Identification of a novel CaMKK substrate

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

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)


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
Issue number1
Publication statusPublished - Jun 24 2011
Externally publishedYes


  • Ca-signaling
  • CaMKK
  • Phosphorylation
  • Syndapin I

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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