Physiological function of calcium/calmodulin-dependent protein kinase cascade

钙调素依赖的蛋白激酶(CaM-Ks)组成了一组不同的酶，参与了细胞内钙离子浓度升高所介导的许多细胞反应。以往的研究表明，两种多功能的CaM-KK，CaM-KK，都是通过上游的CaM-KK(CaM-KK)对激活环Thr残基的磷酸化而激活的，从而大大提高了催化效率。为了研究CaM-KK和CaM-KK级联反应的生理功能，我们试图合成一种有效和特异的CaM-KK抑制剂STO-609。在这项研究中，我们研究了抑制剂STO-609在体外和在完整细胞中对CaM-KK活性的影响。此外，根据CaM-KK亚型对STO-609的明显敏感性是由于ATP结合口袋中的单一氨基酸取代(Val/Leu)所致，我们产生了一个抗STO-609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。我们还用结构性活性的钙/钙调素依赖的蛋白激酶(CaM-KKC)作为MLCK-A的替代激酶，研究了DictyostelialMLCK-A的激活机制，表明该蛋白激酶级联类似于CaM-KKC级联调节DictyostelialMLCK-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.