Regulation of gene expression mediated by Ca^<2+>/calmodulin-dependent protein kinase cascade

Ca^2/钙调蛋白依赖性蛋白激酶级联介导的基因表达调节

• 批准号: 12680637
• 负责人: TOKUMITSU Hiroshi
• 金额: $ 2.43万
• 依托单位: Kagawa Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680637/
• 关键词: Intracellalr Calcium; Calmodulin; CaM-KK; Sgnal Transduction; Protein Kinase cascade; CREB; C.elegans; CaM-kinase; 細胞内カルシウム

Ca^<2+>/calmodulin-dependent protein kinases (CaM-Ks) constitute a diverse group of enzymes, which are involved in many cellular responses mediated by an increase in the concentration of intracellular calcium. Previous studies have demonstrated that two multifunctionla CaM-kinases, CaM-KI and IV, are activated byphosphorylation of an activation loop Thr residue by an upstream CaM-kinase (CaM-KK) resulting in a large increase in catalytic efficiency. In this study, we have found that Ile441 in CaM-KKα is essential for the autoinhibition of CaM-KK and the binding orientation of CaM to CaM-KKα is not critical for relief of the autoinhibition. The unique binding orientation of CaM to CaM-KKα which was originally discovered using NMR analysis, was also confirmed with C.elegans CaM-KK by using X-ray crystallography. In contrast to CaM-KKα, CaM-KKβ-isoform has shown to exhibit enhanced Ca^<2+>/CaM-independent activity which is due to the second regulatory domain (residues 129-151) located in N-terminal of the catalytic domain. This domain inhibits the autoinhibition of CaM-KKβ resulting in generation of its autonomous activity. We also have generated C.elegans carrying CRE-GFP reporter gene and cloned C.elegans CREB. C.elegans CREB-mediated gene expression was induced by C.elegans CaM-K cascade (CaM-KK/CaM-KI) in transfected cells. In living worm, GFP-expression was induced by overexpression of C.elegans CaM-KI 1-295 (constitutively active mutant) in some neurons, which was not observed in CREB-deficient worm. This indicates that CaM-K cascade mediats CREB-dependent transcriptional activation is conserved in C.elegans.

Ca^2+/钙调蛋白依赖性蛋白激酶（CaM-Ks）是一组不同的酶，它们参与由细胞内钙离子浓度增加介导的许多细胞反应。已有的研究表明，两种多功能的钙调素激酶，钙调素激酶Ⅰ（CaM-KI）和Ⅳ（IV），是通过上游钙调素激酶（CaM-KK）磷酸化激活环上的Thr残基而激活的，从而导致催化效率的大幅提高。在本研究中，我们发现CaM-KKα中的Ile 441对于CaM-KK的自抑制是必需的，并且CaM与CaM-KKα的结合方向对于解除自抑制不是关键的。最初使用NMR分析发现的CaM与CaM-KKα的独特结合取向也通过使用X射线晶体学与秀丽隐杆线虫CaM-KK证实。与CaM-KKα相比，CaM-KKβ-亚型显示出增强的Ca^2+/CaM非依赖性活性，这是由于位于催化结构域N端的第二个调节结构域（残基129-151）。该结构域抑制CaM-KKβ的自身抑制，导致其自主活性的产生。我们还制备了携带CRE-GFP报告基因的秀丽隐杆线虫，克隆了秀丽隐杆线虫CREB。在转染的细胞中，秀丽隐杆线虫CaM-K级联（CaM-KK/CaM-KI）诱导了秀丽隐杆线虫CREB介导的基因表达。在活虫中，GFP的表达被C.elegans CaM-KI 1-295（组成型活性突变体）的过表达诱导在一些神经元中，这在CREB缺陷的蠕虫中未观察到。这表明CaM-K级联介导CREB依赖的转录激活在秀丽隐杆线虫中是保守的。

项目成果

Tokumitsu, H.: "Differential Regulatory Mechanism of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase"Biochemistry. 40・46. 13925-13932 (2001)

Tokumitsu, H.：“Ca^<2+>/钙调蛋白依赖性蛋白激酶激酶的差异调节机制”生物化学 40・46（2001）。

Hiroshi Tokumitsu et al.: "Regulatory Mechanism of Ca^<2+>/Calmodulin-dependent Protein Kinase Kinase"The Journal of Biological Chemistry. 275. 20090-20095 (2000)

Hiroshi Tokumitsu 等人：“Ca^2/钙调蛋白依赖性蛋白激酶的调节机制”生物化学杂志。

Hirofumi Korokawa et al.: "Target-induced Conformational Adaptation of Calmodulin Revealed by the Crystal Structure of a Complex with Nematode Ca^<2+>/Calmodulin-dependent Protein Kinase Kinase Peptide"Jouranal of Molecular Biology. 312. 59-68 (2001)

Hirofumi Korokawa等人：“由线虫Ca^2/钙调蛋白依赖性蛋白激酶激酶肽复合物的晶体结构揭示的钙调蛋白的靶标诱导构象适应”分子生物学杂志。

徳光 浩: "Regulatory Mechanism of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase"The Journal of Biological Chemistry. 275. 20090-20095 (2000)

Hiroshi Tokumitsu：“Ca^<2+>/钙调蛋白依赖性蛋白激酶激酶的调节机制”生物化学杂志275。20090-20095（2000）

Kurokawa, H.: "Target-induced Conformational Adaptation of Calmodulin Revealed by the Crystal Structure of a Complex with Nematode Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase"Journal of Molecular Biology. 312. 59-68 (2001)

Kurokawa，H.：“线虫Ca^2/钙调蛋白依赖性蛋白激酶激酶复合物的晶体结构揭示了钙调蛋白的靶标诱导构象适应”分子生物学杂志。