Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish.
We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.
Chou
Chih-Ming
CM
Department of Biochemistry, Taipei Medical University, Taipei, Taiwan. cmchou@tmu.edu.tw
Chen
Yi-Chung
YC
Lee
Ming-Ting
MT
Chen
Gen-Der
GD
Lu
I-Ching
IC
Chen
Shui-Tsung
ST
Huang
Chang-Jen
CJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000196:embryology / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D051767:Early Growth Response Protein 2 / Q000096:biosynthesis; D005091:Exons; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D016678:Genome; D006657:Histones / Q000378:metabolism; D017403:In Situ Hybridization; D007313:Insecta; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051857:Otx Transcription Factors / Q000096:biosynthesis; D051762:PAX2 Transcription Factor / Q000096:biosynthesis; D010766:Phosphorylation; D011494:Protein Kinases / Q000096:biosynthesis*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000502:physiology*
D017931:DNA Primers; D018076:DNA, Complementary; D051767:Early Growth Response Protein 2; D006657:Histones; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D051857:Otx Transcription Factors; D051762:PAX2 Transcription Factor; D012333:RNA, Messenger; C411889:Sh3kbp1 protein, rat; D029961:Zebrafish Proteins; C497662:otx2b protein, zebrafish; C497320:pax2a protein, zebrafish; D012313:RNA; D011494:Protein Kinases; C509136:BSK146 protein, zebrafish; D017346:Protein Serine-Threonine Kinases