title: Generation of autonomous activity of Ca(2+)/calmodulin-dependent protein kinase kinase β by autophosphorylation.
abstract: Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5'-AMP-activated protein kinase, which mediates a variety of Ca(2+) signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKKα and β isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca(2+)/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography-tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in CaMKKβ, but phosphorylation of the equivalent Thr residue (Thr(446)) in the α isoform was not observed. Unlike CaMKKα that has high Ca(2+)/CaM-dependent activity, wild-type CaMKKβ displays enhanced autonomous activity (Ca(2+)/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr(482) with a nonphosphorylatable Ala, without significant changes in Ca(2+)/CaM binding. In addition, a CaMKKα mutant containing the CaMKKβ regulatory domain was shown to be partially phosphorylated at Thr(446), resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the α isoform, CaMKKβ exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr(482), resulting in partial disruption of the autoinhibitory mechanism.
authors.affiliation: Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan. tokumit@med.kagawa-u.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumitsu
authors.forename: Naoya
authors.initials: N
authors.lastname: Hatano
authors.forename: Tomohito
authors.initials: T
authors.lastname: Fujimoto
authors.forename: Saki
authors.initials: S
authors.lastname: Yurimoto
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000737:chemistry
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D013912:Threonine; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; C517701:Camkk1 protein, rat; C517702:Camkk2 protein, rat; D054648:Protein Phosphatase 2