title: Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase.
abstract: To search for the downstream target protein kinases of Ca (2+)/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKalpha catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5'-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr (189) in the activation loop. Phosphorylation of Thr (189) by CaMKKalpha induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr (189) phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKalpha (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKalpha is capable of activating SAD-B through phosphorylation of Thr (189) both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.
authors.affiliation: Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Tomohito
authors.initials: T
authors.lastname: Fujimoto
authors.forename: Saki
authors.initials: S
authors.lastname: Yurimoto
authors.forename: Naoya
authors.initials: N
authors.lastname: Hatano
authors.forename: Naohito
authors.initials: N
authors.lastname: Nozaki
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sueyoshi
authors.forename: Isamu
authors.initials: I
authors.lastname: Kameshita
authors.forename: Akihiro
authors.initials: A
authors.lastname: Mizutani
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mikoshiba
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumitsu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D017931:DNA Primers; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase