Ca 2+/calmodulin-dependent protein kinase phosphatase (CaMKP) is indispensable for normal embryogenesis in zebrafish, Danio rerio

Abstract

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 1272 bp 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, α-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 48 h 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.