Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is indispensable for normal embryogenesis in zebrafish, Danio rerio.
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). However, the biological functions of this enzyme have not been clarified in vivo. To investigate the biological significance of CaMKP during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP (zCaMKP). The isolated cDNA clone possessed an open reading frame of 1272bp encoding 424 amino acids and shared 47% and 48% amino acid identity with rat and human CaMKP, respectively. Interestingly, zCaMKP lacks the Glu cluster corresponding to residues 101-109 in the rat enzyme, and was not activated by polycations such as poly-l-lysine. The recombinant zCaMKP required Mg(2+) rather than Mn(2+) for activity. Furthermore, zCaMKP dephosphorylated CaMKIV but not phosphorylase a, alpha-casein, or extracellular signal-regulating kinase (ERK), suggesting that the enzyme regulates Ca(2+) signaling pathways in zebrafish. Cotransfection of zCaMKP with mammalian CaMKI significantly decreased phospho-CaMKI in ionomycin-stimulated 293T cells. During embryogenesis, the expression of zCaMKP increased gradually after 48h post-fertilization, as demonstrated by Western blotting using an anti-zCaMKP antibody. The knockdown of the zCaMKP gene with morpholino-based antisense oligonucleotides resulted in an increased incidence of embryos with severe morphological and cellular abnormalities, i.e., a significant increase in the number of round-shaped embryos and apoptotic cells in the whole body. A marked decrease in zCaMKP expression was observed in the antisense- but not control oligo-injected embryos. Embryonic death was rescued by coinjection with recombinant rat CaMKP but not with phosphatase-dead mutant (D194A). These results clearly show the significance of zCaMKP during zebrafish embryogenesis.
Sueyoshi
Noriyuki
N
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan. sueyoshi@ag.kagawa-u.ac.jp
Nimura
Takaki
T
Ishida
Atsuhiko
A
Taniguchi
Takanobu
T
Yoshimura
Yukihiro
Y
Ito
Makoto
M
Shigeri
Yasushi
Y
Kameshita
Isamu
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D047108:Embryonic Development / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; C517871:ppm1e protein, zebrafish