PUBLICATION

Expression, characterization, and gene knockdown of zebrafish doublecortin-like protein kinase

Authors
Shimomura, S., Nagamine, T., Nimura, T., Sueyoshi, N., Shigeri, Y., and Kameshita, I.
ID
ZDB-PUB-070523-7
Date
2007
Source
Archives of biochemistry and biophysics   463(2): 218-230 (Journal)
Registered Authors
Keywords
Doublecortin-like protein kinase, Microtubule, Zebrafish, Central nervous systems, Embryogenesis, Gene knockdown, Apoptosis, Protein phosphorylation
MeSH Terms
  • Amino Acid Sequence
  • Animals
  • Catalysis
  • Central Nervous System/embryology
  • Cloning, Molecular
  • Gene Expression
  • Molecular Sequence Data
  • Protein Serine-Threonine Kinases/analysis
  • Protein Serine-Threonine Kinases/genetics
  • Protein Serine-Threonine Kinases/physiology*
  • Sequence Alignment
  • Tissue Distribution
  • Zebrafish/embryology*
  • Zebrafish/genetics
  • Zebrafish/metabolism
  • Zebrafish Proteins/analysis
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/physiology*
PubMed
17498644 Full text @ Arch. Biochem. Biophys.
Abstract
Doublecortin-like protein kinase (DCLK) is a protein Ser/Thr kinase expressed in brain and believed to play crucial roles in neuronal development. To investigate the biological significance of DCLK, we isolated cDNA clones for zebrafish DCLK (zDCLK) and found that there were five splice variants of the kinase. In this study, the catalytic properties of a major isoform of zDCLK, which we designated as zDCLK1, and of an N-terminal truncated mutant retaining the kinase domain were examined by expressing them in Escherichia coli. Mutational analysis of recombinant zDCLK suggested that the kinase was activated not only by phosphorylation at Thr-576 in the activation loop but also by autophosphorylation at the other site(s) in the catalytic domain. zDCLK significantly phosphorylated protein substrates such as myelin basic protein, histones, and synapsin I. Subcellular localization of zDCLK and its N-terminal deletion mutant implicated that microtubule-association of zDCLK is mediated through N-terminal doublecortin like domain of this enzyme. Western blotting analysis and whole mount in situ hybridization revealed that zDCLK was highly expressed in brain and eyes after 24-h post fertilization. Gene knockdown of zDCLK using morpholino-based antisense oligonucleotides induced significant increase of apoptotic cells in the central nervous systems and resulted in the increase of the morphologically abnormal embryos in a dose-dependent manner. These results suggest that zDCLK may play crucial roles in the central nervous systems during the early stage of embryogenesis.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping