Expression, characterization, and gene knockdown of zebrafish doublecortin-like protein kinase.
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.
Shimomura
Sachiko
S
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan.
Nagamine
Tadashi
T
Nimura
Takaki
T
Sueyoshi
Noriyuki
N
Shigeri
Yasushi
Y
Kameshita
Isamu
I
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002490:Central Nervous System / Q000196:embryology; D003001:Cloning, Molecular; D000089742:Doublecortin-Like Kinases; D015870:Gene Expression; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D014018:Tissue Distribution; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*
D029961:Zebrafish Proteins; D000089742:Doublecortin-Like Kinases; C521839:DCLK protein, zebrafish; D017346:Protein Serine-Threonine Kinases