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 原癌基因的种系突变会导致人类遗传性疾病，包括 MEN2A 和 MEN2B。MEN2A 突变诱导 RET 的二硫键连接二聚化，而 MEN2B 突变似乎激活 RET 而无需二聚化。 RET 中的酪氨酸 1062 代表 Shc 接头蛋白的结合位点，对于 RAS/MAPK 和磷脂酰肌醇 3-激酶 (PI3-K)/AKT 信号通路至关重要。在本研究中，我们使用高水平表达 RET 的人神经母细胞瘤和原始神经外胚层肿瘤细胞系，描述了这两条途径如何与酪氨酸 1062 不同。响应 GDNF 刺激，Shc 与 Gab1 和 Grb2 接头蛋白以及 RET 结合，并且 Shc 和 Gab1 在酪氨酸上高度磷酸化。由Shc、Gab1和Grb2组成的复合物形成几乎被苯丙氨酸取代RET中的酪氨酸1062所消除，酪氨酸磷酸化的Gab1还与PI3-K的p85亚基相关，导致PI3-K和AKT激活，而Shc-Grb2-SOS复合物负责RAS/ERK信号通路。这些结果表明，GDNF 激活的 RAS 和 PI3-K 通路主要通过 Shc 与 RET 中的酪氨酸 1062 结合而分叉。此外，使用荧光素酶报告基因检测，我们发现 RAS/ERK 和 PI3-K 信号通路分别对于 GDNF 处理的细胞中 CREB ​​和 NF-kappaB 的激活很重要。

项目成果

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Kawai K、Iwashita T、Murakami H、Hiraiwa N、Yoshiki A、Kusakabe M、Ono K、Iida K、Nakayama A、Takahashi M.：“表达 RET 原癌基因的多发性内分泌肿瘤转基因小鼠的组织特异性癌变

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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