TY - JOUR
T1 - A single amino acid difference between α and β Ca2+/calmodulin-dependent protein kinase kinase dictates sensitivity to the specific inhibitor, STO-609
AU - Tokumitsu, Hiroshi
AU - Inuzuka, Hiroyuki
AU - Ishikawa, Yumi
AU - Kobayashi, Ryoji
PY - 2003/3/28
Y1 - 2003/3/28
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M213183200
DO - 10.1074/jbc.M213183200
M3 - Article
C2 - 12540834
AN - SCOPUS:0038514233
VL - 278
SP - 10908
EP - 10913
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 13
ER -