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

Archives of Biochemistry and Biophysics 488 (2009) 48–59

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Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) is indispensable for normal embryogenesis in zebrafish, Danio rerio q Noriyuki Sueyoshi a,*, Takaki Nimura a, Atsuhiko Ishida b,c, Takanobu Taniguchi b, Yukihiro Yoshimura d, Makoto Ito d, Yasushi Shigeri e, Isamu Kameshita a a

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan c Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan d Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan e National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan b

a r t i c l e

i n f o

Article history: Received 16 March 2009 and in revised form 3 June 2009 Available online 13 June 2009 Keywords: Zebrafish CaM kinase Apoptosis Phosphatase CaMKP

a b s t r a c t Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and regulates multifunctional Ca2+/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-Llysine. The recombinant zCaMKP required Mg2+ rather than Mn2+ for activity. Furthermore, zCaMKP dephosphorylated CaMKIV but not phosphorylase a, a-casein, or extracellular signal-regulating kinase (ERK), suggesting that the enzyme regulates Ca2+ 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. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs)1 play various pivotal roles in Ca2+ signaling pathways involved in the regulation of the neuronal functions such as learning, q The nucleotide sequence reported in this paper has been submitted to the GenBankTM/EBI Data Bank with an Accession No. AB113301. * Corresponding author. Address: Department of Life Sciences, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan. Fax: +81 87 891 3114. E-mail address: [email protected] (N. Sueyoshi). 1 Abbreviations used: CaMK, Ca2+/calmodulin-dependent protein kinase; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; CaMKP, Ca2+/calmodulin-dependent protein kinase phosphatase; AS, antisense; DIG, digoxigenin; hpf, hours postfertilization; MO, morpholino oligos; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; poly(Lys), poly-L-lysine; PP2C, protein phosphatase 2C; RACE, rapid amplification of cDNA ends; WGS, whole genome shotgun; zCaMKP, zebrafish CaMKP; zCaMKP-N, zebrafish CaMKP-N.

0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.06.003

memory, and neuronal cell death [1–5]. CaMKI and CaMKIV have an activation loop containing threonine residues, Thr177 and Thr196, respectively. The phosphorylation of these residues by the upstream kinase, CaMK kinase (CaMKK), activates CaMKI and CaMKIV, whereas CaMKII is activated by the autophosphorylation of Thr286 within the autoinhibitory domain [2–4]. We have identified two novel protein phosphatases, CaMK phosphatase (CaMKP) [6,7] and CaMKP-N [8], that negatively regulate multifunctional CaMKs. The substrate specificity of these phosphatases is much higher than those of the four well-known major classes of Ser/Thr protein phosphatases; PP1, PP2A, PP2B, and PP2C. CaMKP was purified and cloned from rat brain and is known to be a calyculin A-insensitive, Mn2+-dependent, and poly-L-lysine (poly(Lys))-stimulated protein phosphatase that dephosphorylates and regulates both autophosphorylated CaMKII and phosphorylated CaMKI and CaMKIV. CaMKP is localized in the cytoplasm, and is expressed ubiquitously in all tissues,

N. Sueyoshi et al. / Archives of Biochemistry and Biophysics 488 (2009) 48–59

especially in lung, thymus, brain, spleen, uterus, and adrenal gland [7]. Another isoenzyme of CaMKP, CaMKP-N, was found in human [8] and zebrafish [9]. Unlike CaMKP, this homolog is specifically expressed in the brain, and exists inside the nucleus [8,9]. To date, the physiological importance of CaMKP has not been fully clarified. Tan et al. identified CaMKP as a homolog of FEM-2, a product of a gene that participates in sex-determination in Caenorhabditis elegans [10]. Transient expression of either nematode FEM-2, human CaMKP, or rat CaMKP in HeLa cells resulted in apoptosis. In contrast, the expression of PP2C, another member of PPM family protein phosphatases, did not induce apoptosis. These data suggest that CaMKP is involved in apoptotic signaling, although it is unclear how the promotion of apoptosis relates to the intracellular dynamics of CaMKs. To explore the physiological significance of CaMKP, we investigated the molecular mechanism of intracellular regulation of CaMKs by CaMKP using gene knockdown. We used zebrafish as a model animal to investigate the biological significance of CaMKP in vivo. We cloned zebrafish CaMKP (zCaMKP), expressed the enzyme in Escherichia coli, and characterized its enzymatic properties. We also provided the first evidence that zCaMKP dephosphorylates phospho-CaMKI in living cells. Furthermore, we investigated tissue distribution and subcellular localization of zCaMKP, and examined the changes in expression during the developmental stages of embryogenesis. Finally, we conducted a gene knockdown experiment in zebrafish embryos using morpholino-based antisense oligonucleotides techniques. The zCaMKP gene knockdown revealed that CaMKP is indispensable for zebrafish embryogenesis. Materials and methods Materials ATP, Cy3-labeled anti-mouse IgG, bovine serum albumin, poly(Lys), and a-casein from bovine milk were purchased from Sigma Chemicals. Ionomycin calcium salt was purchased from Calbiochem. Goat anti-rabbit IgG conjugated with horseradish peroxidase was obtained from ICN Pharmaceuticals. [c-32P]ATP (5000 Ci/mmol), CNBr-activated Sepharose 4B, and a HiTrap Chelating HP column were obtained from GE Healthcare Bio-Sciences. Recombinant GST-Erk2 (mouse) and recombinant human MEK1 were purchased from Upstate Biotechnology. Phosphorylase b and antiHis6 antibody were purchased from Roche Diagnostics. Anti-Myc antibody was from Invitrogen. Antibody against phospho-CaMKI at Thr177 was prepared as described previously [11]. Recombinant mouse CaMKKa [12] and rat CaMKIa [13] were expressed in E. coli and purified as described previously. Calmodulin was purified from rat testis as described previously [14]. The catalytic subunit of cAMPdependent protein kinase (PKA) was purified as described previously [15]. Recombinant rat CaMKIV(K71R) mutant [16] and rat PP2Ca [17] were expressed in E. coli and purified as described previously.

