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

基本信息

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

项目摘要

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

2 型神经纤维瘤病 (NF2) 是一种常染色体显性遗传性疾病，与良性颅内肿瘤（包括双侧前庭神经鞘瘤和脑膜瘤）的发展密切相关，最近克隆了 NF2 基因，并发现其编码的蛋白质（称为 merlin）。与细胞骨架相关蛋白的 moesin-ezrin-radixin (MER) 家族惊人的相似性阐明 NF2 的生物学功能。蛋白（merlin），我们分析了merlin的细胞定位和信号转导。 1）建立突变体或野生NF2的细胞表达系统，并通过共焦激光扫描显微镜（CLSM）分析其细胞定位。发现其核输出信号 (NES) 依赖性转运机制引导的膜下定位导致 merlin 的核积累 2) 细胞结合蛋白。从牛胸罩提取物中纯化了 merlin，并通过分析其内部氨基酸序列 (19-339) 鉴定为聚 ADP-核糖聚合酶、DNA-PK 亚基 Ku-抗原 85 和 70。 merlin 区域对于它们的相互作用至关重要。免疫沉淀、蛋白质印迹和 CLMS 研究证实了它们的细胞共定位。 3) 当用瘦霉素 B (LMB) 处理细胞时，在 VA13 细胞和小鼠胚胎成纤维细胞 (MEF) 中过表达的 merlin 和 PARP 的亚细胞共定位主要在它们的核区域中观察到。），NES 受体 Crm1 的抑制剂。 merlin 的核积累随着博来霉素诱导的细胞 DNA 损伤而增加。 4) 与野生型细胞形成鲜明对比的是，PARP。即使在 LMB 和博来霉素处理后，基因缺陷的 MEF 也无法在细胞核区域积累 merlin，而这些细胞中 PARP 的过度表达与 merlin 的细胞定位互补。这些结果表明 merlin 是一种细胞质-核穿梭蛋白。 merlin 的肿瘤抑制功能可能通过细胞 merlin 结合蛋白的相互作用与细胞 DNA 修复和相关信号相关。例如 PARP 和 DNA-PK。