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

1. 为阐明多发性1型内分泌肿瘤致病基因蛋白产物menin介导的内分泌细胞增殖调控机制，采用免疫沉淀法探索内分泌细胞中与menin结合的蛋白。用表达Flag-tagged menin的质粒稳定转染大鼠胰岛肿瘤分泌胰岛素的培养细胞。用抗flag抗体对细胞裂解液进行免疫沉淀，得到几种与menin共沉淀的蛋白。质谱分析显示共沉淀蛋白包括热休克蛋白、核糖体蛋白和转录因子样蛋白。哺乳动物细胞双杂交分析表明，后者与menin结合较弱。免疫细胞化学分析表明，该蛋白以斑点状分布于核旁区。Menin先前已被证明具有GTPase活性。用体外转录翻译系统合成的Menin进行GTPase活性测定。体外合成的menin未显示出GTPase活性，提示可能需要翻译后修饰才能具有menin的酶活性。通过1例多发性1型内分泌瘤患者的甲状旁腺肿瘤细胞实验，发现menin介导TGF-β对甲状旁腺细胞的抑制作用。对被迫在培养细胞中表达的各种错义突变menin蛋白的免疫印迹分析表明，几乎所有致病性错义MEN1基因突变都导致menin的快速降解。这些发现提示突变menin的不稳定性参与了MEN1基因错义突变引起的1型多发性内分泌瘤的发病机制。

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.