Mechanism of the Expression of Various Phenotypes in Multiple Endocrine Neoplasia Type 2

2型多发性内分泌肿瘤各种表型的表达机制

• 批准号: 10671112
• 负责人: IMAI Tsuneo
• 金额: $ 2.5万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671112/
• 关键词: ret proto-oncogene; MEN2A; MEN2B; Shc; PI3-K; Gab1; GDNF; MAPK; ret proto-oncogene; PI3-k; tyrasine kinase; MEN 2A; MEN 2B; PTB; SH2; tyrosine kinase

Germ line mutations in ret proto-oncogene result in human hereditary diseases including MEN2A and MEN2B.MEN2A mutations induced disulfide-linked dimerization of the RET whereas MEN2B mutations appear to activate RET without dimerization. Tyrosine 1062 in RET represents a binding site for Shc adaptor proteins and is crucial for both RAS/ MAPK and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, Shc bound to Gab1 and Grb2 adaptor proteins as well as RET, and Shc and Gab1 were highly phosphorylated on tyrosine. The complex formation consisting of Shc, Gab1 and Grb2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine, Tyrosine-phosphorylated Gab1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas Shc-Grb2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through Shc bound to tyrosine 1062 in RET.Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-kappaB in GDNF-treated cells, respectively.

RET原癌基因的生殖系突变导致包括MEN 2A和MEN 2B在内的人类遗传性疾病。MEN 2A突变诱导RET的二硫键连接二聚化，而MEN 2B突变似乎激活RET而不二聚化。RET中的酪氨酸1062代表Shc衔接蛋白的结合位点，并且对于RAS/ MAPK和磷脂酰肌醇3-激酶（PI 3-K）/AKT信号传导途径都是至关重要的。在本研究中，我们的特点是如何从酪氨酸1062这两个途径分歧，使用人神经母细胞瘤和原始神经外胚层肿瘤细胞系高水平表达RET。在GDNF刺激下，Shc与Gab 1和Grb 2衔接蛋白以及RET结合，Shc和Gab 1在酪氨酸上高度磷酸化。酪氨酸磷酸化的Gab 1还与PI 3-K的p85亚基结合，导致PI 3-K和AKT的激活，而Shc-Grb 2-SOS复合物负责RAS/ERK信号通路。这些结果表明，GDNF激活的RAS和PI 3-K信号通路主要通过Shc与RET中的酪氨酸1062位点结合而发生分叉。此外，荧光素酶标记基因分析发现，RAS/ERK和PI 3-K信号通路分别对GDNF处理的细胞中CREB和NF-kappaB的激活起重要作用。

项目成果

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Iwashita T、Murakami H、Kurokawa K、Kawai K、Miyauchi A、Futami H、Qiao S、Ichihara M、Takahashi M.：“用于发展多种内分泌肿瘤 2B 型 RET 突变的二次打击模型。”Biochem Biocphys Res Commun

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Murakami H, Iwashita T, Asai N, Shimono Y, Iwata Y, Kawai K, Takahashi M.: "Enhanced phosphatidylinositol 3-kinase activity and high phosphorylation state of its downstream signalling molecules mediated by Ret with the MEN 2B mutation."Biochem Biophys Res

Murakami H、Iwashita T、Asai N、Shimono Y、Iwata Y、Kawai K、Takahashi M.：“MEN 2B 突变的 Ret 介导的磷脂酰肌醇 3 激酶活性增强及其下游信号分子的高磷酸化状态。”Biochem Biophys