Physiological function of calcium/calmodulin-dependent protein kinase cascade

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。先前的研究已经证明，两种多功能的CaM激酶，CaM-KI和IV，通过上游CaM激酶激酶（CaM-KK）磷酸化活化环Thr残基而被活化，导致催化效率的大幅增加。为了评估CaM-KK和CaM激酶级联的生理功能，我们尝试合成CaM-KK的有效和特异性抑制剂STO-609。在这项研究中，我们表征了抑制剂STO-609对体外和完整细胞中CaM-KK活性的影响。此外，基于CaM-KK异构体对STO-609的不同敏感性是由于ATP结合口袋中的单个氨基酸取代（瓦尔/Leu）的结果，我们已经产生了STO-609抗性CaM-KK突变体，这可能有助于验证STO-609在体内的药理学作用和特异性。我们还通过使用组成型活性的Ca^2+/钙调素依赖性蛋白激酶激酶（CaM-KKc）作为MLCK-A激酶的替代物，研究了网骨藻MLCK-A的激活机制，表明蛋白激酶级联类似于CaM-激酶级联调节网骨藻中的MLCK-A。

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.