Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase

Tomohito Fujimoto, Saki Yurimoto, Naoya Hatano, Naohito Nozaki, Noriyuki Sueyoshi, Isamu Kameshita, Akihiro Mizutani, Katsuhiko Mikoshiba, Ryoji Kobayashi, Hiroshi Tokumitsu

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

To search for the downstream target protein kinases of Ca 2+/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKα catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5′-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr189 in the activation loop. Phosphorylation of Thr189 by CaMKKα induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr189 phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKα (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKα is capable of activating SAD-B through phosphorylation of Thr189 both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.

Original languageEnglish
Pages (from-to)4151-4159
Number of pages9
JournalBiochemistry
Volume47
Issue number13
DOIs
Publication statusPublished - Apr 1 2008
Externally publishedYes

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinases
Phosphotransferases
Chemical activation
Phosphorylation
Protein Kinases
Catalytic Domain
Affinity chromatography
AMP-Activated Protein Kinases
COS Cells
Affinity Chromatography
Proteomics
Purification
Mass spectrometry
Rats
Assays
Brain
Mass Spectrometry

ASJC Scopus subject areas

  • Biochemistry

Cite this

Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase. / Fujimoto, Tomohito; Yurimoto, Saki; Hatano, Naoya; Nozaki, Naohito; Sueyoshi, Noriyuki; Kameshita, Isamu; Mizutani, Akihiro; Mikoshiba, Katsuhiko; Kobayashi, Ryoji; Tokumitsu, Hiroshi.

In: Biochemistry, Vol. 47, No. 13, 01.04.2008, p. 4151-4159.

Research output: Contribution to journalArticle

Fujimoto, T, Yurimoto, S, Hatano, N, Nozaki, N, Sueyoshi, N, Kameshita, I, Mizutani, A, Mikoshiba, K, Kobayashi, R & Tokumitsu, H 2008, 'Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase', Biochemistry, vol. 47, no. 13, pp. 4151-4159. https://doi.org/10.1021/bi702528r
Fujimoto T, Yurimoto S, Hatano N, Nozaki N, Sueyoshi N, Kameshita I et al. Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase. Biochemistry. 2008 Apr 1;47(13):4151-4159. https://doi.org/10.1021/bi702528r
Fujimoto, Tomohito ; Yurimoto, Saki ; Hatano, Naoya ; Nozaki, Naohito ; Sueyoshi, Noriyuki ; Kameshita, Isamu ; Mizutani, Akihiro ; Mikoshiba, Katsuhiko ; Kobayashi, Ryoji ; Tokumitsu, Hiroshi. / Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase. In: Biochemistry. 2008 ; Vol. 47, No. 13. pp. 4151-4159.
@article{dd24aa671c764a519b43136b1a96e518,
title = "Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase",
abstract = "To search for the downstream target protein kinases of Ca 2+/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKα catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5′-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr189 in the activation loop. Phosphorylation of Thr189 by CaMKKα induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr189 phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKα (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKα is capable of activating SAD-B through phosphorylation of Thr189 both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.",
author = "Tomohito Fujimoto and Saki Yurimoto and Naoya Hatano and Naohito Nozaki and Noriyuki Sueyoshi and Isamu Kameshita and Akihiro Mizutani and Katsuhiko Mikoshiba and Ryoji Kobayashi and Hiroshi Tokumitsu",
year = "2008",
month = "4",
day = "1",
doi = "10.1021/bi702528r",
language = "English",
volume = "47",
pages = "4151--4159",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "13",

}

TY - JOUR

T1 - Activation of SAD kinase by Ca2+/calmodulin-dependent protein kinase kinase

AU - Fujimoto, Tomohito

AU - Yurimoto, Saki

AU - Hatano, Naoya

AU - Nozaki, Naohito

AU - Sueyoshi, Noriyuki

AU - Kameshita, Isamu

AU - Mizutani, Akihiro

AU - Mikoshiba, Katsuhiko

AU - Kobayashi, Ryoji

AU - Tokumitsu, Hiroshi

PY - 2008/4/1

Y1 - 2008/4/1

N2 - To search for the downstream target protein kinases of Ca 2+/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKα catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5′-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr189 in the activation loop. Phosphorylation of Thr189 by CaMKKα induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr189 phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKα (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKα is capable of activating SAD-B through phosphorylation of Thr189 both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.

AB - To search for the downstream target protein kinases of Ca 2+/calmodulin-dependent protein kinase kinase (CaMKK), we performed affinity chromatography purification of a rat brain extract using a GST-fused CaMKKα catalytic domain (residues 126-434) as the affinity ligand. Proteomic analysis was then carried out to identify the CaMKK-interacting protein kinases. In addition to identifying the catalytic subunit of 5′-AMP-activated protein kinase, we identified SAD-B as interacting. A phosphorylation assay and mass spectrometry analysis revealed that SAD-B was phosphorylated in vitro by CaMKK at Thr189 in the activation loop. Phosphorylation of Thr189 by CaMKKα induced SAD-B kinase activity by over 60-fold. In transfected COS-7 cells, kinase activity and Thr189 phosphorylation of overexpressed SAD-B were significantly enhanced by coexpression of constitutively active CaMKKα (residues 1-434) in a manner similar to that observed with coexpression of LKB1, STRAD, and MO25. Taken together, these results indicate that CaMKKα is capable of activating SAD-B through phosphorylation of Thr189 both in vitro and in vivo and demonstrate for the first time that CaMKK may be an alternative activating kinase for SAD-B.

UR - http://www.scopus.com/inward/record.url?scp=41449084761&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=41449084761&partnerID=8YFLogxK

U2 - 10.1021/bi702528r

DO - 10.1021/bi702528r

M3 - Article

C2 - 18324781

AN - SCOPUS:41449084761

VL - 47

SP - 4151

EP - 4159

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 13

ER -