Roles of C Kinase Substrate Protein, MARCKS and GAP-43, in Neurotransmitter Release

C 激酶底物蛋白、MARCKS 和 GAP-43 在神经递质释放中的作用

• 批准号: 06680773
• 负责人: TANIGUCHI Hisaaki
• 金额: $ 1.41万
• 依托单位: Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680773/
• 关键词: Mass spectrometry; Protein; Structure; Posttranslational modification; Phosphorylation; Synapse; 中枢神経; 開口放出; Cキナーゼ; MAPキナーゼ; シナプシンI; MARCKS

We have established an LC/MS system, in which a capillary HPLC column is connected on-line to an electrospray mass spectrometer, and studied posttranslational modificaitons of brain-specific phosphoproteins such as MARCKS,GAP-43, synapsin I,and MAP1B.The results obtained revealed novel phosphorylation sites including Ser (Thr) -Pro motif, suggesting that these substrate proteins of PKC,calmodulin-dependent protein kinase II or casein kinase II,are in vivo substrates of so-called proline-directed protein kinase. The in vitro phosphorylation experiments with MAP kinase and Cdk5, which are highly expressed in the brain, demonstrated that these kinases site-specifically phosphorylate the proteins. Other unknown protein kinases with the SP specificity, may also exist in the brain. Phosphorylation of synapsin I affected the bunding activity of actin filaments. These results suggest that the proline-directed protein kinases in addition to PKC and calmodulin-dependent protein kinase II play important roles in the regulation of neurotransmitter release. Furthermore, we have also demonstrated that the phosphorylation of MAP1B is essential for the synapse formation in the cultured cortical neurons. These studies established the importance of the proline-directed protein kinases in the brain funcitons.

我们建立了液相色谱-质谱联用系统，研究了脑特异性磷酸化蛋白MARCKS、GAP-43、突触蛋白I和MAP 1B的翻译后修饰。结果发现了包括Ser（Thr）-Pro基序在内的新的磷酸化位点，提示这些蛋白质可能是PKC的底物蛋白，钙调蛋白依赖性蛋白激酶II或酪蛋白激酶II是所谓的脯氨酸导向蛋白激酶的体内底物。用在脑中高度表达的MAP激酶和Cdk 5进行的体外磷酸化实验表明，这些激酶位点特异性地磷酸化蛋白质。其他未知的具有SP特异性的蛋白激酶也可能存在于脑中。突触蛋白I的磷酸化影响肌动蛋白丝的结合活性。这些结果表明，除了PKC和钙调素依赖性蛋白激酶II外，脯氨酸导向的蛋白激酶在神经递质释放的调节中起重要作用。此外，我们还证明了MAP 1B的磷酸化对于培养的皮层神经元中突触的形成是必不可少的。这些研究确立了脯氨酸导向蛋白激酶在脑功能中的重要性。

项目成果

谷口寿章: "質量分析法による脳Cキナーゼ基質タンパク質の翻訳後修飾の解析" Proc. Jap. Soc. Biomed. Mass Spectrom.19. 21-28 (1994)

Toshiaki Taniguchi：“通过质谱法分析脑 C 激酶底物蛋白”Proc. Biomed.1994。

Taniguchi, H. et al.: "A mass spectrometric study on the in vivo post-translational modification of GAP-43." J. Biol. Chem.269. 22481-22484 (1994)

Taniguchi, H. 等人：“GAP-43 体内翻译后修饰的质谱研究。”

谷口 寿章: "キャピラリーLC/MS法による蛋白質・核酸の修飾の解析" J. Mass Spectrom. Soc. Japan. (印刷中). (1996)

Toshiaki Taniguchi：“通过毛细管 LC/MS 方法分析蛋白质和核酸”J. Mass Spectrom 日本（出版中）。

Muramoto, K. et al.: "A Substrate of ecto-protein kinase is microtubule-associated protein 1B in cortical cell cultures undergoing synaptogenesis." Biochem. Biophys. Res. Commun.205. 1467-1473 (1994)

Muramoto, K. 等人：“胞外蛋白激酶的底物是正在经历突触发生的皮层细胞培养物中的微管相关蛋白 1B。”

Manenti, S. et al.: "Demyristoylation of Myristoylated Alanine-Rich C Kinase Substrate." Biochem. Soc. Trans.23. 561-564 (1995)

Manenti, S. 等人：“肉豆蔻酰化富含丙氨酸 C 激酶底物的去肉豆蔻酰化。”