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in 200 ll of ice cold homogenizing buffer containing 5 mM Tris– HCl buffer (pH 7.5), 0.5 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM b-mercaptoethanol, and homogenized by sonication (BRANSON, Sonifier 250). Crude extracts from adult brains were prepared as above except that three volumes of homogenizing buffer were used. The homogenates were subsequently centrifuged at 20,000g at 2 °C for 10 min, and the supernatant was collected as a crude extract. cDNA cloning of zCaMKP A SMART RACE cDNA amplification kit (Clontech) was used to clone a full-length coding sequence for the zCaMKP using primers based on GenBank zebrafish WGS clones (Accession Nos. 30521310 and 119879949). The 50 -RACE first strand cDNA was primed from the mRNA of adult zebrafish with Superscript II reverse transcriptase using a SMART II oligonucleotide and a 50 -RACE cDNA synthesis primer. The 50 -end of the cDNA was amplified by PCR with gene-specific primer 1 (AS1: 50 -AGT GAT ATT GTC GCT GGA TCC GGC-30 ), a universal primer mix, and a 50 -RACE first strand cDNA template, using the Advantage 2 PCR kit (Clontech). The 30 -RACE first strand cDNA was primed using 30 -RACE cDNA synthesis primers. The 30 -end of the cDNA was obtained by PCR using a universal primer mix, gene-specific primer 2 (S1: 50 -AGC TGC TCA GTT CAC GCC ATT CGA-30 ), and a 30 -RACE first strand cDNA template. The 50 - and 30 -RACE PCR products were cloned into a pGEM-T Easy vector (Promega), and their DNA sequences were subsequently determined. The nucleotide sequences of the 50 -RACE and 30 -RACE products were overlapped and aligned using the DNASIS computer program developed by Hitachi Software Engineering. An open reading frame of 1272 nucleotides was generated. A sense primer (50 -AAA GGG AGT GTC TGC GAA TAA AAC A-30 ) and an antisense primer (50 -ATA AAT GAC CAG GGA TCA GAC GTT C-30 ) were designed from the outside sequences of the open reading frame, respectively. A full-length cDNA was prepared by PCR using these primers and 50 -RACE ready cDNA library as a template with Pyrobest DNA polymerase (TaKaRa BIO). A PCR product was cloned into a pGEM-T Easy vector, and sixteen independent clones were sequenced (pGEMzCaMKP-1 through -16). Plasmid for zCaMKP

Zebrafish, Danio rerio, were maintained at 26 °C and embryos were collected from natural crosses of wild-type fish. Embryos were maintained in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) at 28 °C. Embryos were staged according to hours post-fertilization (hpf) at 28 °C and morphological criteria [18].

The following primers were used for PCR: zCaMKP-S/Nhe (50 AAA GCT AGC ATG AGT GGC GCA GAG AAA GGA GAT GTC-30 ) and zCaMKP-AS/HindIII (50 -GC AAG CTT GAC CCC TGC GGC CGC ACA TGC AGC T-30 ). zCaMKP-S/Nhe contained a NheI restriction site (underlined) and zCaMKP-AS/HindIII contained a HindIII site (double underlined). PCR was performed in a GeneAmp PCR System 2700 (Applied Biosystems) for 30 cycles (each consisting of denaturation at 96 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 2 min) using Pyrobest DNA polymerase and pGEMzCaMKP-3 as a template. After gel purification, the amplified product was digested with NheI and HindIII. The NheI/ HindIII fragment was then cloned into the NheI/HindIII-digested pET-23a(+) (Novagen), sequenced, and designated as pETzCaMKP. For mammalian cells, pczCaMKP was generated by PCR using 50 AAG CTT GGC ATG GCC TCT GGA GCC CCA CAG AA-30 and 50 -GAA TTC CAG ACC CCT GCG GCC GCA CAT GC-30 and pGEMzCaMKP-3 as a template. The PCR fragment was subcloned into pGEM-T Easy and the HindIII/EcoRI fragment was ligated into pcDNA3.1/MycHis(+)B (Invitrogen) to yield pczCaMKP.

Preparation of crude extracts from zebrafish embryos and adult brain

Plasmid for rat CaMKP

Crude extracts of the embryos at each sampling point were prepared as follows: dechorionated embryos (N = 40) were suspended

An insert that included the full-length of the open reading frame was amplified using sense (50 -GC AAG CTT GGC ATG GCC

