Koliou Xeni, Fedonidis Constantinos, Kalpachidou Theodora, Mangoura Dimitra
Basic Research Center, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
J Neurochem. 2016 Jan;136(1):78-91. doi: 10.1111/jnc.13401. Epub 2015 Nov 11.
Neurofibromatosis type-1 (NF-1) is caused by mutations in the tumor suppressor gene NF1; its protein product neurofibromin is a RasGTPase-activating protein, a property that has yet to explain aneuploidy, most often observed in astrocytes in NF-1. Here, we provide a mechanistic model for the regulated nuclear import of neurofibromin during the cell cycle and for a role in chromosome congression. Specifically, we demonstrate that neurofibromin, phosphorylated on Ser2808, a residue adjacent to a nuclear localization signal in the C-terminal domain (CTD), by Protein Kinase C-epsilon (PKC-ε), accumulates in a Ran-dependent manner and through binding to lamin in the nucleus at G2 in glioblastoma cells. Furthermore, we identify CTD as a tubulin-binding domain and show that a phosphomimetic substitution of its Ser2808 results in a predominantly nuclear localization. Confocal analysis shows that endogenous neurofibromin localizes on the centrosomes at interphase, as well as on the mitotic spindle, through direct associations with tubulins, in glioblastoma cells and primary astrocytes. More importantly, analysis of mitotic phenotypes after siRNA-mediated depletion shows that acute loss of this tumor suppressor protein leads to aberrant chromosome congression at the metaphase plate. Therefore, neurofibromin protein abundance and nuclear import are mechanistically linked to an error-free chromosome congression. Concerned with neurofibromin's, a tumor suppressor, mechanism of action, we demonstrate in astrocytic cells that its synthesis, phosphorylation by Protein Kinase C-ε on Ser2808 (a residue adjacent to a nuclear localization sequence), and nuclear import are cell cycle-dependent, being maximal at G2. During mitosis, neurofibromin is an integral part of the spindle, while its depletion leads to aberrant chromosome congression, possibly explaining the development of chromosomal instability in Neurofibromatosis type-1. Read the Editorial Highlight for this article on page 11. Cover Image for this issue: doi: 10.1111/jnc.13300.
1型神经纤维瘤病（NF-1）由肿瘤抑制基因NF1的突变引起；其蛋白质产物神经纤维瘤蛋白是一种RasGTP酶激活蛋白，这一特性尚未解释非整倍体现象，非整倍体在NF-1的星形胶质细胞中最为常见。在此，我们提供了一个机制模型，用于解释神经纤维瘤蛋白在细胞周期中受调控的核输入以及在染色体排列中的作用。具体而言，我们证明，在胶质母细胞瘤细胞中，蛋白激酶C-ε（PKC-ε）使神经纤维瘤蛋白在Ser2808位点（C末端结构域（CTD）中与核定位信号相邻的一个残基）磷酸化后，以依赖Ran的方式积累，并通过在G2期与核纤层蛋白结合而进入细胞核。此外，我们确定CTD是一个微管蛋白结合结构域，并表明其Ser2808位点的磷酸模拟取代导致主要定位于细胞核。共聚焦分析表明，在胶质母细胞瘤细胞和原代星形胶质细胞中，内源性神经纤维瘤蛋白通过与微管蛋白直接结合，在间期定位于中心体，在有丝分裂纺锤体上也有定位。更重要的是，对小干扰RNA介导的缺失后有丝分裂表型的分析表明，这种肿瘤抑制蛋白的急性缺失会导致中期板处染色体排列异常。因此，神经纤维瘤蛋白的丰度和核输入在机制上与无错误的染色体排列相关。关于肿瘤抑制蛋白神经纤维瘤蛋白的作用机制，我们在星形细胞中证明，其合成、蛋白激酶C-ε在Ser2808位点（与核定位序列相邻的一个残基）的磷酸化以及核输入均依赖细胞周期，在G2期达到最大值。在有丝分裂期间，神经纤维瘤蛋白是纺锤体的一个组成部分，而其缺失会导致染色体排列异常，这可能解释了1型神经纤维瘤病中染色体不稳定性的发生。阅读本期第11页关于本文的编辑推荐。本期封面图片：doi: 10.1111/jnc.13300 。
