title: Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells.
abstract: To assess the isoform specificity of the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKKα mutant (Ala292Thr/Leu233Phe) and a CaMKKβ mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wild-type or the STO-609-resistant mutant of CaMKKα was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKKβ mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKKβ is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.
authors.affiliation: †Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Fujiwara
authors.affiliation: †Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Hiraoka
authors.affiliation: ‡Carna Biosciences, Inc., Kobe 650-0047, Japan.
authors.forename: Tomohito
authors.initials: T
authors.lastname: Fujimoto
authors.affiliation: †Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kanayama
authors.affiliation: †Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Magari
authors.affiliation: †Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumitsu
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry*; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002477:Cells / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053644:Naphthalimides / Q000737:chemistry*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D051381:Rats
chemical_list: D001562:Benzimidazoles; D007527:Isoenzymes; D053644:Naphthalimides; D047428:Protein Kinase Inhibitors; C458525:STO 609; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; C517686:Camk4 protein, rat; D055372:AMP-Activated Protein Kinases