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calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish
Archives of Biochemistry and Biophysics 457 (2007) 205–216 www.elsevier.com/locate/yabbi
Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebraWsh 夽 Takaki Nimura a, Noriyuki Sueyoshi a,¤, Atsuhiko Ishida b, Yukihiro Yoshimura c, Makoto Ito c, Hiroshi Tokumitsu d, Yasushi Shigeri e, Naohito Nozaki f, Isamu Kameshita a a
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan b Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan c Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan d Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan e National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan f Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka 238-8580, Japan Received 28 August 2006, and in revised form 27 September 2006 Available online 6 November 2006
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 signiWcance of CaMKP-N during zebraWsh embryogenesis, we cloned and characterized zebraWsh CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebraWsh whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed fulllength 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 signiWcantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebraWsh 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 signiWcant morphological abnormalities in zebraWsh 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 zebraWsh. © 2006 Elsevier Inc. All rights reserved. Keywords: ZebraWsh; CaM kinase; Apoptosis; Phosphatase; CaMKP; CaMKP-N
The increase of Ca 2+ concentration in response to extracellular stimuli activates various Ca 2+/calmodulin (CaM)-dependent enzymes involved in cellular signaling, such as Ca2+/CaM-dependent protein kinases (CaMKs). Multifunctional CaMKs, CaMKI, II, and IV, phosphorylate a broad range of substrates, and participate in vari夽 The nucleotide sequence reported in this paper has been submitted to the GenBank™/EBI Data Bank with an Accession No. AB113302. * Corresponding author. Fax: +81 87 891 3114. E-mail address: [email protected] (N. Sueyoshi).
0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.09.034
ous cellular processes [1–4]. CaMKIV occurs predominantly in the nuclei of brain and thymus, and phosphorylates a number of proteins including synapsin I, microtubule associated protein 2, tau protein, myosin light chain, tyrosine hydroxylase, cAMP-response element binding protein (CREB), and so on [5–7]. It is reported that transcriptional regulation by CaMKIV through phosphorylation of CREB plays important roles in the regulation of memory and neuronal plasticity [8,9]. CaMKIV is strongly activated through phosphorylation of a threonine residue located within the region called the
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“activation loop” by another CaMK1, CaMK kinase (CaMKK) [10,11]. CaMKIV, once activated, should then be dephosphorylated by protein phosphatases in order to downregulate its activity prior to the next stimulus, and it is reported that various Ser/Thr protein phosphatases dephosphorylate CaMKIV in vitro [12]. We previously puriWed and isolated a Mn2+-dependent, okadaic acid/calyculin A-insensitive protein phosphatase with a molecular weight of 54,000, and designated it CaMK phosphatase (CaMKP) [13]. CaMKP is a member of PPM family phosphatases, and speciWcally dephosphorylates phosphorylated CaMKs including CaMKIV in vitro [13– 15]. Another protein phosphatase having some homology with CaMKP was found in the human database and was named as CaMKP-N, since it had similar biochemical properties to CaMKP and showed nuclear localization [16]. CaMKP-N is also a Mn2+-dependent protein phosphatase and dephosphorylates CaMKIV in vitro [16,17]. However, the physiological signiWcance of CaMKP-N has yet to be determined. In the present study, we employed zebraWsh as a model animal to analyze the biological functions of CaMKP-N in vivo. We cloned zebraWsh CaMKP-N (zCaMKP-N) and demonstrated that the nuclear localization signal (NLS) is localized at the C-terminal region of zCaMKP-N. We also provide the Wrst evidence that CaMKP-N dephosphorylates phospho-CaMKIV in living cells. Furthermore, a gene knockdown experiment of zCaMKP-N revealed that CaMKP-N is indispensable for some of the processes in zebraWsh embryogenesis. Materials and methods Materials Ionomycin calcium salt was obtained from Calbiochem. Cy3-labeled anti-mouse IgG, ATP, and bovine serum albumin were purchased from Sigma. Anti-His6 antibody and anti-CaMKIV antibody were from Roche and BD Biosciences, respectively. Horseradish peroxidase (HRP)-labeled anti-mouse IgG and HRP-labeled anti-rabbit IgG were from ICN Pharmaceuticals. Calmodulin (CaM) was puriWed from rat testis as described previously [18].
cDNA cloning of zebraWsh CaMKP-N SMART RACE cDNA AmpliWcation kit (Clontech) was used to clone a full-length coding sequence for zCaMKP-N using primers based on a GenBank zebraWsh whole genome shotgun (WGS) clones (Accession Nos.: 132560840 and 45566463). 5⬘-RACE Wrst strand cDNA was primed from mRNA of adult zebraWsh with SuperscriptII reverse transcriptase using a SMART II oligonucleotide and a 5⬘-RACE cDNA synthesis primer. The 5⬘-end of the cDNA was ampliWed by PCR with gene-speciWc primer 1 (AS1: 5⬘-GTCTCTGAGGAACACTACGATTACCGT-3⬘) and a univer-
1 Abbreviations used: CaM, calmodulin; CaMK, Ca2+/calmodulin-dependent protein kinase; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; CaMKP, Ca2+/calmodulin-dependent protein kinase phosphatase; CaMKP-N, nuclear Ca2+/calmodulin-dependent protein kinase phosphatase; NLS, nuclear localization signal; PBS, phosphate-buVered saline; PP2C, protein phosphatase 2C.
sal primer mix and 5⬘-RACE Wrst strand cDNA as a template, using an Advantage 2 PCR kit (Clontech). The 3⬘-RACE Wrst strand cDNA was primed using 3⬘-RACE cDNA synthesis primer. The 3⬘-end of the cDNA was obtained by PCR using a universal primer mix and gene-speciWc primer 2 (S1: 5⬘-CTGGCGCGACTTGTCTTCAATAAG-3⬘), and 3⬘RACE Wrst strand cDNA as a template. The 5⬘- and 3⬘-RACE PCR products were cloned into pGEM-T Easy vector (Promega), and their DNA sequences determined. The nucleotide sequences of the products of 5⬘RACE and 3⬘-RACE were overlapped and aligned using the DNASIS computer program developed by Hitachi Software Engineering. An open reading frame of 1899 nucleotides was generated. A sense primer (5⬘AGACTTCTTGCCCGGCTCCA-3⬘) and an antisense primer (5⬘ACGTCCACCGCAGGAGAGTG-3⬘) were designed from the outside sequences of the open reading frame. A full-length cDNA was prepared by PCR using these primers and 3⬘-RACE ready cDNA library as a template with Pyrobest DNA polymerase (TaKaRa). The PCR product was cloned into a pGEM-T Easy vector, and Wve independent clones were sequenced (pGEMzCaMKPN-1, -2, -3, -4, -5).
Construction of plasmids In the case of zCaMKP-N(WT), the following primers were used for PCR with pGEMzCaMKPN-5 as a template: 5⬘-upstream primer (5⬘GCTAGCATGGCCGGCTCTGCCAACGA-3⬘) and 3⬘-downstream primer (5⬘-CTCGAGGACCCTTTTGCTATGGGTTACATGAGG-3⬘). The NheI (underlined)–XhoI (double underlined) fragment was inserted into the NheI–XhoI sites of pET-23a(+) (Novagen) to generate plasmid pETzCaMKPN(WT). Mutagenesis on Asp-188 was performed by the inverse PCR method [19], with the following primers: D188A-upstream primer, 5⬘-GCCGGTCACGGCGGGGTGGACGCT-3⬘ and D188Adownstream primer, 5⬘-AAACACGGCAAAGTAGGCCTGCTCTTC-3⬘ (underline shows the site of mutation), and pETzCaMKPN(WT) as a template. The 5⬘-ends of the PCR fragment were then phosphorylated by T4 polynucleotide kinase (Nippon Gene) and self-ligated by T4 DNA ligase (Nippon Gene), and the recombinant plasmid obtained was designated as pETzCaMKPN(D188A). To express zCaMKPN and its mutants in mammalian cells, the following plasmids were prepared. pczCaMKPN; the DNA fragment coding C-terminal myc/His6-tagged full-length zCaMKPN was ampliWed with PCR using primers (5⬘-AAGCTTGCGATGGCC GGCTCTGCCAA-3⬘ and 5⬘-CTCGAGACCCTTTTGCTATGGGTTA CATGAGG-3⬘), and pETzCaMKPN(WT) as a template, the HindIII (underlined)–XhoI (double underlined) fragment was inserted into HindIII–XhoI sites of pcDNA3.1(+)/myc-His B (Invitrogen). pczCaMKPN(D188A) was prepared by the same procedure except that pETzCaMKPN(D188A) was used as a template. pczCaMKPN(1–574); the DNA fragment coding residues 1–574 of zCaMKP-N, lacking the putative nuclear localization signal, was ampliWed with PCR using primers (5⬘-AAGCTTGCGATGGCCGGCTCTGCCAA-3⬘ and 5⬘-CTCGAG CCCTCCTGAGAGAAGAGCGCAGC-3⬘) and inserted into HindIII (underlined)–XhoI (double underlined) sites of pcDNA3.1(+)/myc-His B. zCaMKP-N(448–633) was ampliWed with PCR using EGFP-zPN-U1342/ ERI primer (5⬘-GAATTCTGATGGAGGTGCGGAGAATGG-3⬘) and LHis6/SalI primer (5⬘-TTTGTCGACTCAGTGGTGGTGGTGGTGG TG-3⬘), and pETzCaMKPN(WT) as a template. zCaMKP-N(448–574) was ampliWed with PCR using EGFP-zPN-U1342/ERI primer and LHis6/ SalI primer and pETzCaMKPN(1–574) as a template. zCaMKP-N(489– 633) was ampliWed with PCR using EGFP-zPN-U1465/ERI primer (5⬘and LHis6/SalI GAATTCTATCACAGGGCCGGATGTGCA-3⬘) primer and pETzCaMKPN(WT) as a template. EcoRI (underlined)–SalI (double underlined) fragment was inserted into the EcoRI–SalI sites of pEGFP-C1 (Clontech) to generate plasmid pEGFP-zCaMKPN(448–633), (448–574), and (489–633), respectively. In the case of rat CaMKIV(WT), the following primers were used for PCR: CaMKIV(WT)-5⬘ upstream primer (5⬘-GATATCGTTATGCTCA AAGTCACGGTGCC-3⬘) and CaMKIV(WT)-3⬘ downstream primer (5⬘The EcoRV CTCGAGTACTCTGGCAGAATAGCATCCTG-3⬘). (underlined)–XhoI (double underlined) fragment was inserted into the EcoRV–XhoI sites of pcDNA3.1(+)/myc-His B to generate plasmid
T. Nimura et al. / Archives of Biochemistry and Biophysics 457 (2007) 205–216 pcrCaMKIV(WT). Mutagenesis on Lys-71 was performed by the inverse PCR method [19], with the following primers: CaMKIV(K71R)-5⬘ upstream primer, 5⬘-CGCGTGTTAAAGAAAACAGTGGACAAGA-3⬘ and CaMKIV(K71R)-3⬘ downstream primer, 5⬘-GAGAGCATAGGGC TTCTGGG-3⬘ (underline shows the site of mutation) and pETrCaMKIV(WT) as a template. The 5⬘-ends of the PCR fragment were phosphorylated by T4 polynucleotide kinase and self-ligated by T4 DNA ligase, and the recombinant plasmid thus obtained was designated as pETrCaMKIV(K71R).
Expression of proteins in Escherichia coli and puriWcation In the case of zCaMKP-N, pETzCaMKPN(WT or D188A) was introduced into E. coli strain Rosetta(DE3) (Novagen). The transformed bacteria were grown at 37 °C to an A600 of 0.5, and then isopropyl--Dthiogalactopyranoside was added to a Wnal concentration of 0.25 mM. After 6 h at 25 °C, the bacteria were harvested by centrifugation and suspended in buVer A (20 mM Tris–HCl (pH 7.5) containing 150 mM NaCl, and 0.05% Tween 40). After sonication, cell debris was removed by centrifugation (20,000g) at 4 °C for 10 min, and supernatant obtained was loaded on a HiTrap Chelating HP column (Amersham Biosciences) preequilibrated with buVer A. The column was washed with buVer A, buVer A containing 20 mM imidazole, buVer A containing 50 mM imidazole, and then eluted with buVer A containing 200 mM imidazole. The active fraction was dialyzed against buVer B (20 mM Tris–HCl (pH 7.5) containing 0.05% Tween 40 and 14.2 mM 2-mercaptoethanol) and used for phosphatase assay. In the case of CaMKIV(K71R), the pETrCaMKIV(K71R) construct was introduced into E. coli strain BL21(DE3) (Novagen). The transformed bacteria were grown at 37 °C for 12 h, harvested by centrifugation, and suspended in buVer A. CaMKIV(K71R) expressed in E. coli was extracted and puriWed with a HiTrap Chelating HP column as described above. The active fraction was dialyzed against buVer A and stored in aliquots at ¡80 °C until use.
Production of antibodies An antibody against zCaMKP-N was produced by immunizing rabbits with the antigenic peptide, CYEDRMDSFTDRTSLS, corresponding to the amino acid residues 474–488 of zCaMKP-N. Immunization was carried out essentially according to a previously described procedure [20]. Monoclonal antibody to detect phospho-CaMKIV at Thr-196 was prepared as described [21].
SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting SDS–PAGE was performed essentially according to the method of Laemmli [22] on slab gels consisting of a 10% acrylamide separation gel and a 3% stacking gel. The resolved proteins were electrophoretically transferred to nitrocellulose membranes (Protran BA85, Schleicher and Schuell) and immuno-reactive protein bands were detected essentially according to the method described previously [23].
Protein phosphatase assay Protein phosphatase assay was carried out using a phosphopeptide pp10 (YGGMHRQETpVDC), which contains the amino acid sequence around the autophosphorylation site of CaMKII, as a substrate [24]. The reaction mixture (50 l) contained 50 mM Tris–HCl (pH 8.0), 2 mM MgCl2 (or MnCl2), 0.1 mM EGTA, 0.01% Tween 20, 40 M pp10, and an appropriate amount of zCaMKP-N. The reaction was started by adding zCaMKP-N and incubated at 30 °C for 10 min. Phosphate released in the mixture was determined by malachite green assay [25]. Protein phosphatase activity of CaMKP-N was also determined using phosphorylated CaMKIV mutant. Recombinant CaMKIV(K71R) (40 g/ ml) was phosphorylated by CaMKK (5 g/ml) at 30 °C for 30 min in a
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reaction mixture comprising 40 mM Hepes–NaOH (pH 8.0), 5 mM Mg(CH3COO)2, 2 mM dithiothreitol, 0.1 mM EGTA, 0.1 mM ATP, 1 mM CaCl2, and 1 M CaM, and the reaction stopped by adding 2 mM EGTA. Dephosphorylation of a phosphoprotein substrate was carried out at 30 °C for 30 min in a reaction mixture containing 20 mM Tris–HCl (pH 7.5), 2 mM MnCl2, 0.05% Tween 40, 20 g/ml phospho-CaMKIV(K71R), and an appropriate amount of zCaMKP-N. The reaction was started by the addition of zCaMKP-N and terminated by the addition of an equal amount of SDS sample buVer. The sample was then subjected to SDS– PAGE and analyzed by Western blotting analysis using phospho-CaMKIV antibody.
Cell culture and transfection Mouse neuroblastoma, Neuro2a, was cultured in Dulbecco’s modiWed Eagle’s medium (DMEM, Sigma) containing 10% heat inactivated fetal calf serum. Cells were grown at 37 °C in a humidiWed incubator with a 5% CO2/95% air atmosphere. Transfection of Neuro2a cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Neuro2a cells were plated at 4 £ 104 in a 35-mm dish in 2 ml of DMEM containing 10% fetal calf serum. After 48 h of culture, cells were incubated for 24 h in 1 ml of DMEM containing 5% fetal calf serum, 6 l of Lipofectamine 2000, and 5 g 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 M ionomycin in DMEM at 37 °C. After stimulation, the medium was removed and 150 l of SDS sample buVer added to stop the reaction. Samples were boiled for 5 min, electrophoresed on SDS–polyacrylamide gel, and analyzed by Western blotting.
Immunocytochemistry of zCaMKP-N Transfected cells were cultured on cover glass and treated with 3.7% formaldehyde in phosphate-buVered saline (PBS) for 20 min. After being rinsed with PBS, cells were permeabilized with 0.1% Triton X-100 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% bovine serum albumin in PBS at 4 °C for 16 h followed by incubation with Cy3-labeled anti-mouse IgG at room temperature for 2 h. The nuclear chromatin region was stained with DAPI [4⬘,6-diamidino-2-phenylindole] (DOJINDO, Japan) at 2 g/ml. Stained cells were observed under a Xuorescence microscope (model BX51, Olympus, Japan) and images were obtained using a Cool SNAP CCD camera (Roper ScientiWc).
Fish maintenance ZebraWsh, Danio rerio, were maintained at 26 °C and embryos collected from natural crosses of wild-type Wsh. Collected embryos were maintained in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) at 26 °C. Embryos were staged according to hours post-fertilization (hpf) at 26 °C and morphological criteria [26].
Whole mount in situ hybridization analysis Whole mount in situ hybridization analysis was carried out as described in [27]. Embryos were incubated in E3 medium containing 0.1 mM phenylthiourea to block melanin production. The sense (as negative control) and the antisense RNA probes labeled with digoxigenin(DIG)-UTP were synthesized using the DIG RNA Labeling mix (Roche) from zCaMKP-N cDNA (346–1215) subcloned into pGEM-T Easy. The hybridization was detected by anti-DIG antibody conjugated to alkaline phosphatase and NBT/BCIP as a substrate.
Morpholino oligo injections Morpholino-based oligonucleotides (GENE TOOLS) were solubilized in sterilized water at a concentration of 5 g/l, and diluted to
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working concentrations in sterilized water before injection. ZebraWsh embryos were injected at one- to four-cell stage with morpholino oligonucleotides using a micromanipulator and microinjector (Narishige) according to the method of Nasevicius and Ekker [28]. Injected embryos were cultured in E3 medium at 26 °C. The sequences of mor-
pholino oligonucleotides used in the present study were; zCaMKP-N antisense morpholino oligo (AS-MO), 5⬘-GCCATCGCTTCTC ATTCCTGGAGAG-3⬘ and zCaMKP-N control morpholino oligo (5 mis-MO), and 5⬘-GCGATGGCTTCTGATTCCTCGACAG -3⬘ (underlines show the sites of mismatch).
