Physiological function of calcium/calmodulin-dependent protein kinase cascade

Ca^<2+>/钙调素依赖性蛋白激酶（CaM-Ks）构成了一组不同的酶，它们参与许多由细胞内钙浓度增加介导的细胞反应。先前的研究表明，两种多功能cam -激酶，CaM-KI和IV，是由上游cam -激酶激酶（CaM-KK）磷酸化一个激活环苏氨酸残基激活的，从而导致催化效率的大幅提高。为了评估CaM-KK和cam激酶级联的生理功能，我们试图合成一种有效的特异性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异构体对STO-609的明显敏感性是由于atp结合囊中的单个氨基酸取代（Val/Leu）的结果，我们已经产生了一个具有STO-609抗性的CaM-KK突变体，这可能有助于验证STO-609的体内药理作用和特异性。我们还研究了Dictyostelium MLCK-A的激活机制，使用组成型活性Ca^<2+>/钙调素依赖性蛋白激酶（CaM-KKc）作为MLCK-A激酶的替代物，表明蛋白激酶级联调节Dictyostelium MLCK-A类似于cam -激酶级联。

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.