the observation that an ezrin variant lacking its actin-binding domain failed to enhance nuclear translocation of β-DG, while disruption of the actin cytoskeleton led to a reduction in β-DG nuclear localization. Finally, we show that ezrin-mediated cytoskeletal reorganization enhances nuclear translocation of the cytoplasmic but not the transmembranal fraction of β-DG. This is the first study showing that cytoskeleton reorganization can modulate nuclear translocation of β-DG, with the implication that β-DG can respond to cytoskeleton-driven changes in cell morphology by translocating from the cytoplasm to the nucleus to orchestrate nuclear processes in response to the functional requirements of the cell.

β-肌营养不良蛋白聚糖（β-dystroglycan, β-DG）是多种蛋白复合物的组成成员，能够靶向定位于真核细胞的多个位点，这些复合物包括跨膜肌营养不良蛋白关联复合物，以及定位于核被膜、含有emerin、核纤层蛋白A/C和B1的复合物。我们注意到，β-DG的被输入蛋白α2/β1（importin α2/β1）识别的核定位信号（nuclear localization signal, NLS）同时也是与细胞骨架相互作用的埃兹蛋白（ezrin）的结合位点，因此首次开展研究以探究埃兹蛋白的结合是否会调控β-DG的核转位。
出乎意料的是，我们在C2C12成肌细胞中发现，埃兹蛋白非但不会抑制，反而会增强β-DG的核转位。过表达磷酸化模拟型活化埃兹蛋白变体（Ez-T567D），或是通过激活Rho信号通路活化内源性埃兹蛋白，均可诱导形成富含肌动蛋白的表面突起，同时如定量显微镜成像、亚细胞分级分离/蛋白质免疫印迹分析所示，β-DG的核转位水平显著升高。
与之相反，过表达非磷酸化失活型埃兹蛋白变体（Ez-T567A）或是抑制Rho信号通路，则会降低β-DG的核转位水平，同时伴随细胞表面突起的缺失。进一步研究发现，缺失肌动蛋白结合结构域的埃兹蛋白变体无法增强β-DG的核转位，而破坏肌动蛋白细胞骨架则会降低β-DG的核定位水平，这表明肌动蛋白细胞骨架在埃兹蛋白增强β-DG核转位的过程中发挥了作用。
本研究还证实，埃兹蛋白介导的细胞骨架重排仅会增强胞质组分β-DG的核转位，而非跨膜组分β-DG。本研究首次揭示细胞骨架重排可调控β-DG的核转位，这意味着β-DG能够响应细胞形态的细胞骨架依赖性变化，从胞质移位至细胞核，以协调核内过程以满足细胞的功能需求。