Fish maintenance

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TCT GGA GCC CCA CAG AA-30 ) and antisense (50 -AT GGA TCC GCT TCT CTG TGA GGT ATT GAT CTC AAG-30 ) primers and rat brain 50 -RACE ready cDNA library as a template. The amplified fragment was treated with HindIII and BamHI and subcloned into pcDNA3.1/ Myc-His(+)B. The recombinant plasmid was purified and designated pcrCaMKP. Plasmid for rat CaMKI Plasmid was obtained by PCR with a sense primer (50 -GGT ACC GCC ATG CCA GGG GCA GTG GAA GGC-30 ) and an antisense primer (50 -CCG CGG GTC CAT GGC CCT AGA GCT TGG GG-30 ) and rat brain 50 -RACE ready cDNA library as a template. The PCR fragment was cleaved with corresponding restriction enzymes and ligated into pcDNA3.1/Myc-His(+)B to yield pcrCaMKI-MH. Expression and purification of recombinant CaMKPs Escherichia coli BL21(DE3) cells transformed with pETzCaMKP or pETrCaMKP [13] were grown at 25 °C for 16 h in 5 ml of medium A (LB medium containing 100 lg/ml ampicillin) with shaking. The culture was then transferred to a 500-ml flask containing 100 ml of medium A and incubated with shaking at 25 °C for 24 h without induction. Under these conditions, the His6-tagged zCaMKP was expressed in soluble form in large amounts. The cells were harvested by centrifugation and suspended in 10 ml of buffer A (20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, 0.05% Tween 40, and 1 mM PMSF). After sonication, cell debris were removed by centrifugation (8000g for 10 min), and the supernatant was loaded onto a HiTrap Chelating HP column (1 ml; GE Healthcare) pre-equilibrated with buffer A. The column was subsequently washed with 10 ml of buffer A, 10 ml of buffer A containing 20 mM imidazole, and 10 ml of buffer A containing 50 mM imidazole. Following this, the column was eluted with buffer A containing 200 mM imidazole. The active fractions were pooled and used for the characterization of the enzyme. Protein phosphatase assay using phosphopeptide as a substrate A protein phosphatase assay was carried out using phosphopeptide pp10 (YGGMHRQETpVDC) as a substrate [13]. The reaction mixture (50 ll) contained 50 mM Tris–HCl (pH 8.5), 2 mM MgCl2, 0.1 mM EGTA, 0.01% Tween 20, 40 lM pp10, and an appropriate amount of zCaMKP or rat PP2Ca. In the case of rat CaMKP, MgCl2 was replaced with MnCl2. The reaction was initiated by adding phosphatase and incubated at 30 °C for 6 min. The inorganic phosphate released in the mixture was determined using a malachite green assay as described previously [16]. One enzyme unit was defined as the amount capable of catalyzing the release of 1 lmol of phosphate/min from pp10 under the condition described above. Protein phosphatase assay using phosphoproteins as substrates Phosphorylase b and a-casein were phosphorylated by phosphorylase kinase (Sigma) and PKA, respectively, as described previously [6]. GST-ERK2 and CaMKIV(K71R) were phosphorylated by active MEK1 and CaMKKa, respectively, as described previously [16]. Dephosphorylation of the phosphoproteins was analyzed as described previously [6,16]. The concentrations of phosphoproteins presented in the text represent the concentrations of 32P bound to the substrate proteins. Production and purification of antibody directed to zCaMKP We used 8-week old Japanese white rabbits (Japan SLC Inc.) for antibody production. Approximately 200 lg of a purified prepara-

tion of recombinant zCaMKP, emulsified with an equal volume of Freund’s complete adjuvant (DIFCO Laboratories), was injected at multiple intradermal sites. Two weeks later the rabbits were injected with the same dose of zCaMKP, emulsified in Freund’s incomplete adjuvant (DIFCO Laboratories). Two intravenous boosters of 200 lg each of zCaMKP in phosphate-buffered saline (PBS) were given to the rabbits at 2-week intervals. The antiserum were harvested 1 week after the final injection. The antibody was purified by affinity chromatography on zCaMKP-coupled CNBr-Sepharose 4B according to the manufacturers protocol. Cell culture and transfection Human embryonic kidney, 293T, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat inactivated fetal calf serum. Cells were grown at 37 °C in a humidified incubator with a 5% CO2/95% air atmosphere. Transfection of 293T cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 293T cells were plated at 4  104 in a 35 mm dish in 2 ml of DMEM containing 10% fetal calf serum. After 24 h of culture, the cells were incubated for 24 h in 1 ml of DMEM containing 5% fetal calf serum, 6 ll of Lipofectamine 2000, and 3 lg of plasmid DNA for transfection. Transfected cells were cultured in serum free DMEM for 6 h to starve the cells, and then stimulated by 1 lM ionomycin in DMEM at 37 °C. After stimulation, the medium was removed and 200 ll of SDS-sample buffer was added to stop the reaction. The samples were boiled for 5 min, electrophoresed on SDS–polyacrylamide gel, and analyzed by Western blotting. Immunocytochemistry Mouse neuroblastoma, Neuro2a, was cultured on a glass cover slip and transfected by Lipofectamine 2000. The cells were treated with 3.7% formaldehyde in PBS for 20 min, then rinsed with PBS. Following this, the cells were permeabilized with 0.1% Triton X100 in PBS for 5 min. After treatment with 1% bovine serum albumin in PBS, the samples were incubated with anti-myc antibody (Invitrogen) diluted 1:1000 with 1% BSA in PBS at 4 °C for 16 h followed by incubation with Cy3-labeled anti-mouse IgG at room temperature for 2 h. Stained cells were observed under a confocal laser-scanning microscope (TCS SP, Leica). SDS–PAGE and Western blotting SDS–PAGE was performed essentially according to the method of Laemmli [19] on slab gels consisting of a 10% acrylamide separation gel and a 3% stacking gel. The resolved proteins were electrophoretically transferred to a nitrocellulose membrane (Protran BA85, Schleicher and Schuell) and incubated with an anti-zCaMKP antibody at a dilution of 1:500 in 5% skim milk in Tris-buffered saline (pH 7.5) containing 0.05% Tween 20. The membranes were then treated with anti-rabbit IgG conjugated with horseradish peroxidase (1:2000 dilution). The immunoreactive protein bands were visualized with a chemiluminescent substrate (SuperSignal West Dura, Pierce). Whole mount in situ hybridization Whole mount in situ hybridization analysis was carried out as described previously [20]. Embryos were incubated in E3 medium containing 0.1 mM phenylthiourea to block melanin production. The sense (as a negative control) and the antisense RNA probes, labeled with digoxigenin(DIG)-UTP, were synthesized using the DIG

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RNA Labeling mix (Roche) and zCaMKP cDNA (418–1173) subcloned into pGEM-T Easy as a template. The hybridization was detected by anti-DIG antibody conjugated to alkaline phosphatase using NBT-BCIP as a substrate. Morpholino injections Morpholino oligonucleotides (MO), stable nucleic acids analog, were purchased from GeneTools and solubilized in sterilized water

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at a concentration of 5 lg/ll. The resulting stock solution was diluted to working concentrations in sterilized water before injection into one- to four-cell stage embryos. Injected embryos were cultured in E3 medium at 26 °C until use. We used the following morpholino oligonucleotides sequences (see Fig. 7A): zCaMKP AS–MO, 50 -CTC CTT TCT CTG CGC CAC TCA TGC T-30 ; zCaMKP 5mis-MO, 50 CTG CTT TGT CTG CGG CAC TGA TCC T-30 (underlines show the sites of mismatch). MO injection was performed according to the method of Nasevicius and Ekker [21].

Fig. 1. Nucleotide and deduced amino acid sequences of zCaMKP. The deduced amino acid sequence is shown as a one-letter code below the nucleotide sequence. The amino acid residues are numbered beginning with the initiation Met. The termination codon is denoted by an asterisk. The double underline indicates a PP2C motif (PROSITE entry No. PS01032).

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Acridine orange staining

Results

Embryos were stained with the vital dye, acridine orange (Sigma), to examine the occurrence of apoptosis. Embryos were dechorionated and placed in 5 lg/ml of acridine orange in E3 medium. The embryos were treated with acridine orange for 30 min then washed with E3 medium and viewed using a green filter under a fluorescence microscope.

cDNA cloning of zCaMKP

Other methods The nucleotide sequence of both strands was determined by the dideoxynucleotide chain termination method using a BigDye Terminator Cycle Sequencing Ready Reaction Kit Ver.3 (Applied Biosystems) and a DNA Sequencer (model 3100, Applied Biosystems). Protein concentrations were determined by the method of Bensadoun and Weinstein using bovine serum albumin as a standard [22].

(A)

To isolate a cDNA clone of the CaMKP isoform in zebrafish we conducted a BLAST search against the zebrafish whole genome shotgun (WGS) sequence database using the tblastn program. When the amino acid sequence of rat CaMKP was used as a probe, two DNA sequences (Accession Nos. 30521310 and 119879949) corresponding to the central and C-terminal amino acid sequences of the putative zCaMKP were found. To obtain the 50 - and 30 -terminal segments of the cDNA, we performed 50 - and 30 -RACE using two gene specific primers derived from the WGS clones. A full-length cDNA was obtained by PCR using a set of primers, which had been designed from the outside sequences of the open reading frame. Sixteen clones sequenced were classified into four groups. The most dominant one was Type-1 (10 clones), and minor validations

1 33

288

382

Rat PP2C 28% (catalytic) 1

162

406

450

Rat CaMKP 47% (full length) 1

150

394 424

Zebrafish CaMKP 55% (catalytic) 1

156

633

400

Zebrafish CaMKP-N

(B)

: Phosphate binding : Metal binding Fig. 2. Alignment of the deduced amino acid sequence of zCaMKP with other mammalian phosphatases. (A) Schematic illustration of the primary structures of rat PP2Ca (Accession No. J04503), rat CaMKP (Accession No. AB0123634), zCaMKP, and zCaMKP-N (Accession No. NP_001018354). Catalytic domains and acidic amino acid clusters are shown by gray and black boxes, respectively. (B) Catalytic domains of zCaMKP (residues 150–394), zCaMKP-N (residues 156–400), rat CaMKP (residues 162–406), and rat PP2Ca (residues 33–288), were aligned using CLUSTAL W. Identical amino acids are indicated by white letters on a black background. Gaps inserted into the sequences are indicated by dots. The seven amino acid residues shown by the arrows and an arrowhead indicate the critical residues for binding of metal ions and a phosphate group, respectively [24].

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were a change of Arg to Ser at position 411 (Type-2, 2 clones), Glu to Gly at 361 (Type-3, 2 clones), Glu to Asp at 97, Leu to Ile at 199, Arg to Gly at 367 (Type-4, 2 clones) of the Type-1. Fig. 1 shows the nucleotide and deduced amino acid sequences of zCaMKP (Type-1). The open reading frame of 1272 nucleotides encoded a polypeptide of 424 amino acids containing a PP2C motif (PROSITE entry No. PS01032) (Fig. 1. double underlined). From the deduced amino acid sequences, the molecular weight and pI of the zCaMKP were calculated to be 46,412 and 5.40, respectively. The similarity in sequence to other mammalian homologs was analyzed using

1

(A)

2

CLUSTAL W software [23]. Fig. 2A shows schematic illustration of the primary structures of rat PP2Ca, rat CaMKP, zCaMKP, and zebrafish CaMKP-N (zCaMKP-N). These four enzymes showed significant sequence homologies in their catalytic domain, but no sequence homologies in the N-terminal and the C-terminal regions. Fig. 2B shows the alignment of the amino acid sequence of the homologous region of zCaMKP (residues 150–394) with those of zCaMKP-N (residues 156–400), rat CaMKP (residues 162–406), and rat PP2Ca(residues 33–288). On the basis of sequence alignments all of the amino acids involved in metal binding [24] are

(B) Phosphatase Activity (pmol/min/mg protein)

(kDa)

66 45 36

1400 1200 1000 800 600 400 200

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0

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100 80 60 40 20 0 (2 mM) Mg 2+ Mg 2+ Mg 2+ Mg 2+ Mg 2+ Mg 2+ Mg 2+ (2 mM)

+ + + + + + Mn 2+ Ca 2+ Co 2+ Cu 2+ Ni 2+ EDTA

Fig. 3. Enzymatic properties of zCaMKP. (A) Electrophoretic analysis of purified zCaMKP (lane 1) and rat CaMKP (lane 2). His6-tagged CaMKPs expressed in E. coli were purified using a HiTrap Chelating HP column. Purified CaMKPs (0.25 lg) were subjected to 10% SDS–PAGE and stained with Coomassie brilliant blue. (B) The effect of pH on zCaMKP. Activity was measured using 60 ng of zCaMKP and pp10 as a substrate following the method described under ‘‘MATERIALS AND METHODS”. Open square, Tris–HCl buffer, pH 7.0–9.0, containing 2 mM MgCl2; closed square, Gly–NaOH buffer, pH 8.0–10.0, containing 2 mM MgCl2. (C) The effect of divalent cations and EDTA on recombinant zebrafish (black) and rat (hatched) CaMKPs. Activity was measured using 60 ng of enzyme and pp10 as a substrate following the method described in Materials and methods, except that the reaction was performed in the presence of 2 mM metal ions or EDTA. (D) Activation of zCaMKP and rat CaMKP by Mg2+ and Mn2+. Closed square, zCaMKP with MgCl2; closed circle, zCaMKP with MnCl2; open square, rat CaMKP with MgCl2; open circle, rat CaMKP with MnCl2. (E) Inhibition of zCaMKP by metal ions and EDTA. Activity was measured as described in (C), except that the reaction was carried out in the presence of either 2 mM MgCl2 and 2 mM metal ions or EDTA.

