Study on signal transduction mechanism of p120 RasGAP piotein

p120 RasGAP蛋白信号转导机制研究

• 批准号: 13672270
• 负责人: MORIOKA Hiroshi
• 金额: $ 2.3万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672270/
• 关键词: RasGAP; RhoGAP; Biacore; SH2 domain; SH3 domain; phosphorylated tyrosine; signal transduction; domain engineering; Ras; GTPase活性; Proline Rich Region

GTPase-activating proteins (GAPs) play an important role in signal tiansduction pathways regulated by small GTP-binding proteins RasGAP is composed of several domains. The C-terminus shows GAP activity as a negative regulator of Ras. The N-terminus contains SH2-SH3-SH2 that appears to be involved in interactions with signaling proteins, and functions as a Ras effector. These two SH2 domains have been found to associate stably with a tyrosine-phosphorylated protein, p190 RhoGAP, which is thought to regulate actin cytoskeleton dynamics. Several studies suggest that the interaction between Ras-GAP and Rho-GAP is regulated by phosphorylation of tyrosine residues, Tyr1087 and/or Tyr1105 in the middle domain of Rho-GAP, however, mechanisms for RasGAP-RhoGAP complex formation have not yet been determined.To investigate these regulation mechanisms for molecular interactions, seven recombinant proteins composed of SH2(n)-SH3-SH2(c) domains of RasGAP have been prepared and subjected to binding assays against three kinds of tyrosine-phosphorylated synthetic peptides of p190 RhoGAP by means of biosensor (BIAcore) respectively. Results obtained from biosensor analysis revealed that both SH2(n) and SH2(c) domain could bind to each tyrosine-phosphorylated peptide. In addition, the protein containing both SH2(n) and SH2(c) domains synergistically bound to the dual tyrosine-phosphorylated peptide.

GTP酶激活蛋白(GAP)在小分子GTP结合蛋白调控的信号转导通路中发挥重要作用，RasGAP由多个结构域组成。C末端是RAS的负性调节因子，具有GAP活性。N-末端含有SH2-SH3-SH2，它似乎参与了与信号蛋白的相互作用，并发挥了RAS效应的功能。这两个SH2结构域被发现与酪氨酸磷酸化蛋白p190 RhoGAP稳定相关，p190 RhoGAP被认为调节肌动蛋白细胞骨架的动力学。一些研究表明，Ras-GAP和Rho-GAP之间的相互作用受Rho-GAP中间结构域酪氨酸残基、Tyr1087和/或Tyr1105的磷酸化调节，但RasGAP-RhoGAP复合体的形成机制尚未确定。为了研究这些分子相互作用的调节机制，我们制备了7种由RasGAP的SH2(N)-SH3-SH2(C)结构域组成的重组蛋白，并利用生物传感器(Biacore)分别与p190 RhoGAP的三种酪氨酸磷酸化合成肽进行了结合分析。生物传感器分析结果表明，SH2(N)和SH2(C)结构域都能与每个酪氨酸磷酸化肽结合。此外，含有SH2(N)和SH2(C)结构域的蛋白质与双酪氨酸-磷酸化肽协同结合。

项目成果

Kurosaki, Y.: "Pyrimidine dimer formation and oxidative damage in M13 bacteriophage inactivation by ultraviolet C irradiation."Photochem.Photobiol.. 78. 349-354 (2003)

Kurosaki, Y.：“紫外线 C 照射导致 M13 噬菌体失活中的嘧啶二聚体形成和氧化损伤。”Photochem.Photobiol.. 78. 349-354 (2003)

Sakurai S., Kitano K., Okada K., Hamada K., Morioka H., Hakoshima T: "Preparation and crystallization of human flap endonuclease FEN-1 in complex with proliferating-cell nuclear antigen, PCNA."Acta Cryst.. D59. 933-935 (2003)

Sakurai S.、Kitano K.、Okada K.、Hamada K.、Morioka H.、Hakoshima T：“人瓣核酸内切酶 FEN-1 与增殖细胞核抗原 PCNA 复合物的制备和结晶。”Acta Cryst..

Kobayashi H.: "DNA binding mode of antibody fragments specific for TT photo-dimers."Phosphorus Sulfur and Silicon. 177. 1553-1556 (2002)

Kobayashi H.：“TT 光二聚体特异性抗体片段的 DNA 结合模式。”磷硫和硅。

Suzuki N., Ichikawa N., Kasai, S., Yamada M., Nishi N., Morioka H., Yamashita H., Kitagawa Y., Utani A., Hoffman M.P., Nomizu M: "Syndecan binding sites in the laminin al chain G domain."Biochemistry. 42. 12625-12633 (2003)

Suzuki N.、Ichikawa N.、Kasai, S.、Yamada M.、Nishi N.、Morioka H.、Yamashita H.、Kitakawa Y.、Utani A.、Hoffman M.P.、Nomizu M：“层粘连蛋白中的 Syndecan 结合位点

Suzuki N.: "Syndecan binding sites in the laminin al chain G domain."Biochemistry. 42. 12625-12633 (2003)

Suzuki N.：“层粘连蛋白α1链G结构域中的Syndecan结合位点。”生物化学。