Study on cellular signal transduction of Neurofibromatosis Type 2 tumor suppressor gene product (Merlin)

神经纤维瘤病2型抑癌基因产物（Merlin）的细胞信号转导研究

• 批准号: 12671369
• 负责人: ARAKI Norie
• 金额: $ 2.5万
• 依托单位: Kumamoto University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671369/
• 关键词: Neurofibromatosis Type 2; NF2; Merlin; Tumor suppressor gene; Poly (ADP-ribose) polymerase (PARP); Ku-antigen; DNA-Pks; 神経線維腫瘍2型; poly (ADP-ribose)polymerase (PARP); CD44; poly(ADP-ribose)polymerase(PARP)

The neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited disorder, and strongly associated with development of benign intracranial tumors including bilateral vestibular schwannomas and meningiomas. NF2 gene was recently cloned, and the protein it encodes (termed as merlin) was found to be striking similarity to the moesin-ezrin-radixin (MER) family of cytoskeleton-associated proteins. To elucidate the biological function of NF2 protein (merlin), we analyzed the cellular localization and signal transduction of merlin. 1) Cellular expression systems of mutants or wild NF2 were established, and their cellular localization was analyzed by con focal lazer scanning microscopy (CLSM). The wild merlin showed cytoplasmic and submembranous localization that was found to be directed by its nuclear export signal (NES) dependent transport mechanism. The N-terminal mutation resulted in nuclear accumulation of merlin. 2) Cellular binding proteins of merlin were purified from bovine bra … More in extracts and identified as poly ADP-ribose polymerase, DNA-PK subunits Ku-antigen 85 and 70 by the analysis of their internal ammo-acid sequences. The N-terminal (19-339) region of merlin is essential for their interaction. The immuno-precipitation, western blotting, and CLMS study confirmed their cellular co-localization. NF2 mutations impair the merlin-related complex formation and their cellular signal transport. 3) Subcellular co-localizations of merlin and PARP, overexpressed in VA13 cells and mouse embryonic fibroblast (MEF), were observed mainly in their nuclear region when cells were treated with leptomycin B (LMB), an inhibitor of NES receptor Crm1. This nuclear accumulation of merlin increased with cellular DNA damages induced by bleomycin. 4) In marked contrast to wild type cells, PARP gene deficient MEF could not accumulate merlin in their nuclear region, even after the treatments of LMB and bleomycin, whereas the overexpression of PARP in those cells worked complementary to the cellular localization of merlin. These results suggest that merlin is a cytoplasmic-nuclear shuttling protein directed by its NES-dependent transport pathway being associated with PARP. The tumor suppressive function of merlin may be linked to cellular DNA-repair and related signals via the interaction of cellular merlin binding proteins such as PARP and DNA-PKs. Less

神经纤维瘤病2型（NF 2）是一种常染色体显性遗传疾病，与良性颅内肿瘤包括双侧前庭神经鞘瘤和脑膜瘤的发生密切相关。NF 2基因是最近克隆的，它编码的蛋白质（称为merlin）被发现与细胞凋亡相关蛋白中的膜突蛋白-埃兹蛋白-根蛋白（MER）家族具有惊人的相似性。为了阐明NF 2蛋白（merlin）的生物学功能，我们分析了merlin的细胞定位和信号转导。1)建立了突变体和野生型NF 2的细胞表达系统，并用共聚焦激光扫描显微镜（CLSM）分析了突变体和野生型NF 2的细胞定位。野生型merlin蛋白在细胞质和膜下的定位与其依赖于核输出信号（内斯）的转运机制有关。N-末端突变导致merlin在核中积累。2)... class ='class10'>牛血清中分离纯化了merlin的细胞结合蛋白 ...更多信息 经氨基酸序列分析，鉴定为聚ADP核糖聚合酶、DNA-PK亚基Ku抗原85和70。Merlin蛋白的N端（19-339）区域是它们相互作用的关键。免疫沉淀、蛋白质印迹和CLMS研究证实了它们的细胞共定位。NF 2突变损害merlin相关复合物的形成及其细胞信号传递。3)merlin和PARP在VA 13细胞和小鼠胚胎成纤维细胞（MEF）中过表达，当细胞用内斯受体Crm 1抑制剂leptomycin B（LMB）处理时，观察到merlin和PARP的亚细胞共定位主要在其核区域。随着博来霉素诱导的细胞DNA损伤，merlin的核积累增加。4)与野生型细胞形成鲜明对比的是，PARP基因缺陷的MEF不能在其核区域积累merlin，即使在LMB和博来霉素处理后，而在这些细胞中PARP的过表达与merlin的细胞定位互补。这些结果表明，merlin是一个细胞质-核穿梭蛋白指导其NES依赖的运输途径与PARP。merlin的肿瘤抑制功能可能通过细胞merlin结合蛋白如PARP和DNA-PK的相互作用与细胞DNA修复和相关信号相关。少

项目成果

Araki, N., et al.: "Proteomic analysis of tumor suppressor related proteins in central nervous system"J. Chromatogr. B. (in press). (2002)

Araki, N., et al.：“中枢神经系统肿瘤抑制相关蛋白的蛋白质组学分析”J.

Araki,N., et.al.: "Proteomic analysis of tumor suppressor related proteins in brain."Protein Science. 9,(1). 162 (2000)

Araki,N., et.al.：“大脑中肿瘤抑制相关蛋白的蛋白质组学分析。”蛋白质科学。

Tokuo, H., Yunoue. S., Feng. L., Kimoto. M., Tsuji. H., Ono, T., Saya, H., and Araki, N.: "Phosphorylation of neurofibromin by cAMP dependent protein kinase is regulated via a cellular association of NG, NG-dimethylarginine dimethylaminohydrolase"FEBS Let

德雄，H.，汤上。

Araki,N., et.al.: "New Development in Glycomedicine, Study of autoantibody against advanced glycation endproducts of the Maillard reaction."Elsevier Science B.V(in press). (2001)

Araki,N., et.al.：“糖医学的新发展，针对美拉德反应晚期糖基化终产物的自身抗体的研究。”Elsevier Science B.V（出版中）。

Araki N., Morimasa T., Sakai T., Kamo M., Tokuoh H., Yunoue S., Miyazaki K., Abe K., Saya H., Tsugita A.: "Comparative analysis of brain proteins from p53 deficient mice by two-dimensional electrophoresis"Electrophoresis. 21. 1880-1889 (2000)

Araki N.、Morimasa T.、Sakai T.、Kamo M.、Tokuoh H.、Yunoue S.、Miyazaki K.、Abe K.、Saya H.、Tsugita A.：“p53 缺陷小鼠脑蛋白的比较分析