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conserved (open arrowheads in Fig. 2B). Interestingly, zCaMKP lacks a Glu cluster corresponding to residues 101–109 in rat enzyme (Fig. 2A), which is essential for the stimulation by polycations such as poly(Lys) [25]. Expression and purification of the recombinant zCaMKP The recombinant zCaMKP was purified from the lysate of the recombinant E. coli carrying pETzCaMKP by chromatography on a HiTrap Chelating HP column, as described under ‘‘MATERIALS AND METHODS”. The final preparation of the enzyme yielded a single protein band corresponding to 47 kDa on SDS–PAGE with Coomassie brilliant blue staining (Fig. 3A). This band corresponded to the band obtained on Western blotting using anti-His6 tag monoclonal antibodies (data not shown). The molecular size of the enzyme estimated by SDS–PAGE was in good agreement with the molecular weight of zCaMKP (Mr = 46,412) calculated from the nucleotide sequence. In a typical experiment, 2.5 mg (1.93 U) of recombinant zCaMKP was obtained from 50-ml E. coli culture. Enzymatic properties of the recombinant zCaMKP The pH optimum of zCaMKP was approximately, pH 8.5, with Gly–NaOH buffer containing 2 mM MgCl2 when pp10 was used as a substrate (Fig. 3B). Activation of zCaMKP by 2 mM MgCl2 was larger than that by 2 mM MnCl2 (Fig. 3C and D). In contrast, rat CaMKP is activated by MnCl2 but not by MgCl2 [6]. Other divalent metal ions and EDTA had no effect on zCaMKP activation (Fig. 3C). However, we observed strong inhibition with 2 mM Ca2+, Co2+, Cu2+, Ni2+, and EDTA in the presence of 2 mM Mg2+ when pp10 was used as a substrate (Fig. 3E). To compare zCaMKP with other well-known protein phosphatases, we examined the effects of various protein phosphatase inhibitors on zCaMKP activity. Orthovanadate, a potent tyrosine phosphatase inhibitor that also inhibits PP1 and PP2A at a millimolar level, had no effect on the activity of zCaMKP (data not shown). In addition, neither okadaic acid nor calyculin A, potent and specific inhibitors for PP1 and PP2A, inhibited the activity of zCaMKP (data not shown).

(A)

Substrate specificity of the recombinant zCaMKP Mammalian CaMKPs are stimulated by poly(Lys) and are highly specific for multifunctional CaMKs [6]. The Glu cluster, corresponding to residues 101–109 in the rat enzyme, is essential for stimulation by poly(Lys) [25]. Given that zCaMKP lacks the Glu cluster we did not expect poly(Lys) to be involved in the activation of zCaMKP. Our results support this hypothesis; CaMKIV(K71R) was not significantly dephosphorylated by zCaMKP even in the presence of poly(Lys) (Fig. 4A, lanes 5 and 6) whereas the same amount of rat CaMKP profoundly dephosphorylated CaMKIV(K71R) in a poly(Lys)-dependent manner (Fig. 4A, lanes 3 and 4). The specific activity of zCaMKP and rat CaMKP was similar when using a phosphopeptide substrate (pp10) (data not shown). These data suggest that poly(Lys) does not affect the activity of zCaMKP. To assess the basal activity of zCaMKP, we used 50-fold higher concentrations of zCaMKP for the dephosphorylation reactions to examine the dephosphorylation of CaMKIV(K71R) and other phosphoproteins in the absence of poly(Lys). As shown in Fig. 4B, none of the phosphorylated proteins, such as GST-ERK, phosphorylase a, and casein, were significantly dephosphorylated by zCaMKP. In contrast, CaMKIV(K71R), which had been phosphorylated by CaMKK, was strongly dephosphorylated even in the absence of poly(Lys) when added simultaneously with these phosphoproteins. Similar results were obtained using rat CaMKP (data not shown). Therefore, our results strongly suggest that zCaMKP and rat CaMKP are specific protein phosphatases for multifunctional CaMKs irrespective of the presence of poly(Lys). Dephosphorylation of CaMKs by CaMKP in living cells Previously, we reported that rat CaMKP and zebrafish CaMKP-N are exclusively localized in the cytosol and nucleus, respectively [7,9]. To examine subcellular localization of zCaMKP, myc-tagged zCaMKP was transiently expressed in mouse neuroblastoma Neuro2a cells and detected by indirect immunofluorescence. In contrast to rat CaMKP (Fig. 5A) and zCaMKP-N (Fig. 5C), zCaMKP was detected in both the cytosol and the nucleus (Fig. 5B). Next,

(B)

Rat CaMKP - - + + - zCaMKP - - - - + + - + - + - + Poly(Lys)

GST-ERK Phosphorylase a Casein zCaMKP Poly(Lys)

+ -

+ + -

+ -

+ + -

+ -

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Fig. 4. Substrate specificity of zCaMKP. (A) CaMKIV(K71R) (5 nM), which had been phosphorylated by CaMKKa (5 nM), and GST-ERK2, which had been phosphorylated by activated MEK1, were incubated at 30 °C with rat CaMKP (0.3 lg/ml) (lanes 3 and 4) or zCaMKP (0.3 lg/ml) (lanes 5 and 6) in the presence or absence of 10 lg/ml poly(Lys), as described in Materials and methods. After incubation for 1 min, the reaction was terminated, and aliquots were analyzed by SDS–PAGE followed by autoradiography. (B) Approximately 5 nM of either GST-ERK2 (lanes 1, 2, 7 and 8), phosphorylase a (lanes 3, 4, 9 and 10), or casein (lanes 5, 6, 11 and 12), was incubated for 1 min at 30 °C with (lanes 2, 4, 6, 8, 10 and 12) or without (lanes 1, 3, 5, 7, 9 and 11) 15 lg/ml zCaMKP in the presence of CaMKIV(K71R) (5 nM) as described in Materials and methods. After the reaction was terminated, aliquots were analyzed by SDS–PAGE, followed by autoradiography. The arrows indicate the position corresponding to the CaMKIV(K71R). Western blot analysis using CaMKIV-specific antibody revealed that the bands shown by the arrowheads were proteolytic fragments generated from the CaMKIV protein during the purification (data not shown).