Fig. 1. Nucleotide and deduced amino acid sequences of zebraWsh CaMKP-N. The amino acid residues are numbered beginning with the Wrst Met and are shown with a one-letter code below the nucleotide sequence. The termination codon is denoted by an asterisk. The solid underline and double underline indicate an acidic amino acid cluster and a PP2C motif (PROSITE entry No. PS01032), respectively. A predicted NLS is boxed.
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Acridine orange staining Dechorionated embryos were stained with the vital dye, acridine orange (Sigma) to examine the occurrence of apoptosis. After treatment
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with 2 g/ml of the dye in E3 medium for 30 min, embryos were washed with E3 medium. The visualization and photography was performed using Xuorescence microscope, SMZ1500 (Nikon, Japan), and COOLPIX4500 (Nikon, Japan).
Fig. 2. Alignment of deduced amino acid sequence of zCaMKP-N with other mammalian phosphatases. (A) Amino acid sequence of zCaMKP-N was aligned with rat CaMKP-N and human CaMKP-N using CLUSTAL W. Identical amino acids are shaded, and gaps inserted into the sequences are indicated by dots. The acidic amino acid clusters are underlined. A predicted nuclear localization signal is double underlined. (B) Alignment of catalytic domains of zCaMKP-N (Accession No.: AB113302), rat CaMKP (Accession No.: AB023634), and rat PP2C (Accession No.: J04503). Catalytic domains of zCaMKP-N(residues 156–405), rCaMKP(residues 162–411), and rPP2C(residues 33–293) are aligned using CLUSTAL W. The seven amino acid residues shown by the arrows indicate the critical residues for the binding of metal ions. (C) Schematic illustration of the primary structures of rat PP2C, rat CaMKP, rat CaMKP-N, and zebraWsh CaMKP-N. Catalytic domains and acidic amino acid clusters are shown by gray and black boxes, respectively. The dark gray with asterisk indicates the position of the PP2C motif. Amino acid identity between indicated catalytic domains are also shown.
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Other methods Protein concentrations were determined by the method of Bensadoun and Weinstein using bovine serum albumin as a standard [29]. Nucleotide sequences were determined on both strands by the dideoxynucleotide chain termination method with a BigDye Terminator Cycle Sequencing Ready Reaction Kit Ver. 3.1 (Applied Biosystems) and a DNA Sequencer (model 3100, Applied Biosystems).
Results cDNA cloning of zebraWsh CaMKP-N In earlier papers, cDNA clones for human CaMKP-N [16] and rat CaMKP-N [17] were isolated and recombinant enzymes characterized in vitro. However, the physiological functions of these mammalian CaMKP-Ns are not well understood. To investigate the physiological signiWcance of CaMKP-N in vivo, we used zebraWsh as a model animal in this study. To isolate a cDNA clone for zCaMKP-N, BLAST search against the zebraWsh whole genome shotgun sequence database was carried out using the tblastn program. By using the amino acid sequence of human CaMKP-N as a probe, two DNA sequences (Accession Nos.: 132560840 and 45566463) were found to contain the central part and the C-terminal parts, respectively, of zCaMKP-N. To obtain the 5⬘- and 3⬘-terminal segments of the cDNA, 5⬘- and 3⬘-RACE were performed using two gene speciWc primers derived from the sequences of the whole genome shotgun clones. The nucleotide sequences of the products of 5⬘-RACE and 3⬘-RACE were overlapped, and a set of primers was designed from the outside sequences of the open reading frame. A full-length cDNA was ampliWed by PCR using these primers and a 3⬘-RACE ready cDNA library as a template, and then subcloned into pGEM-T Easy vector. The nucleotide sequence of the cDNA clone for zCaMKP-N was determined by sequencing Wve independent clones. The nucleotide sequence and the deduced amino acid sequence of zCaMKP-N are shown in Fig. 1. The open reading frame of 1899 nucleotides encoded a polypeptide of 633 amino acids containing a protein phosphatase 2C (PP2C) motif (PROSITE entry No. PS01032) (Fig. 1, double underlined). From the deduced amino acid sequences, the molecular mass, and pI of zCaMKP-N were calculated to be 71,036 and 4.97, respectively. The similarity in the sequence of zCaMKP-N to those of other mammalian homologues was analyzed using CLUSTAL W software [30]. An alignment of the deduced amino acid sequence of zCaMKP-N with those of rat and human enzymes is shown in Fig. 2A. The number of amino acid residues of zCaMKP-N(633 amino acids) is much smaller than those of rat CaMKP-N (750 amino acids) or human CaMKP-N(757 amino acids); zCaMKP-N exhibited 49% and 48% identities with those of rat and human enzymes, respectively. An alignment of the catalytic domain of zCaMKP-N with those of rat CaMKP (Accession No.: AB023634) and rat PP2C (Accession No.: J04503) is
shown in Fig. 2B. Seven amino acid residues in zCaMKPN, corresponding to residues Arg-33, Glu-37, Asp-38, Asp60, His-62, Asp-239, and Asp-282 of PP2C, were found to be conserved in PPM family phosphatases. These highly conserved amino acids have been reported to be essential residues for catalytic functions such as metal ion binding in the PP2C family [31]. Structures of zCaMKP-N, rat CaMKP-N, rat CaMKP, and rat PP2C are schematically illustrated in Fig. 2C. A characteristic feature of rat CaMKP and rat CaMKP-N is an acidic amino acid cluster, which is essential for stimulation by poly L-lysine [17,32], at the N-terminal region of the molecule. Although an acidic amino acid cluster is also found in zCaMKP-N, it is localized in the C-terminal region of the catalytic domain (Fig. 2C). Characterization of zCaMKP-N To determine whether zCaMKP-N possessed protein phosphatase activity, His6-tagged recombinant zCaMKPN was expressed in E. coli and puriWed using a HiTrap Chelating HP column. The puriWed zCaMKP-N showed a major protein band of approximately 80 kDa on SDS–
Fig. 3. PuriWcation of recombinant zCaMKP-N and determination of its activity. (A) Electrophoretic analysis of puriWed zCaMKP-N. His6-tagged zCaMKP-N expressed in E. coli was puriWed using a HiTrap Chelating HP column. PuriWed zCaMKP-N (0.5 g protein) was subjected to 10% SDS–PAGE and stained with Coomassie brilliant blue (left panel), and detected by Western blotting analysis with anti-His6 antibody (right panel). (B) PuriWed zCaMKP-N (8 g/ml) was assayed using 40 M pp10 as a substrate in the presence of 2 mM MgCl2 or 2 mM MnCl2, or in its absence, in the standard reaction mixture. Phosphate released in the reaction mixture was determined as described in “Materials and methods.” Data are means § SD values from three independent determinations.
