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）构成了潜水酶，这些酶涉及许多由细胞内钙浓度升高介导的许多细胞反应。先前的研究表明，通过上游CAM-激酶（CAM-KK）激活了两个多函数CAM-激酶，即CAM-KI和IV，从而激活了激活环的THR住宅，从而大大提高了催化效率。在这项研究中，我们发现CAM-KKα中的ILE441对于CAM-KK的自身抑制至关重要，CAM与CAM-KKα的结合方向对于缓解自身抑制并不是至关重要的。最初使用NMR分析发现的CAM与CAM-KKα的独特结合方向也通过使用X射线晶体学证实了C.Elegans CAM-KK。与CAM-KKα相反，CAM-KKβ-异型类型已显示出增强的Ca^<2+>/与CAM非依赖性活性，这是由于第二个调节域（残基129-151），位于催化域的N末端。该结构域抑制了CAM-KKβ的自身抑制作用，从而产生其自主活性。我们还产生了携带CRE-GFP记者基因并克隆的C.Elegans Creb的C.Elegans。 C.Elegans CREB介导的基因表达是通过翻译细胞中的C.Elegans Cam-K-K-K-cascade（CAM-KK/CAM-KI）诱导的。在活蠕虫中，通过某些神经元中的C.Elegans cam-ki 1-295（组成型活性突变体）过表达诱导GFP表达，这在CREB缺陷蠕虫中未观察到。这表明CAM-K级联体介导CREB依赖性转录激活在C.Elegans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。

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。

徳光浩,村松正明,伊倉光彦,小林良二: "Regulatory Mechanism of Ca^<2+>/Calmodulin-dependent Protein Kinase Kinase"The Journal of Biological Chemistry. 275・26. 20090-20095 (2000)

Hiroshi Tokumitsu、Masaaki Muramatsu、Mitsuhiko Ikura、Ryoji Kobayashi：“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・26（2000）。