Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

Shota Takabatake, Satomi Ohtsuka, Takeyuki Sugawara, Naoya Hatano, Naoki Kanayama, Masaki Magari, Hiroyuki Sakagami, Hiroshi Tokumitsu

Research output: Contribution to journalArticle

Abstract

Background: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. Methods: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. Results: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. Conclusion: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. General significance: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation.

Original languageEnglish
Pages (from-to)672-680
Number of pages9
JournalBiochimica et Biophysica Acta - General Subjects
Volume1863
Issue number4
DOIs
Publication statusPublished - Apr 1 2019

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Calcium-Calmodulin-Dependent Protein Kinases
Phosphotransferases
Phosphorylation
Protein Kinases
AMP-Activated Protein Kinases
Protein Kinase Inhibitors
HeLa Cells
Cerebellum
Enzymes
Cyclic AMP-Dependent Protein Kinases

Keywords

  • Calmodulin
  • CaMKK
  • Intracellular Ca
  • Phosphorylation
  • PKA
  • Signal transduction

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling. / Takabatake, Shota; Ohtsuka, Satomi; Sugawara, Takeyuki; Hatano, Naoya; Kanayama, Naoki; Magari, Masaki; Sakagami, Hiroyuki; Tokumitsu, Hiroshi.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1863, No. 4, 01.04.2019, p. 672-680.

Research output: Contribution to journalArticle

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T1 - Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

AU - Takabatake, Shota

AU - Ohtsuka, Satomi

AU - Sugawara, Takeyuki

AU - Hatano, Naoya

AU - Kanayama, Naoki

AU - Magari, Masaki

AU - Sakagami, Hiroyuki

AU - Tokumitsu, Hiroshi

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AB - Background: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. Methods: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. Results: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. Conclusion: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. General significance: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation.

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KW - PKA

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