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Fig. 5. Dephosphorylation of CaMKs by zCaMKP in mammalian cells. (A–C) Subcellular localization of myc-tagged rat CaMKP (A), zCaMKP (B), and zCaMKP-N (C). Neuro2a cells were transiently transfected with above phosphatases and the expressed proteins were stained by means of indirect immunofluorescence with anti-myc antibody. (D) pcrCaMKI-MH was cotransfected with pczCaMKP or pcrCaMKP into 293T cells. Cells expressed the indicated proteins with myc-tag were stimulated by 1 lM ionomycin and lysed with SDS-sample buffer at the indicated times. The cell lysates (40 lg protein) were subjected to SDS–PAGE and then analyzed by Western blotting using a phosphoCaMKI antibody (upper panel). Triplicated samples independently prepared under the same conditions were shown. An asterisk shows non-specific binding of the antibody. The lysates were also analyzed by Western blotting using anti-myc antibodies to detect myc-tagged CaMKI and CaMKPs. (lower panel). (E) The Western blotting using a phospho-CaMKI antibody was quantified by Scion Image. The 100% phosphorylation implicates the phosphorylation level of CaMKI in the absence of CaMKP and ionomycin. Each data point shows the mean ± SE (n = 3) of separate experiments.

we examined whether CaMKP dephosphorylates phospho-CaMKs in living cells. CaMKI was transiently transfected into 293T cells alone or in combination with either rat CaMKP or zCaMKP. The transfected cells were stimulated with ionomycin, a calcium ionophore, and the phosphorylation level of CaMKI was analyzed by Western blotting using phospho-CaMKI specific antibody. As shown in Fig. 5D, the phosphorylation level of CaMKI was markedly increased when the cells transfected with CaMKI alone were treated with ionomycin (lanes 1–6). Conversely, phosphorylation was significantly attenuated when the cells cotransfected with CaMKP and CaMKI were treated under the same conditions (lanes

7–12). Quantitative analysis of immunoreactive bands was carried out using Scion Image software and summarized in Fig. 5E. These data suggest that zCaMKP as well as rat CaMKP significantly dephosphorylates phospho-CaMKI in the living cells. Similar results were obtained from cells cotransfected with CaMKP and CaMKII (data not shown). Temporal and spatial expression of zCaMKP We examined the expression of zCaMKP during the developmental stages of embryogenesis by Western blotting with anti-

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zCaMKP antibody. As shown in Fig. 6A, zCaMKP was detected in the embryos at 48 hpf but not at 36 hpf. Expression of zCaMKP peaked 84 hpf, and was constant between 84 and 120 hpf. To investigate the tissue distribution of zCaMKP in zebrafish we conducted a Western blotting analysis of the various tissue extracts. When the tissue extract was prepared from adult zebrafish and detected by immunoblotting, a strong signal corresponding to the size of zCaMKP was detected in the brain and eyes. A significant immunoreactive band was also detected in other tissues such as heart, testis, skeletal muscle, and fins (Fig. 6B). Apart from prominent expression in the brain and eyes, these results are largely in agreement with the tissue localization of mammalian CaMKP, which is ubiquitously expressed in various tissues [7]. The tissue distribution of the zCaMKP gene transcript was also confirmed by whole mount in situ hybridization. We observed a strong zCaMKP expression signal in the brain and eyes of embryos at 48 hpf (Fig. 6D and E). The signal was not observed when the sense probe was used instead of the antisense probe (Fig. 6C).

zCaMKP antibody (Fig. 7G, lane 5). The knockdown of the zCaMKP gene with morpholino-based antisense oligonucleotides led to an increase in the number of embryos with severe morphological abnormalities. The knockdown phenotype was characterized by roundshaped embryos with a smaller mid- and hindbrain (Fig. 7D) in comparison with the control phenotype (Fig. 7B). We speculated that the observed abnormality might be due to abnormal induction of apoptosis during embryogenesis. Therefore, we further investigated the effects of zCaMKP AS–MO-mediated knockdown on apoptotic cell death using acridine orange staining. Acridine orange is a vital dye reported to stain apoptotic cells but not necrotic cells in Drosophila [26]. As expected, a large number of apoptotic cells were detected in the whole body of antisense-injected embryos (Fig. 7E, white punctate spots). To test the specificity of the phenotype, we made another morpholino oligo (zCaMKP 5mis-MO) in which a five-base mismatch was introduced into zCaMKP AS–MO (Fig. 7A). Injection with zCaMKP 5mis-MO resulted in a normal phenotype without significant apoptotic cells (Fig. 7C and F) and no inhibition of zCaMKP expression (Fig. 7G, lane 4). Quantitative analysis of apoptotic cells were shown in Fig. 7H. To clarify whether phosphatase activity of zCaMKP is a prerequisite for normal embryogenesis, we prepared a phosphatasedead mutant of zCaMKP(D194A), and examined whether coinjection of the mutant phosphatase with zCaMKP AS–MO could rescue the abnormal phenotype. As shown in Fig. 8A, zCaMKP(D194A) could not dephosphorylate phospho-CaMKI under the conditions where zCaMKP(WT) completely dephosphorylated it. As expected, coinjection of recombinant rat CaMKP(WT) with zCaMKP AS–MO reduced the gross abnormality rate from 100% to 46% (Fig. 8C). However, coinjection of the phosphatase-dead mutant failed to rescue the phenotype (Fig. 8D). These results clearly demonstrated that catalytic activity of zCaMKP is critical for the normal development of zebrafish.

Gene knockdown of zCaMKP during zebrafish embryogenesis During embryogenesis, the expression of zCaMKP increased gradually after 48 hpf (Fig. 6A). This led us to analyze the role of zCaMKP in zebrafish embryogenesis. In an attempt to understand more precisely how zCaMKP is related to embryonic development, we performed functional gene knockdown experiments in zebrafish using antisense morpholino-modified oligonucleotides that were targeted to the zCaMKP gene (zCaMKP AS–MO, Fig. 7A). Morpholinos have been reported to act as effective and specific translational inhibitors in the zebrafish embryo [21]. Indeed, injection of zCaMKP AS–MO to 1–4 cell stage embryos at a concentration of 1 lg/ll (about 0.8 ng injected) resulted in knockdown of zCaMKP protein as demonstrated by Western blotting using affinity-purified anti-

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Fig. 6. Expression of zCaMKP in zebrafish. (A) Expression of zCaMKP during zebrafish embryogenesis. Crude extracts (60 lg) from zebrafish embryos at the indicated stages and adult brain extract (60 lg) were subjected to 10% SDS–PAGE and transferred onto a nitrocellulose membrane for detection with affinity-purified anti-zCaMKP antibody. (B) Tissue distribution of zCaMKP. Protein extracts (60 lg) of the brain (lane 1), eye (lane 2), heart (lane 3), testis (lane 4), skeletal muscle (lane 5), fin (lane 6), ovary (lane 7), and gill (lane 8) were prepared from adult zebrafish, subjected to 10% SDS–PAGE, and detected by Western blotting analysis with affinity-purified anti-zCaMKP antibody. Purified His6-tagged zCaMKP (2 ng) are shown in lane 9. An asterisk indicates the non-specific band. (C–E) Localization of the zCaMKP gene transcript in zebrafish embryos. Zebrafish embryos at 48 hpf were analyzed by whole mount in situ hybridization with DIG-labeled RNA probes and detected by anti-DIG antibody conjugated to alkaline phosphatase. Gene transcript of zCaMKP was detected as a blue signal. (C) Sense RNA probe as a negative control. (D) and (E), antisense RNA probe for zCaMKP. (E) shows a dorsal view of (D).

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Fig. 7. Gene knockdown of zCaMKP during embryogenesis. (A) Sequences of zCaMKP mRNA around the 50 initiation Met (double underlined), AS–MO and 5mis-MO used in this study. Underlines in the 5mis-MO indicate mismatch nucleotides compared to AS–MO. (B–D) Phenotypes of the control (B) and embryos injected with 5mis-MO (C) or AS–MO (D) at 72 hpf. Morpholino oligos were injected into 1–4 cell stage embryos at a concentration of 1 lg/ll (approximately 0.8 ng/embryo). Acridine orange staining of embryos injected with AS–MO (E) or 5mis-MO (F). Embryos were stained with acridine orange at 48 hpf and observed by stereoscopic microscopy as described in Materials and methods. (G) Detection of zCaMKP in the AS–MO injected embryos. Lysates were prepared at 72 hpf from zebrafish embryos injected with AS–MO, 5mis-MO, or from embryos without injection. Protein extracts were subjected to 10% SDS–PAGE and detected by Western blotting with affinity-purified anti-zCaMKP antibody. Lane 1, recombinant zCaMKP (2 ng); lane 2, crude extract (60 lg) from adult zebrafish brain; lane 3, crude extract (120 lg) from embryos without injection; lane 4, crude extract (120 lg) from 5mis-MO injected embryos; lane 5, crude extract (120 lg) from AS–MO injected embryos. (H) Apoptotic cells per embryo at 24 hpf were counted using a green filter under a fluorescence microscope. Data represent the mean ± SE of 30 embryos.

Discussion In an attempt to clarify physiological functions of CaMKP on the basis of its molecular properties, we chose zebrafish as a model animal, and cloned the zebrafish homolog of CaMKP. The cDNA clone obtained shared relatively high degrees of sequence homology with mammalian CaMKPs; it showed 47% and 48% homology with rat CaMKP and human CaMKP, respectively, in their catalytic domains. The clone had some prominent structural features, including (1) a short C-terminal region adjacent to the catalytic domain that had low sequence homology with mammalian CaMKP and (2) it lacked an acidic amino acid cluster that is responsible for activation by poly(Lys) in rat CaMKP (Fig. 2A). The enzymatic properties of zCaMKP, including the metal requirements, were similar to those of PP2Ca. However, as shown in Fig. 4B, the zebrafish homolog was highly specific to phosphorylated CaMKs even in the absence of poly(Lys). We observed minimal dephosphorylation of phosphopro-

teins, other than phospho-CaMKIV. Furthermore, embryonic death caused by knockdown of zCaMKP was rescued by coinjection with rat CaMKP (Fig. 8C), suggesting that these enzymes are functionally identical. Thus, we concluded that the cDNA clone obtained in this study was that of the zebrafish homolog of CaMKP. PPM family protein phosphatases require divalent metal ions such as Mg2+ and Mn2+ for their activity. Although rat CaMKP was found to be stimulated only by Mn2+, zCaMKP could be stimulated either by Mg2+ and Mn2+ as in case of PP2Ca when phosphopeptide was used as a substrate (Fig. 3C and D). Metal requirement shows different patterns depending on the substrates used for the phosphatase assay. When para-nitrophenyl phosphate was used as a substrate, both CaMKP and PP2C exclusively require Mn2+, but not Mg2+, for their activity (data not shown). Six critical amino acids for the binding of metal ions (shown by open arrowheads in Fig. 2B) are highly conserved in PPM family enzymes, including PP2Ca, rCaMKP, zCaMKP-N and zCaMKP. Therefore, differences in

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Fig. 8. Phenotypic rescue by microinjection of rat CaMKP. (A) Phosphatase activity in wild-type (WT) and point mutant (D194A) rat CaMKP was determined using phosphoCaMKI as a substrate. Phospho-CaMKI was incubated in a standard reaction mixture (50 ll) containing 5 lg/ml of CaMKP at 30 °C for 30 min. The reaction was stopped by the addition of SDS-sample buffer. The samples were subjected to SDS–PAGE and analyzed by Western blotting using antibodies as indicated. (B–D) Knockdown of zCaMKP and rescue by the active CaMKP. Zebrafish embryos were injected with AS–MO (0.8 ng/embryo) (B). AS–MO (0.8 ng/embryo) was coinjected with either rat CaMKP-WT (0.45 ng/ embryo) (C) or rat CaMKP-D194A (0.45 ng/embryo) (D). Embryos are shown at 72 hpf.

metal requirements of these phosphatases could not be simply explained by these critical amino acids. Differences in neighboring residues around the critical amino acids may produce structural changes in the catalytic pocket. Subtle environmental changes in the active site may affect catalytic properties such as metal requirement of the phosphatases. Therefore, precise structural analysis in the active site will reveal the molecular basis of metal requirements of the phosphatases. We have to wait for the results of X-ray crystarographic analysis of CaMKPs, which is now in progress in this laboratory. Rat CaMKP is strongly activated by polycations such as poly(Lys) in vitro, specifically dephosphorylating phosphorylated CaMKs to deactivate them [6]. However, there is no direct evidence that CaMKP dephosphorylates and deactivates CaMKs in cells. To date, a report by Harvey et al. [27] is the only investigation to address this issue. They found that the phosphorylation level of vimentin, a CaMKII substrate, was significantly attenuated in the cells overexpressing human CaMKP. We used phospho-specific antibody against phosphorylated CaMKI and showed that co-expression of zCaMKP or rat CaMKP with CaMKI markedly suppressed ionomycin-stimulated phosphorylation of CaMKI in 293T cells (Fig. 5D and E). To our knowledge, this is the first report directly showing that CaMKP can dephosphorylate CaMKs in the cells. Previously, we showed that an acidic amino acid cluster in the N-terminal domain of rat CaMKP is responsible for stimulation by polycations [13,25]. It should be noted that even zCaMKP, which could not be activated by polycations due to lack of the acidic amino acid cluster, effectively dephosphorylated CaMKs in the cells. This observation suggests that basal CaMKP activity, which is not activated by activators such as polycations, is sufficient for dephosphorylation of phosphorylated CaMKs in the cells. Alternatively, there might exist another type of activator in the cells which substitutes for polycations or which can activate CaMKP independently of the acidic amino acid cluster. We are now trying to identify such activators by two-hybrid techniques using rat CaMKP and zCaMKP as baits.

In an effort to further explore the physiological functions of CaMKP, we examined both the temporal expression of zCaMKP during zebrafish embryogenesis and the distribution of CaMKP in zebrafish tissue. CaMKP expression was first detected by Western blotting analysis at 48 hpf. This result was in agreement with the timing based on in situ hybridization (Fig. 6C). One of the prominent features of the tissue distribution of zCaMKP is its striking enrichment in the brain and eyes (Fig. 6B). This observation is consistent with the data obtained by whole mount in situ hybridization in zebrafish embryos (Fig. 6C). CaMKP may play an important role in the visual and central nervous systems. We also observed modest but significant expression of CaMKP in other tissues such as the heart, skeletal muscle, and fins. Our results suggest that tissue distribution of CaMKP is largely similar to that in rats [7]. This is in contrast to the tissue distribution of zebrafish CaMKP-N, which is expressed exclusively in the central nervous system [9]. Therefore, CaMKP-N is likely to be involved in some functions specific to the central nervous system such as higher order neuronal functions, whereas CaMKP is likely to be involved in ubiquitous cellular functions. The relative abundance of CaMKP expression in each tissue may vary, depending on the stage of development. Previously, we showed that CaMKP-N is essential for development of the central nervous system in zebrafish using antisense knockdown techniques. Therefore, the observation that CaMKP is expressed at 48 hpf, a relatively early stage during zebrafish development, led us to examine whether CaMKP also plays an essential role in zebrafish embryogenesis. Microinjection of antisense morpholino-modified oligonucleotide targeted to the zCaMKP gene into the embryo resulted in severe morphological abnormalities of the embryos with numerous apoptotic cells stained with acridine orange in the whole body (Fig. 7). Thus, our data strongly suggested that CaMKP is essential for normal development of the zebrafish embryo. Knockdown of CaMKP may induce apoptosis of cells throughout the whole body wherever CaMKP is expressed, leading to severe morphological abnormality of the tissue. This is in clear contrast to the knockdown of

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CaMKP-N. In this instance apoptotic cells, stained with acridine orange, are only observed in the central nervous system, corresponding to the expression of CaMKP-N, and are associated with mild morphological abnormality [9]. Thus, it is likely that knockdown of CaMKP/CaMKP-N results in apoptosis in the cells that express each phosphatase. The details regarding the molecular mechanism by which knockdown of CaMKP/CaMKP-N induces apoptosis are currently unknown. Tan et al. [10] reported that over-expression of human CaMKP or rat CaMKP in HeLa cells results in apoptosis, although the molecular mechanism for this effect also has not yet been clarified. There are many reports that CaMKII and/or CaMKIV is involved in apoptosis [28–30]. Very recently, Fahrmann et al. [31] reported that calyculin A, a PP1/PP2A inhibitor, induces apoptosis of MDCK cells with concomitant activation of CaMKII. Furthermore, KN-93, a CaMKII inhibitor, can rescue calyculin A-induced apoptosis. Taken together, it appears that the regulation of CaMK activity levels and the timing of expression are important for determining cell survival. CaMKP and CaMKP-N may regulate cellular apoptosis by modulating CaMK activities. It is well known that intracellular signal transduction is achieved on the basis of a subtle balance between phosphorylation and dephosphorylation. Similarly, CaMK activities are finely regulated by both phosphorylation and dephosphorylation [32,33]. The appropriate regulation of CaMK activity, including CaMKII, is essential for normal cellular function. The autophosphorylation status of CaMKII is closely related to learning and memory ability. To ensure normal learning and memory, CaMKII activity must be appropriately regulated. Dysregulation of CaMKs can cause various diseases [33]. CaMKP and CaMKP-N may act as critical negative regulators of CaMKs involved in important biological processes such as apoptosis and memory. Acknowledgments This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, National Project ‘‘Knowledge Cluster Initiative” (2nd stage, ‘‘Sapporo Biocluster Bio-S”) granted by the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Smoking Research Foundations, the Naito Foundation, the Sumitomo Foundation, the fund for Kagawa University Young Scientists, and an AIST Research Grant.

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