A single amino acid difference between α and β Ca²⁺/calmodulin-dependent protein kinase kinase dictates sensitivity to the specific inhibitor, STO-609

We recently developed STO-609, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase kinase (CaM-KK), and we demonstrated that CaM-KKβ is more sensitive to STO-609 than the CaM-KKα isoform (Tokumitsu H., Inuzuka H., Ishikawa Y., Ikeda M., Saji I., and Kobayashi R. (2002) J. Biol. Chem. 277, 15813-15818). By using catalytic chimera and point mutants of both isoforms, we demonstrated that Val²⁶⁹ in CaM-KKβ/Leu²³³ in CaM-KKα confers a distinct sensitivity (∼10-fold) to STO-609 on CaM-KK isoforms. Various mutations of Val²⁶⁹ in CaM-KKβ indicate that substitution by hydrophobic residues with bulky side chains significantly decreases drug sensitivity and that the V269F mutant is the most effective drug-resistant enzyme (∼80-fold higher IC₅₀ value). These findings are consistent with a result obtained with a full-length mutant expressed in COS-7 cells. Furthermore, suppression of CaM-KK-mediated CaM-KIV activation in transfected HeLa cells by STO-609 treatment was completely abolished by the co-expression of the CaM-KKβ V269F mutant. Based on the results that the distinct sensitivity of CaM-KK isoforms to STO-609 is because of 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.