title: Identification of a novel CaMKK substrate.
abstract: Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5'-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N(6)-(1-methylbutyl)-ATP and genetically engineered CaMKKα mutant, CaMKKα (Phe(230)Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKKα (Phe(230)Gly), but not with wild-type kinase, in the presence of N(6)-(1-methylbutyl)-ATP and Ca(2+)/CaM, induced significant threonine phosphorylation of a 50kDa protein as well as CaMKI phosphorylation at Thr(177). The 50kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKKα and β isoforms at Thr(355)in vitro. Phosphorylation of HA-Syndapin I at Thr(355) in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca(2+)-signaling pathways that are independent of downstream protein kinases.
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: Naoya
authors.initials: N
authors.lastname: Hatano
authors.forename: Naohito
authors.initials: N
authors.lastname: Nozaki
authors.forename: Saki
authors.initials: S
authors.lastname: Yurimoto
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; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D007527:Isoenzymes; C117738:Pacsin1 protein, rat; D011994:Recombinant Proteins; D013912:Threonine; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase