Identification of an endogenous substrate of zebrafish doublecortin-like protein kinase using a highly active truncation mutant

Sachiko Shimomura, Tadashi Nagamine, Naoya Hatano, Noriyuki Sueyoshi, Isamu Kameshita

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Doublecortin-like protein kinase (DCLK), a Ser/Thr protein kinase predominantly expressed in brain and eyes, is believed to play crucial roles in neuronal functions. However, the regulatory mechanisms for DCLK activation and its physiological targets are still unknown. In the present study, we found that a deletion mutant consisting of the catalytic domain of zebrafish DCLK, zDCLK(377-677), exhibited the highest activity among various mutants. Since fully active zDCLK(377-677) showed essentially the same substrate specificity as wild-type zDCLK, we used it to search for physiological substrates of zDCLK. When a zebrafish brain extract was resolved by isoelectric focusing and then phosphorylated by zDCLK(377-677), a highly basic protein with a molecular mass of ∼90 kDa was detected. This protein was identified as synapsin II by mass spectrometric analysis. Synapsin II was found to interact with the catalytic domain of zDCLK and was phosphorylated at Ser-9 and Ser-58. When synaptosomes were isolated from zebrafish brain, both synapsin II and zDCLK were found to coexist in this preparation. Furthermore, synapsin II in the synaptosomes was efficiently phosphorylated by zDCLK. These results suggest that zDCLK mediates its neuronal functions through phosphorylation of physiological substrates such as synapsin II.

Original languageEnglish
Pages (from-to)711-722
Number of pages12
JournalJournal of Biochemistry
Volume147
Issue number5
DOIs
Publication statusPublished - Jan 1 2010
Externally publishedYes

Fingerprint

Synapsins
Protein Kinases
Substrates
Brain
Synaptosomes
Zebrafish
Catalytic Domain
Phosphorylation
Isoelectric Focusing
Molecular mass
Substrate Specificity
Proteins
Chemical activation
zebrafish doublecortin-like protein kinase

Keywords

  • autoinhibitory domain
  • catalytic domain
  • doublecortin-like protein kinase
  • synapsin IIsynaptosomes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Medicine(all)

Cite this

Identification of an endogenous substrate of zebrafish doublecortin-like protein kinase using a highly active truncation mutant. / Shimomura, Sachiko; Nagamine, Tadashi; Hatano, Naoya; Sueyoshi, Noriyuki; Kameshita, Isamu.

In: Journal of Biochemistry, Vol. 147, No. 5, 01.01.2010, p. 711-722.

Research output: Contribution to journalArticle

Shimomura, Sachiko ; Nagamine, Tadashi ; Hatano, Naoya ; Sueyoshi, Noriyuki ; Kameshita, Isamu. / Identification of an endogenous substrate of zebrafish doublecortin-like protein kinase using a highly active truncation mutant. In: Journal of Biochemistry. 2010 ; Vol. 147, No. 5. pp. 711-722.
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