Functional Analysis of ret Oncogene with Multiple Endocrine Neoplasia Type 2

2型多发性内分泌肿瘤ret癌基因的功能分析

• 批准号: 08671354
• 负责人: IMAI Tsuneo
• 金额: $ 1.41万
• 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671354/
• 关键词: ret proto-oncogene; MEN2A; MEN2B; Shc; Tyrosine kinase; tywsine Kinase; SHC; チロシンキナーゼ

Analysis of the intracellular signaling pathway through Ret activated by multiple endocrine neoplasia (MEN) 2A and 2B mutations.The ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A or 2B mutation can transform NIH3T3 cells with high efficiencies as a consequence of its constitutive activation. We analyzed the intracellular signaling pathway through Ret activated by MEN 2A,2B mutations, and glial-cell-line-derived neurotrophic factor (GDNF). The results showed that all of them induce a signal transducing complex consisting of Ret, Shc, and Grb2 proteins. In addition, GDNF clearly activated a Ras-MAPK pathway in human neuroblastoma cells. Ret is expressed mainly as two isoforms that differ in the carboxy-terminal sequence : a long isoform (1114 amino acids) and a short isoform (1072 amino acids). The long isoform contains the consensus sequence for binding of the Shc PTB domain but not of its SH2 domain, whereas the short isoform has the consensus sequences for binding of both domains. In vitro binding assay revealed that the long isoform of the MEN2A-Ret protein and both isoforms of the MEN2B-Ret protein bound preferentially to the Shc PTB domain. On the other hand, the short isoform of MEN2A-Ret bound to the PTB and SH2 domains. In neuroblastoma cells expressing both isoforms of Ret, its activation by GDNF also resulted in the binding of both domains. GDNF and MEN2A mutations activate Ret by inducing its dimerization, whereas the MEN2B mutation increases Ret catalytic activity without dimerization. Our results thus suggest that Ret dimerization might be required for binding of the Shc SH2 domain to the short isoform.

多内分泌肿瘤(MEN)2A和2B突变激活的Ret激活的细胞内信号通路分析带有多内分泌肿瘤(MEN)2A或2B突变的ret原癌基因由于其结构性激活，可以高效地转化NIH3T3细胞。我们通过MEN 2A、2B突变激活的Ret和胶质细胞系衍生神经营养因子(GDNF)分析了细胞内信号转导途径。结果表明，它们都能诱导出由Ret、Shc和Grb2蛋白组成的信号转导复合体。此外，GDNF明显激活了人神经母细胞瘤细胞中的Ras-MAPK通路。RET主要表达为两种在羧基末端序列不同的异构体：长异构体(1114个氨基酸)和短异构体(1072个氨基酸)。长异构体包含与Shc PTB结构域结合的共同序列，但不包含与其SH2结构域结合的共同序列，而短异构体具有两个结构域结合的共同序列。体外结合分析表明，MEN2A-Ret蛋白的长异构体和MEN2B-Ret蛋白的两个异构体优先与Shc PTB结构域结合。另一方面，MEN2A-Ret的短亚型结合在PTB和SH2结构域上。在表达这两种Ret异构体的神经母细胞瘤细胞中，GDNF激活Ret也导致这两个结构域的结合。GDNF和MEN2A突变通过诱导Ret的二聚化来激活Ret，而MEN2B突变在没有二聚化的情况下增加了Ret的催化活性。因此，我们的结果表明，Shc SH2结构域与短异构体的结合可能需要Ret二聚化。

项目成果

Asai N.Murakami H.Iwashita T,Takahashi M.: "A mutation at tyrosine 1062 in MEN2A-Ret and MEN2B-Ret impairs their transforming activity and association with shc adaptor proteins." Journal of Biological Chemistry. 271. 17644-17649 (1996)

Asai N.Murakami H.Iwashita T、Takahashi M.：“MEN2A-Ret 和 MEN2B-Ret 中酪氨酸 1062 的突变会损害它们的转化活性以及与 shc 接头蛋白的关联。”

浅井直也: "A mutation at Tyrosine 1062 in MEN2A-Ret and MEN2B-Ret impairs their trasforming activity and association with Shc adaptor proteins" The Journal of Biological Chemistry. Vol.271No.30. 17644-17649 (1996)

Naoya Asai：“MEN2A-Ret 和 MEN2B-Ret 中酪氨酸 1062 的突变损害了它们的转化活性以及与 Shc 接头蛋白的关联”《生物化学杂志》第 271 卷 17644-17649（1996 年）。

岩下寿秀: "Mechanism of Ret dysfunciton by Hirschsprung mutations affecting its extracelluler domanin" Oxford University Press. Vol.5No.10. 1577-1580 (1996)

Toshihide Iwashita：“先天性巨结肠突变影响其细胞外域的 Ret 功能障碍的机制”，牛津大学出版社，第 5 卷，第 1577-1580 期（1996 年）。

Masaki Wada et al.: "Detection of Ret Homodimers in MEN 2A-Associated Pheochromocytoma" Biochemical and Biophysical Research Communications. 218. 606-609 (1996)

Masaki Wada 等人：“MEN 2A 相关嗜铬细胞瘤中 Ret 同二聚体的检测”生物化学和生物物理研究通讯。

Mikinao Oiwa et al.: "Characterization of Ret-Sch-Grb2 Complex Induced by GDNF,MEN2A,and MEN2B Mutations." Biochemical and Biophysical Research Communications. 237. 747-751 (1997)

Mikinao Oiwa 等人：“GDNF、MEN2A 和 MEN2B 突变诱导的 Ret-Sch-Grb2 复合物的表征”。