Aguilar Areli, Wagstaff Kylie M, Suárez-Sánchez Rocío, Zinker Samuel, Jans David A, Cisneros Bulmaro
*Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico.
*Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico
FASEB J. 2015 May;29(5):1842-58. doi: 10.1096/fj.14-257147. Epub 2015 Jan 30.
Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased accumulation of cells in S phase and, significantly, altered localization of lamins A/C, B1, and B2 with accompanying gross nuclear morphology defects. Because α-DB interacts specifically with lamin B1 in vivo and in vitro, nuclear α-DB would appear to play a key role in nuclear shape maintenance through association with the nuclear lamina.
尽管α-肌营养不良蛋白（DB）组装进肌营养不良蛋白相关蛋白复合体中，该复合体对细胞骨架组织至关重要，但它也在细胞核中被发现。在此，我们首次描绘了负责α-DB核靶向的核输入途径，以及核α-DB在决定核形态方面的重要性。我们使用该蛋白的各种截短版本以及定点诱变，绘制了α-DB锌指结构域（ZZ）内核定位信号的关键残基。下拉、免疫沉淀和AlphaScreen分析表明，输入蛋白（IMP）α2/β1异二聚体与α-DB的ZZ结构域高亲和力相互作用。使用针对特定输入蛋白的抗体进行的体外核输入分析，以及使用小干扰RNA（siRNA）或显性负性输入蛋白构建体进行的体内研究，证实了IMPα2/β1在α-DB核转运中的关键作用。敲低α-DB表达扰乱了C2C12成肌细胞的细胞周期进程，S期细胞积累减少，并且显著改变了核纤层蛋白A/C、B1和B2的定位，伴有明显的核形态缺陷。由于α-DB在体内和体外都与核纤层蛋白B1特异性相互作用，核α-DB似乎通过与核纤层结合在维持核形状中起关键作用。