T. Nimura et al. / Archives of Biochemistry and Biophysics 457 (2007) 205–216
PAGE (Fig. 3A, left panel) and Western blotting analysis using anti-His6 antibody (Fig. 3A, right panel). Maximal enzyme activity was observed when Mn2+ was added in the reaction mixture, but about two-third enzyme activity was also observed with Mg2+ (Fig. 3B). The phosphatase activity was not aVected by additions of 10 M okadaic acid or 100 nM calyculin A (data not shown), suggesting that zCaMKP-N is also a member of the PPM family phosphatases. Subcellular localization of zCaMKP-N In a previous paper, we reported that the human CaMKP-N was exclusively localized in the cell nucleus [16]. To examine subcellular localization of zCaMKP-N, myctagged zCaMKP-N was transiently expressed in Neuro2a cells and detected by indirect immunoXuorescence. As revealed by Xuorescence microscopic analysis, zCaMKPN(WT) was exclusively detected in the nucleus (Fig. 4B, upper panels), as in case of human enzyme. A predicted nuclear localization signal (NLS), RKKRRLDVLPLRR, was found in the C-terminal region (residues 575–587) of zCaMKP-N (Fig. 4A). To examine whether this region is responsible for nuclear localization, a C-terminal truncated mutant, zCaMKP-N(1–574), lacking the putative NLS was also transiently expressed in Neuro2a cells. In contrast to zCaMKP-N(WT), Xuorescence microscopic analysis
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indicated that zCaMKP-N(1–574) was localized in nuclei and cytoplasm (Fig. 4B, lower panels). Then, to investigate further that RKKRRLDVLPLRR (residues 575–587) sequence is NLS, EGFP-fused C-terminal region of zCaMKP-N mutants were expressed in Neuro2a cells (Fig. 4C). EGFP-zCaMKP-N(448–633 and 489–633) with 575–587 residues localized in nuclei, while EGFPzCaMKP-N(448–574) without the sequence localized in nuclei and cytosol (Fig. 4D). These results suggest that zCaMKP-N is clearly localized in the nucleus as in cases of rat and human enzymes, and that the NLS of this enzyme exists at the C-terminal region (575–587) of the enzyme. Dephosphorylation of CaMKIV by zCaMKP-N First we examined whether zCaMKP-N dephosphorylates phospho-CaMKIV in vitro. In this experiment, we constructed an expression vector for a point mutant of zCaMKP-N that showed no phosphatase activity. This mutant, zCaMKP-N(D188A), was prepared by changing Asp-188 that corresponds to Asp-60 of PP2C (essential for metal binding, see Fig. 2B) to Ala. The His6-tagged zCaMKP-N(D188A) mutant expressed in E. coli was detected at the same position (approximately 80 kDa) as that of zCaMKP-N(WT) by Western blotting analysis using anti-His6 antibody (Fig. 5A). Since the zCaMKPN(D188A) mutant showed essentially no phosphatase
Fig. 4. Nuclear localization of zCaMKP-N. (A) Schematic illustration of wild type (WT) zCaMKP-N and its C-terminal deletion mutant, zCaMKP-N(1– 574). The phosphatase domain is shaded in gray and the predicted NLS is shaded in black (red letters are basic amino acids). (B) Subcellular localization of zCaMKP-N(WT) and zCaMKP-N(1–574). Neuro2a cells were transfected with myc-tagged zCaMKP-N(WT) or myc-tagged zCaMKP-N(1–574) and transiently expressed myc-tagged proteins were stained by means of indirect immunoXuorescence with anti-myc antibody (left panels), and visualized by Xuorescence microscopy. Staining images with DAPI are shown in the central panels. Merged images are also shown in the right panels. (C) Schematic illustration of EGFP-fused C-terminal region of zCaMKP-N(448–633), (448–574), and (489–633). Black boxes indicate predicted NLS(575–587, see A). (D) Subcellular localization of EGFP- zCaMKP-N(448–633), (448–574), and (489–633). Neuro2a cells were transfected with above mutants and the expressed proteins were visualized by Xuorescence microscopy (left panels). Staining images with DAPI are shown in the central panels. Merged images are also shown in the right panels. (For interpretation of the references to color in this Wgure legend, the reader is referred to the web version of this paper.)
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Fig. 5. Dephosphorylation of CaMKIV by zCaMKP-N. (A) Wild type (WT) and point mutant (D188A) of zCaMKP-Ns (0.5 g protein) were expressed in E. coli, subjected to SDS–PAGE, and detected by Western blotting analysis using anti-His6 antibody. (B) Phosphatase activities of WT and D188A of CaMKP-Ns (8 g/ml) were determined by the standard malachite green assay as described in “Materials and methods.” Data are means § values from three independent experiments. (C) Phosphatase activities of zCaMKP-N(WT) and zCaMKP-N(D188A) were determined using phospho-CaMKIV(K71R) as a substrate. PhosphoCaMKIV(K71R) (20 g/ml) was incubated in a standard reaction mixture (20 l) containing varying amounts of WT or D188A mutant of zCaMKP-N (0.625, 1.25, 2.5, 5, and 10 g/ml) at 30 °C for 30 min, stopped the reaction by the addition of SDS sample buVer, and subjected to SDS– PAGE. Phospho-CaMKIV(K71R) was detected by Western blotting analysis using phospho-CaMKIV monoclonal antibody as described in “Materials and methods.”
activity (Fig. 5B), this point mutant can be used as a phosphatase-dead zCaMKP-N. When CaMKIV(K71R) phosphorylated by CaMKK was incubated with varying amounts of zCaMKP-N, CaMKIV(K71R) was clearly dephosphorylated in a dose-dependent manner (Fig. 5C). In contrast, the CaMKP-N(D188A) mutant did not dephosphorylate CaMKIV(K71R) at all, even at the highest dose (Fig. 5C). These results indicated that zCaMKP-N(WT) but not zCaMKP-N(D188A) eYciently dephosphorylates phospho-CaMKIV(K71R) in vitro. Next, we examined whether zCaMKP-N dephosphorylates phospho-CaMKIV in vivo. Neuro2a cells were transiently transfected with CaMKIV or cotransfected with CaMKIV and zCaMKP-N, and stimulated by ionomycin, a calcium ionophore. The phosphorylation level of CaMKIV was then analyzed by Western blotting analysis using a phospho-CaMKIV speciWc antibody. The time course of phosphorylation of CaMKIV after ionomycin treatment is shown in Fig. 6A. CaMKIV was phosphorylated by ionomycin treatment, while CaMKIV remained unphosphorylated in cells cotransfected with zCaMKP-N (Fig. 6A). The phosphorylation level of CaMKIV in cells transfected with only CaMKIV was markedly increased, while in cells coex-
Fig. 6. Dephosphorylation of CaMKIV by zCaMKP-N in Neuro2a cells. (A) pcrCaMKIV(WT) (2.5 g) was cotransfected with or without pczCaMKPN (2.5 g) to Neuro2a cells and the total amount of DNA was adjusted to 5 g with the empty vector. Cells were stimulated by 1 M ionomycin, and lysed with SDS sample buVer at the indicated times. The cell lysates (10 g protein) were subjected to SDS–PAGE and then analysed by Western blotting analysis using both CaMKIV and phospho-CaMKIV antibodies. (B) Neuro2a cells were transfected with pcrCaMKIV in the absence or presence of pczCaMKPN(WT) or pczCaMKPN(D188A) as in (A); cells were then stimulated with 1 M ionomycin for 10 min. After stimulation, the cells were lysed with SDS sample buVer and the cell lysates (10 g protein) subjected to SDS–PAGE and analyzed by Western blotting using CaMKIV, phospho-CaMKIV, and zCaMKP-N antibodies. An asterisk indicates a possible proteolytic product of zCaMKP-N. (C) Neuro2a cells were transiently transfected with myc-tagged zCaMKPN(D188A). These cells were stained by means of indirect immunoXuorescence with anti-myc antibody (left panel) or DAPI staining (central panel), and visualized by Xuorescence microscopy. A merged image is also shown in the right panel. These data are representative of at least three independent experiments with similar results.
pressed with zCaMKP-N(WT) it was signiWcantly suppressed (Fig. 6B, lanes 2 and 3). In contrast, the phosphorylation level of CaMKIV in cells coexpressed with zCaMKP-N(D188A) mutant was not suppressed at all (Fig. 6B, lane 4). The zCaMKP-N(D188A) mutant was found to be localized in the nucleus in transformed Neuro2a cells, as in case of zCaMKP-N(WT) (Fig. 6C). Therefore, inability of zCaMKP-N(D188A) to dephosphorylate phospho-CaMKIV was not due to mislocalization of the mutant. These results indicated that phospho-CaMKIV
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could be eYciently dephosphorylated by zCaMKP-N in the nucleus of living cells. Localization of zCaMKP-N gene transcript In order to elucidate the role of zCaMKP-N in the early development of zebraWsh, we examined the temporal and spatial patterns of zCaMKP-N gene expression using whole mount in situ hybridization. Positive signals were seen in brain and neural tube after 48 hpf, especially in telencephalon, diencephalon, epiphysis, and cerebellum but not mesencephalon (Figs. 7B–D). These results were in good agreements with tissue localization of mammalian CaMKP-N, which is highly expressed in central nervous system (CNS) [16,17]. Gene knockdown of zCaMKP-N Morpholino-mediated knockdown of genes in zebraWsh embryos has become a routine and eYcient method to provide information about gene function in vivo [28]. To investigate the biological signiWcance of CaMKP-N in zebraWsh embryogenesis, we carried out a gene knockdown experiment using an antisense morpholino oligonucleotide designed on the basis of the sequence at the 5⬘-untranslated region that included the initiation Met of zCaMKP-N mRNA. ZebraWsh embryos at 1–4 cell stage were injected with an antisense morpholino oligo (AS-MO) or a 5-bases mismatch morpholino oligo (5mis-MO) (Fig. 8A). These embryos were cultured at 26 °C and observed by stereoscopic microscopy. Injection of the AS-MO in a concentration of 1 g/l (approximately 0.8 ng injection per egg) led to an increase in the number of embryos with morphological and cellular abnormalities. On the other hand, embryos injected with the 5mis-MO at the same concentration developed normally. Acridine orange is known to stain apoptotic cells, but not necrotic cells [33]. Therefore, we can examine
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whether apoptosis occurs in the embryos with morphological abnormalities by acridine orange staining. The cells in the brain and spinal cord of the embryos injected AS-MO exhibited typical apoptotic images by acridine orange staining analysis (Fig. 8B, lower panels), but not embryos injected 5mis-MO (Fig. 8B, upper panels). In good agreement with the results of in situ hybridization, apoptotic cells were observed in telencephalon, diencephalon, epiphysis, cerebellum, and spinal cord but not mesencephalon (Fig. 8B, lower panel). At 72 hpf, a signiWcant increase of round-shaped embryos was observed (Fig. 8C). Among about 100 embryos tested, less than 2% and 7% were abnormal in embryos with no injection and 5mis-MO-injected embryos, respectively. In contrast, more than 80% and 95% of embryos were abnormal when 0.5 g/l (0.4 ng/embryo) and 1 g/l (0.8 ng/embryo) of AS-MO were injected, respectively (Fig. 8D). These results suggest that zCaMKPN is indispensable for normal development, especially in CNS, of zebraWsh embryos. Discussion We previously found a protein phosphatase having a certain homology with CaMKP in the human database and named it CaMKP-N [16]. CaMKP-N has similar biochemical properties to CaMKP, but in contrast to CaMKP it localizes exclusively in cell nuclei. To clarify the physiological signiWcance of CaMKP-N, we cloned a CaMKP-N homologue of zebraWsh and analyzed its biological functions in this study. The cDNA of zCaMKP-N possessed an open reading frame of 1899 bp encoding 633 amino acids, and exhibited identities of 49% and 48% with rat CaMKPN and human CaMKP-N, respectively. The catalytic domain of zCaMKP-N was highly homologous to mammalian CaMKP-Ns, and the PP2C motif, which is highly conserved among PPM family phosphatases, was found in this region (Fig. 2).
Fig. 7. Localization of zCaMKP-N gene transcript in zebraWsh embryos. ZebraWsh embryos (48 hpf) were analyzed by whole mount in situ hybridization with DIG-labeled RNA probes. The hybridization was detected by anti-DIG antibody conjugated to alkaline phosphatase. (A) Sense RNA probe, (B) antisense RNA probe. (C) Represents an enlarged view of (B). (D) Dorsal view of (C). Red arrows indicate forebrain, in which strong signal was detected.
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Fig. 8. Gene knockdown of zCaMKP-N during zebraWsh embryogenesis. (A) Sequences of zCaMKP-N mRNA around the 5⬘ 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) Acridine orange staining of zebraWsh embryos injected with morpholino oligos. AS-MO (lower panels) or 5mis-MO (upper panels) was injected into 1–4 cell stage embryos at a concentration of 1 g/l (about 0.8 ng/ embryo). At 48 hpf, embryos were stained with acridine orange and observed by stereoscopic microscopy as described in “Materials and methods”. (C) Phenotypes of zebraWsh embryos injected with AS-MO (right panel) or 5mis-MO (left panel) at 72 hpf. (D) Frequency of abnormal phenotypes in embryos injected with 5mis-MO (1 g/l) or AS-MO (0.5 or 1 g/l), and control embryos without injection.
When human GFP-CaMKP-N fusion protein was transfected in COS-7 cells, it clearly localizes in the nucleus [16]. Although typical NLS was not found in human CaMKPN, two NLSs, NLS1 and NLS2, were identiWed in its C-terminal region by mutagenesis analysis [34]. In the case of zCaMKP-N, typical NLS was found in the C-terminal region of zCaMKP-N(residues 575–587) (Fig. 4A). When zCaMKP-N was transiently expressed in Neuro2a cells, zCaMKP-N was exclusively localized in the nucleus (Fig. 4B). In contrast to wild type zCaMKP-N, its truncation mutant, zCaMKP-N(1–574), devoid of the C-terminal region including the putative NLS localized in nuclei and cytoplasm (Fig. 4B). In addition, EGFP fusion proteins with residues 448–633 and with residues 489–633, both of which contained the putative NLS, 575–587, translocated
to nuclei, whereas the fusion protein with residues 448–574, which was devoid of the putative NLS, showed homogenous distribution throughout the cytoplasm and nuclei (Fig. 4D). These results suggest that the region 575–587 actually functions as NLS for zCaMKP-N. To examine the enzymatic properties of zCaMKP-N, the recombinant protein was expressed in E. coli and puriWed. PuriWed enzyme exhibited not only Mn2+-dependent but also Mg 2+-dependent protein phosphatase activity (Fig. 3B), and the activity was not aVected by 10 M okadaic acid or 100 nM calyculin A (data not shown). These characteristic properties are in good agreement with those of the PPM family phosphatases including mammalian CaMKP and CaMKP-N [12,13,16]. However, in contrast to zCaMKP-N, human CaMKP-N, as well as rat CaMKP, appeared to be much less active in the presence of Mg2+ than in the presence of Mn2+ [13,16,35]. As shown in Fig. 5C, zCaMKP-N could eYciently dephosphorylate CaMKIV in vitro, as well as could human or rat CaMKP-N [16,17]. Based on the structural features, the subcellular distribution, and the enzymatic properties of zCaMKP-N as described above, we concluded that zCaMKP-N is a zebraWsh homologue of mammalian CaMKP-N. Since both CaMKP-N and CaMKIV are expressed in brain and are speciWcally localized in the nucleus [12,16,17], and since CaMKP-N eYciently dephosphorylates CaMKIV in vitro [16,17], it is highly likely that one of the potential physiological substrates for CaMKP-N is CaMKIV that is phosphorylated by CaMKK. To conWrm this, we examined whether CaMKIV could be dephosphorylated by zCaMKP-N in living cells. The cDNA of CaMKIV was transfected in Neuro2a cells with cDNA of zCaMKPN(WT) or CaMKP-N(D188A), and the phosphorylation level of the transiently expressed CaMKIV was assessed using a phospho-CaMKIV speciWc antibody (Fig. 6). As shown in Fig. 6, CaMKIV was markedly phosphorylated by endogenous CaMKK in response to the increase in the intracellular Ca2+. However, coexpression of wild type zCaMKP-N, but not the inactive mutant zCaMKPN(D188A), signiWcantly attenuated the ionomycin-stimulated phosphorylation of CaMKIV. These results provide the Wrst demonstration that CaMKP-N dephosphorylates CaMKIV in living cells. Thus, zCaMKP-N is suggested to be a potential downregulator for CaMKIV in vivo. To further explore the biological functions of CaMKPN, gene expression of zCaMKP-N in zebraWsh embryos was investigated by whole mount in situ hybridization analysis. In good agreement with the tissue localization of mammalian CaMKP-N [16,17], zCaMKP-N mRNA is predominantly expressed in brain, especially in telencephalon, diencephalon, epiphysis, and cerebellum but not mesencephalon as shown in Fig. 7. During development, zCaMKP-N mRNA was detected in embryos at 24 hpf and gradually increased after 29 hpf (data not shown), suggesting that zCaMKP-N may be involved in the control of neuronal development in the CNS of zebraWsh embryos. We
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further provided evidence about the function of zCaMKPN by morpholino-knockdown experiment in zebraWsh embryos. The injection of the antisense morpholino oligonucleotide, which was designed to functionally knockdown zCaMKP-N, into zebraWsh embryos led to a marked increase of embryos with morphological abnormalities (Fig. 8D). Within the injection of AS-MO, abnormality was also detected at the cellular level when embryos were stained with acridine orange, which was used to stain apoptotic cells, but not necrotic cells, in Drosophila [33]. Dead cells, possibly apoptotic cells, were detected in telencephalon, diencephalon, epiphysis, cerebellum, and spinal cord but not mesencephalon (Fig. 8B, lower panels). On the other hand, few dead cells were detected in 5mis-MOinjected embryos (Fig. 8B, upper panels). Whole mount in situ hybridization using the zCaMKP-N gene suggests that expression of zCaMKP-N mRNA corresponded roughly to the region where dead cells were detected (Fig. 7). These results indicated that zCaMKP-N is related to apoptotic events, especially in the CNS of zebraWsh embryos. It is reported that overexpression of CaMKP results in apoptotic cell death [36], but the mechanism of this phenomenon