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Open Access Highly Accessed Research article

In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5-triphosphate as a phosphate donor

Saki Yurimoto1, Tomohito Fujimoto1, Masaki Magari2, Naoki Kanayama2, Ryoji Kobayashi1 and Hiroshi Tokumitsu12*

Author affiliations

1 Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa, 761-0793, Japan

2 Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, 3-1-1, Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan

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Citation and License

BMC Biochemistry 2012, 13:27  doi:10.1186/1471-2091-13-27

Published: 5 December 2012

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.

Keywords:
Calmodulin; CaMKK; Phosphate donor; GTP; Phosphorylation