Physiological function of calcium/calmodulin-dependent protein kinase cascade

钙/钙调蛋白依赖性蛋白激酶级联的生理功能

• 批准号: 14580649
• 负责人: TOKUMITSU Hiroshi
• 金额: $ 2.56万
• 依托单位: Kagawa University(Faculty of Medicine)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580649/
• 关键词: Calmodulin; Intracellular calcium; Protein kinase; CaM-kinase; CaM-KK; Protein kinase inhibitor; STO-609; MLCK-A; 細胞内情報伝達; リン酸化酵素カスケード; CREB

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 multifunctional CaM-kinases, CaM-KI and IV, are activated by phosphorylation of an activation loop Thr residue by an upstream CaM-kinase kinase (CaM-KK) resulting in a large increase in catalytic efficiency. In order to evaluate the physiological functions of CaM-KK and of the CaM-kinase cascade, we attempted to synthesize a potent and specific inhibitor of CaM-KK, STO-609. In this study, we characterize the effects of the inhibitor STO-609 on CaM-KK activity both in vitro and in intact cells. Furthermore, based on the results that the distinct sensitivity of CaM-KK isoforms to STO-609 is due to a single amino acid substitution (Val/Leu) in the ATP-binding pocket, we have generated an STO-609-resistant CaM-KK mutant, which might be useful for validating the pharmacological effects and specificity of STO-609 in vivo. We also have examined the activation mechanism of Dictyostelium MLCK-A by using constitutively active Ca^<2+>/calmodulin-dependent protein kinase kinase (CaM-KKc) as a surrogate MLCK-A kinase, indicating that the protein kinase cascade regulates MLCK-A in Dictyostelium analogous to CaM-kinase cascade.

Ca 2+ /钙调蛋白依赖性蛋白激酶(CaM-K)构成多种酶，其参与由细胞内钙浓度增加介导的许多细胞反应。先前的研究表明，两种多功能 CaM 激酶 CaM-KI 和 IV 通过上游 CaM 激酶激酶 (CaM-KK) 磷酸化激活环 Thr 残基而被激活，从而大大提高催化效率。为了评估 CaM-KK 和 CaM 激酶级联的生理功能，我们尝试合成一种有效且特异性的 CaM-KK 抑制剂 STO-609。在这项研究中，我们描述了抑制剂 STO-609 在体外和完整细胞中对 CaM-KK 活性的影响。此外，基于CaM-KK亚型对STO-609的独特敏感性是由于ATP结合袋中的单个氨基酸取代（Val/Leu）所致，我们生成了STO-609抗性CaM-KK突变体，这可能有助于验证STO-609在体内的药理作用和特异性。我们还通过使用组成型活性Ca^2+/钙调蛋白依赖性蛋白激酶激酶(CaM-KKc)作为替代MLCK-A激酶，研究了盘基网柄菌MLCK-A的激活机制，表明蛋白激酶级联调节盘基网柄菌中的MLCK-A，类似于CaM激酶级联。

项目成果

Kimura Y.et al.: "A CaMK cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans."EMBO Reports. 3. 962-966 (2002)

Kimura Y.等人：“CaMK 级联激活秀丽隐杆线虫神经元中 CRE 介导的转录。”EMBO 报告。

Tokumitsu H.et al.: "Regulatory mechanism of dictyostelium myosin light chain kinase A."The Journal of Biological Chemistry. 279. 42-50 (2004)

Tokumitsu H.等人：“盘基网柄菌肌球蛋白轻链激酶 A 的调节机制”。《生物化学杂志》。

Inuzuka H. et al.: "Transcriptional regulation of nuclear orphan receptor, NOR-1, by Ca^<2+>/calmodulin-dependent protein kinase cascade."FEBS Letters. 522. 89-92 (2002)

Inuzuka H.等人：“Ca 2+ /钙调蛋白依赖性蛋白激酶级联对核孤儿受体NOR-1的转录调节。”FEBS Letters。

Suizu F. et al.: "Characterization of Ca^<2+>/calmodulin-dependent protein kinase I as a myosin light chain kinase in vitro and in vivo."Biochemical Journal. 367. 335-345 (2002)

Suizu F.等人：“Ca 2+ /钙调蛋白依赖性蛋白激酶I作为肌球蛋白轻链激酶的体外和体内表征。”生物化学杂志。

Tokumitsu H. et al.: "A single amino acid difference between α and β Ca^<2+>/calmodulin-dependent protein kinase kinase dictates sensitivity to the specific inhibitor, STO-609."The Journal of Biological Chemistry. 278. 10908-10913 (2003)

Tokumitsu H. 等人：“α 和 β Ca^2+/钙调蛋白依赖性蛋白激酶激酶之间的单个氨基酸差异决定了对特定抑制剂 STO-609 的敏感性。”《生物化学杂志》278。 10908-10913 (2003)