EXAMINATION OF REGULATROY MECHANISMS FOR CELL FUNCTIONS BY THE GOLGI APPARATUS

高尔基体细胞功能调节机制的研究

• 批准号: 15570156
• 负责人: NAKAMURA Nobuhiro
• 金额: $ 2.37万
• 依托单位: KANAZAWA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570156/
• 关键词: GOLGI APPARATUS; PHOSPHORYLATION; KINASE; GROWTH SIGNAL

We have shown that a 277th amino acid residue of GRASP65 (S277) is phosphorylated in interphase cells and the phosphorylation signal is markedly enhanced by the growth factor treatment including EGF. ERK is activated by the EGF induced growth factor signal and the activated ERK phosphorylates S277 directly. We further found that S277 is heavily phosphorylated during M phase and analyzed the molecular mechanism for this up regulation of the phosphorylation. The amino acid sequence around S277 (PGSPG) is well conserved among mammalian CTRASP65 homologues. This sequence is well fitted with the target sequence of cdk1/cyclinB. S277 was strongly phosphorylated by a cytoplasmic extract of M phase cells and this was completely inhibited by roscovitine, a cdk specific inhibitor. S277 was also phosphorylated by an ERK inactive cytoplasmic extract of M phase cells that was prepared in the presence of U0126, a MEK inhibitor. These results strongly suggested that cdk1/cyclinB, and not ERK, is responsible for the phosphorylation of S277 in M phase. Surprisingly, the mitotic entry was strongly inhibited by the microinjection of purified GRASP65 without N-terminal myristoylation (Δm-GRASP65). This was not observed by the microinjection of Δm-GRASP65 in which S277 was changed with alanine. These results suggested that Δm-GRASP65 interact with some cytoplasmic factors and inhibits the mitotic entry. We have found that Plk1 specifically binds to phosphorylated S277 region of GRASP65 and there are some cytoplasmic factors that bind to unphosphorylated S277 region of GRASP65.

我们已经表明，GRASP 65的第277个氨基酸残基（S277）在间期细胞中被磷酸化，并且磷酸化信号被包括EGF在内的生长因子处理显著增强。ERK被EGF诱导的生长因子信号激活，激活的ERK直接磷酸化S277。我们进一步发现S277在M期被高度磷酸化，并分析了这种磷酸化上调的分子机制。S277（PGSPG）周围的氨基酸序列在哺乳动物CTRASP 65同源物中是非常保守的。该序列与cdk 1/cyclinB的靶序列吻合良好。S277被M期细胞的胞质提取物强烈磷酸化，并且这被cdk特异性抑制剂roscovitine完全抑制。S277也被M期细胞的ERK非活性胞质提取物磷酸化，所述提取物是在MEK抑制剂U 0126存在下制备的。这些结果强烈地表明，cdk 1/cyclinB，而不是ERK，是负责在M期的S277的磷酸化。令人惊讶的是，通过显微注射纯化的GRASP 65而没有N-末端豆蔻酰化（Δ m-GRASP 65）强烈抑制有丝分裂进入。通过显微注射Δ m-GRASP 65（其中S277被丙氨酸改变）未观察到这一点。这些结果表明Δ m-GRASP 65与细胞质中的某些因子相互作用，抑制有丝分裂进入。我们发现Plk 1与GRASP 65的磷酸化S277区域特异性结合，并且有一些细胞质因子与GRASP 65的未磷酸化S277区域结合。

项目成果

Structural integrity of the Golgi is temperature sensitive in conditional-lethal mutants with no detectable GM130

• DOI: 10.1034/j.1600-0854.2003.00080.x
• 发表时间: 2003-04-01
• 期刊: TRAFFIC
• 影响因子: 4.5
• 作者: Vasile, E;Perez, T;Krieger, M
• 通讯作者: Krieger, M

Yoshimura, S. et al.: "Dynamics of Golgi matrix proteins after a block of ER to Golgi transport"J.Biochem.. 135. 201-216 (2004)

Yoshimura, S. 等人：“ER 至高尔基体运输受阻后高尔基体基质蛋白的动力学”J.Biochem.. 135. 201-216 (2004)

Vasile, E., Perez, T., Nakamura, N., Krieger, M.: "Structural Integrity of the Golgi is Temperature Sensitive in Conditional-Lethal Mutants with No Detectable GM130"Traffic. 4. 254-272 (2003)

Vasile, E.、Perez, T.、Nakamura, N.、Krieger, M.：“在未检测到 GM130 的条件致死突变体中，高尔基体的结构完整性对温度敏感”。

Identification of a five-pass transmembrane protein family localizing in the Golgi apparatus and the ER.

• DOI: 10.1016/j.bbrc.2003.10.197
• 发表时间: 2003-12
• 期刊: Biochemical and biophysical research communications
• 影响因子: 3.1
• 作者: A. Shakoori;G. Fujii;S. Yoshimura;M. Kitamura;K. Nakayama;Takashi Ito;H. Ohno;N. Nakamura

Shakoori, A. et al.: "Identification of a five-pass transmembrane protein family localizing in the Golgi apparatus and the ER"Biochem.Biophys.Res.Commun.. 312. 850-857 (2003)

Shakoori, A. 等人：“定位于高尔基体和内质网的五次跨膜蛋白家族的鉴定”Biochem.Biophys.Res.Commun.. 312. 850-857 (2003)