title: In vitro substrate phosphorylation by Ca²⁺/calmodulin-dependent protein kinase kinase using guanosine-5'-triphosphate as a phosphate donor.
abstract: BACKGROUND
  Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases - including CaMKI, CaMKIV, and AMPK- to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
  
  
  RESULTS
  Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKIα (Thr177) and of AMPK (Thr172) in vitro. Kinetic analysis indicated that the Km values of CaMKK isoforms for GTP (400-500 μM) were significantly higher than those for ATP (~15 μM), and a 2- to 4-fold decrease in Vmax was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of ~45 kDa and ~35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
  
  
  CONCLUSIONS
  These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.
authors.affiliation: Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Yurimoto
authors.forename: Tomohito
authors.initials: T
authors.lastname: Fujimoto
authors.forename: Masaki
authors.initials: M
authors.lastname: Magari
authors.forename: Naoki
authors.initials: N
authors.lastname: Kanayama
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tokumitsu
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D053644:Naphthalimides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001562:Benzimidazoles; D053644:Naphthalimides; D020033:Protein Isoforms; D011994:Recombinant Proteins; C458525:STO 609; D006160:Guanosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases