title: A single amino acid difference between alpha and beta Ca2+/calmodulin-dependent protein kinase kinase dictates sensitivity to the specific inhibitor, STO-609.
abstract: We recently developed STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK), and we demonstrated that CaM-KK beta is more sensitive to STO-609 than the CaM-KK alpha 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 Val(269) in CaM-KK beta/Leu(233) in CaM-KK alpha confers a distinct sensitivity ( approximately 10-fold) to STO-609 on CaM-KK isoforms. Various mutations of Val(269) in CaM-KK beta 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 ( approximately 80-fold higher IC(50) 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-KK beta 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.
authors.affiliation: Department of Signal Transduction Sciences, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. tokumit@kms.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumitsu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Inuzuka
authors.forename: Yumi
authors.initials: Y
authors.lastname: Ishikawa
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D019556:COS Cells; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D053644:Naphthalimides; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D053644:Naphthalimides; D011994:Recombinant Proteins; C458525:STO 609; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase