Studies on the controlling mechanism of endocrine cell proliferation mediated by the MEN1 gene product menin

MEN1基因产物menin介导的内分泌细胞增殖调控机制研究

基本信息

批准号：
17590283
负责人：
TSUKADA Toshihiko
金额：
$ 2.43万
依托单位：
National Cancer Center Research Institute and Research Center for Innovative Oncology, National Cancer Center Hospital East
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

1. To elucidate the controlling mechanism for endocrine cell proliferation mediated by menin, the protein product of the causative gene for multiple endocrine neoplasia type 1, proteins binding to menin in endocrine cells were explored by immunoprecipitation. Insulin-secreting culture cells derived from a rat pancreatic islet tumor were stably transfected with plasmid expressing Flag-tagged menin. Immunoprecipitation of the cell lysate with anti-Flag antibody yeilded several proteins coprecipitated with menin. Mass spectrometric analyses revealed that the coprecipitated proteins included heat shock proteins, ribosomal proteins and transcription factor-like proteins. The latter has been shown to bind weakly to menin by the mammalian cell two-hybrid analysis. Immunocytochemical analyses demonstrated that this menin-binding protein is localized to juxtanuclear area in a spotted pattern.2. Menin has previously been demonstrated to have GTPase activity. Menin synthesized by the in vitro transcription and translation system was tested for GTPase activity. The in vitrosynthesized menin failed to show GTPase activity, suggesting that the post-translational modification may be required to have the enzyme activity of menin.3. By the experiments with parathyroid tumor cells derived from a patient of multiple endocrine neoplasia type 1, menin has been shown to mediate cell growth-inhibiting effects of TGF-β on parathyroid cells.4. Immunoblotting analyses of various missense mutant menin proteins that are forced to be expressed in culture cells demonstrated that almost all pathogenic missense MEN1 gene mutations caused rapid degradation of menin. These findings suggest that instability of the mutant menin is involved in the pathogenesis of multiple endocrine neoplasia type 1 caused by the missense mutations of the MEN1 gene.

1。为了阐明由Menin介导的内分泌细胞增殖的控制机制，Menin是多种内分泌肿瘤1型的致病基因的蛋白质产物，通过免疫原化探索了内分泌细胞中蛋白与梅宁的蛋白质结合。用大鼠胰岛肿瘤衍生的胰岛素分泌培养细胞稳定地用质粒表达标记为标记的梅宁。用抗FLAG抗体对细胞裂解物的免疫沉淀，与梅宁共沉淀的几种蛋白质。质谱分析表明，共沉淀的蛋白包括热休克蛋白，核糖体蛋白和转录因子样蛋白。通过哺乳动物两性杂交分析，后者已显示出与梅宁弱结合。免疫细胞化学分析表明，这种梅宁结合蛋白在斑点模式下定位于近核区域。2。梅宁先前已被证明具有GTPase活性。测试了通过体外转录和翻译系统合成的Menin的GTPase活性。玻璃合成的梅宁未能表现出GTPase活性，这表明可能需要进行翻译后修饰才能具有MENIN的酶活性。3。通过衍生自多内分泌肿瘤1型患者的甲状旁腺肿瘤细胞的实验，已证明Menin可介导TGF-β对甲状旁腺细胞的细胞生长抑制作用。4。在培养细胞中被迫表达的各种错义突变蛋白的免疫印迹分析表明，几乎所有致病的Men1基因突变都会导致梅宁迅速降解。这些发现表明，突变体的不稳定性参与由Men1基因的错义突变引起的多种内分泌肿瘤1型的发病机理。