Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish

Takaki Nimura, Noriyuki Sueyoshi, Atsuhiko Ishida, Yukihiro Yoshimura, Makoto Ito, Hiroshi Tokumitsu, Yasushi Shigeri, Naohito Nozaki, Isamu Kameshita

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

Original languageEnglish
Pages (from-to)205-216
Number of pages12
JournalArchives of Biochemistry and Biophysics
Volume457
Issue number2
DOIs
Publication statusPublished - Jan 15 2007

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinases
Ionomycin
Phosphoprotein Phosphatases
Zebrafish
Phosphoric Monoester Hydrolases
Genes
Morpholinos
Antisense Oligonucleotides
Enzymes
Brain
Nucleotides
Complementary DNA
Cells
Amino Acids
Proteins
Embryonic Structures
Gene Knockdown Techniques
Nuclear Localization Signals
Neural Tube
Firearms

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish. / Nimura, Takaki; Sueyoshi, Noriyuki; Ishida, Atsuhiko; Yoshimura, Yukihiro; Ito, Makoto; Tokumitsu, Hiroshi; Shigeri, Yasushi; Nozaki, Naohito; Kameshita, Isamu.

In: Archives of Biochemistry and Biophysics, Vol. 457, No. 2, 15.01.2007, p. 205-216.

Research output: Contribution to journalArticle

Nimura, T, Sueyoshi, N, Ishida, A, Yoshimura, Y, Ito, M, Tokumitsu, H, Shigeri, Y, Nozaki, N & Kameshita, I 2007, 'Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish', Archives of Biochemistry and Biophysics, vol. 457, no. 2, pp. 205-216. https://doi.org/10.1016/j.abb.2006.09.034
Nimura, Takaki ; Sueyoshi, Noriyuki ; Ishida, Atsuhiko ; Yoshimura, Yukihiro ; Ito, Makoto ; Tokumitsu, Hiroshi ; Shigeri, Yasushi ; Nozaki, Naohito ; Kameshita, Isamu. / Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish. In: Archives of Biochemistry and Biophysics. 2007 ; Vol. 457, No. 2. pp. 205-216.
@article{dd0793126565478bbd9dedd721f00335,
title = "Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish",
abstract = "Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.",
author = "Takaki Nimura and Noriyuki Sueyoshi and Atsuhiko Ishida and Yukihiro Yoshimura and Makoto Ito and Hiroshi Tokumitsu and Yasushi Shigeri and Naohito Nozaki and Isamu Kameshita",
year = "2007",
month = "1",
day = "15",
doi = "10.1016/j.abb.2006.09.034",
language = "English",
volume = "457",
pages = "205--216",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish

AU - Nimura, Takaki

AU - Sueyoshi, Noriyuki

AU - Ishida, Atsuhiko

AU - Yoshimura, Yukihiro

AU - Ito, Makoto

AU - Tokumitsu, Hiroshi

AU - Shigeri, Yasushi

AU - Nozaki, Naohito

AU - Kameshita, Isamu

PY - 2007/1/15

Y1 - 2007/1/15

N2 - Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

AB - Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

UR - http://www.scopus.com/inward/record.url?scp=33845993559&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33845993559&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2006.09.034

DO - 10.1016/j.abb.2006.09.034

M3 - Article

C2 - 17169323

AN - SCOPUS:33845993559

VL - 457

SP - 205

EP - 216

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -