Identification of a novel CaMKK substrate

Tomohito Fujimoto, Naoya Hatano, Naohito Nozaki, Saki Yurimoto, Ryoji Kobayashi, Hiroshi Tokumitsu

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates specific downstream protein kinases including CaMKI, CaMKIV and 5′-AMP-activated protein kinase. In order to examine the variety of CaMKK-mediated signaling pathways, we searched for novel CaMKK substrate(s) using N6-(1-methylbutyl)-ATP and genetically engineered CaMKKα mutant, CaMKKα (Phe230Gly), that was capable of utilizing this ATP analogue as a phosphate donor. Incubation of rat brain extracts with recombinant CaMKKα (Phe230Gly), but not with wild-type kinase, in the presence of N6-(1-methylbutyl)-ATP and Ca2+/CaM, induced significant threonine phosphorylation of a 50kDa protein as well as CaMKI phosphorylation at Thr177. The 50kDa CaMKK substrate was partially purified by using serial column chromatography, and was identified as Syndapin I by LC-MS/MS analysis. We confirmed that recombinant Syndapin I was phosphorylated by CaMKKα and β isoforms at Thr355 in vitro. Phosphorylation of HA-Syndapin I at Thr355 in transfected HeLa cells was significantly induced by co-expression of constitutively active mutants of CaMKK isoforms. This is the first report that CaMKK is capable of phosphorylating a non-kinase substrate suggesting the possibility of CaMKK-mediated novel Ca2+-signaling pathways that are independent of downstream protein kinases.

Original languageEnglish
Pages (from-to)45-51
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume410
Issue number1
DOIs
Publication statusPublished - Jun 24 2011
Externally publishedYes

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinases
Phosphotransferases
Substrates
Phosphorylation
Adenosine Triphosphate
Protein Kinases
Protein Isoforms
Column chromatography
AMP-Activated Protein Kinases
Threonine
HeLa Cells
Chromatography
Rats
Brain
Phosphates

Keywords

  • Ca-signaling
  • CaMKK
  • Phosphorylation
  • Syndapin I

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Identification of a novel CaMKK substrate. / Fujimoto, Tomohito; Hatano, Naoya; Nozaki, Naohito; Yurimoto, Saki; Kobayashi, Ryoji; Tokumitsu, Hiroshi.

In: Biochemical and Biophysical Research Communications, Vol. 410, No. 1, 24.06.2011, p. 45-51.

Research output: Contribution to journalArticle

Fujimoto, Tomohito ; Hatano, Naoya ; Nozaki, Naohito ; Yurimoto, Saki ; Kobayashi, Ryoji ; Tokumitsu, Hiroshi. / Identification of a novel CaMKK substrate. In: Biochemical and Biophysical Research Communications. 2011 ; Vol. 410, No. 1. pp. 45-51.
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