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

Saki Yurimoto, Tomohito Fujimoto, Masaki Magari, Naoki Kanayama, Ryoji Kobayashi, Hiroshi Tokumitsu

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 K m 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 V max 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 Ca 2+/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.

Original languageEnglish
Article number27
JournalBMC Biochemistry
Volume13
Issue number1
DOIs
Publication statusPublished - 2012

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinases
Phosphorylation
Guanosine Triphosphate
Phosphotransferases
Phosphates
Substrates
Protein Isoforms
AMP-Activated Protein Kinases
Calcium-Calmodulin-Dependent Protein Kinase Kinase
triphosphoric acid
guanosine kinase
In Vitro Techniques
Adenosine Triphosphate
Signal transduction
Protein Kinases
Assays
Signal Transduction
Brain
Nucleotides
Chemical activation

Keywords

  • Calmodulin
  • CaMKK
  • GTP
  • Phosphate donor
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5′-triphosphate as a phosphate donor. / Yurimoto, Saki; Fujimoto, Tomohito; Magari, Masaki; Kanayama, Naoki; Kobayashi, Ryoji; Tokumitsu, Hiroshi.

In: BMC Biochemistry, Vol. 13, No. 1, 27, 2012.

Research output: Contribution to journalArticle

@article{529b05e4380b40a485b6d7ed2d3e3299,
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 K m 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 V max 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 Ca 2+/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, GTP, Phosphate donor, Phosphorylation",
author = "Saki Yurimoto and Tomohito Fujimoto and Masaki Magari and Naoki Kanayama and Ryoji Kobayashi and Hiroshi Tokumitsu",
year = "2012",
doi = "10.1186/1471-2091-13-27",
language = "English",
volume = "13",
journal = "BMC Biochemistry",
issn = "1471-2091",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5′-triphosphate as a phosphate donor

AU - Yurimoto, Saki

AU - Fujimoto, Tomohito

AU - Magari, Masaki

AU - Kanayama, Naoki

AU - Kobayashi, Ryoji

AU - Tokumitsu, Hiroshi

PY - 2012

Y1 - 2012

N2 - 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 K m 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 V max 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 Ca 2+/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.

AB - 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 K m 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 V max 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 Ca 2+/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.

KW - Calmodulin

KW - CaMKK

KW - GTP

KW - Phosphate donor

KW - Phosphorylation

UR - http://www.scopus.com/inward/record.url?scp=84870347155&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84870347155&partnerID=8YFLogxK

U2 - 10.1186/1471-2091-13-27

DO - 10.1186/1471-2091-13-27

M3 - Article

C2 - 23216827

AN - SCOPUS:84870347155

VL - 13

JO - BMC Biochemistry

JF - BMC Biochemistry

SN - 1471-2091

IS - 1

M1 - 27

ER -