remains to be elucidated. Although it is also currently unclear why the antisense knockdown of zCaMKP-N caused cellular apoptosis leading to morphological abnormalities of the brain and spinal cord, there have been several reports that CaMKIV is important for regulating apoptotic events [37–40]. Since at least one of the potential targets of CaMKP-N is CaMKIV, as discussed above, it is likely that CaMKP-N also plays a pivotal role in the regulation of cell death and survival through regulating CaMKIV activity. Two opposite eVects of CaMKIV on apoptosis are reported; some reports reveal that activation of CaMKIV prevents cells from apoptosis [38,41], while others show that activation of CaMKIV leads to the promotion of apoptotic cell death [39,40]. Understanding the molecular mechanisms whereby knockdown of zCaMKPN results in apoptosis may shed light on a novel pathway for regulation of apoptotic events. The results presented in this study clearly indicate that CaMKP-N is involved in the regulation of the phosphorylation level of CaMKIV in cells, and that it has important functions in zebraWsh embryogenesis. Further studies will be necessary to further explore the role of CaMKP-N in embryogenesis, including the molecular mechanisms regulating cell death and survival. Acknowledgments We sincerely thank Dr. Naotaka Tanaka and Dr. Kaoru Takegawa for helpful advises on microscopic observation. This work was supported by Grant-in-Aid for ScientiWc Research from the Ministry of Education, Science, Sports and Culture of Japan, and by grants from the Kato Memorial Bioscience Foundation, the Smoking Research Foundation, the Akiyama Foundation, and AIST Research Grant.
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Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebraWsh 夽 Takaki Nimura a, Noriyuki Sueyoshi a,¤, Atsuhiko Ishida b, Yukihiro Yoshimura c, Makoto Ito c, Hiroshi Tokumitsu d, Yasushi Shigeri e, Naohito Nozaki f, Isamu Kameshita a a
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan b Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan c Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan d Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan e National Institute of Advanced Industrial Science and Technology, Osaka 563-8577, Japan f Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka 238-8580, Japan Received 28 August 2006, and in revised form 27 September 2006 Available online 6 November 2006
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 signiWcance of CaMKP-N during zebraWsh embryogenesis, we cloned and characterized zebraWsh CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebraWsh whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed fulllength 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 signiWcantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebraWsh 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 signiWcant morphological abnormalities in zebraWsh 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 zebraWsh. © 2006 Elsevier Inc. All rights reserved. Keywords: ZebraWsh; CaM kinase; Apoptosis; Phosphatase; CaMKP; CaMKP-N
The increase of Ca 2+ concentration in response to extracellular stimuli activates various Ca 2+/calmodulin (CaM)-dependent enzymes involved in cellular signaling, such as Ca2+/CaM-dependent protein kinases (CaMKs). Multifunctional CaMKs, CaMKI, II, and IV, phosphorylate a broad range of substrates, and participate in vari夽 The nucleotide sequence reported in this paper has been submitted to the GenBank™/EBI Data Bank with an Accession No. AB113302. * Corresponding author. Fax: +81 87 891 3114. E-mail address: [email protected] (N. Sueyoshi).
0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.09.034
ous cellular processes [1–4]. CaMKIV occurs predominantly in the nuclei of brain and thymus, and phosphorylates a number of proteins including synapsin I, microtubule associated protein 2, tau protein, myosin light chain, tyrosine hydroxylase, cAMP-response element binding protein (CREB), and so on [5–7]. It is reported that transcriptional regulation by CaMKIV through phosphorylation of CREB plays important roles in the regulation of memory and neuronal plasticity [8,9]. CaMKIV is strongly activated through phosphorylation of a threonine residue located within the region called the
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“activation loop” by another CaMK1, CaMK kinase (CaMKK) [10,11]. CaMKIV, once activated, should then be dephosphorylated by protein phosphatases in order to downregulate its activity prior to the next stimulus, and it is reported that various Ser/Thr protein phosphatases dephosphorylate CaMKIV in vitro [12]. We previously puriWed and isolated a Mn2+-dependent, okadaic acid/calyculin A-insensitive protein phosphatase with a molecular weight of 54,000, and designated it CaMK phosphatase (CaMKP) [13]. CaMKP is a member of PPM family phosphatases, and speciWcally dephosphorylates phosphorylated CaMKs including CaMKIV in vitro [13– 15]. Another protein phosphatase having some homology with CaMKP was found in the human database and was named as CaMKP-N, since it had similar biochemical properties to CaMKP and showed nuclear localization [16]. CaMKP-N is also a Mn2+-dependent protein phosphatase and dephosphorylates CaMKIV in vitro [16,17]. However, the physiological signiWcance of CaMKP-N has yet to be determined. In the present study, we employed zebraWsh as a model animal to analyze the biological functions of CaMKP-N in vivo. We cloned zebraWsh CaMKP-N (zCaMKP-N) and demonstrated that the nuclear localization signal (NLS) is localized at the C-terminal region of zCaMKP-N. We also provide the Wrst evidence that CaMKP-N dephosphorylates phospho-CaMKIV in living cells. Furthermore, a gene knockdown experiment of zCaMKP-N revealed that CaMKP-N is indispensable for some of the processes in zebraWsh embryogenesis. Materials and methods Materials Ionomycin calcium salt was obtained from Calbiochem. Cy3-labeled anti-mouse IgG, ATP, and bovine serum albumin were purchased from Sigma. Anti-His6 antibody and anti-CaMKIV antibody were from Roche and BD Biosciences, respectively. Horseradish peroxidase (HRP)-labeled anti-mouse IgG and HRP-labeled anti-rabbit IgG were from ICN Pharmaceuticals. Calmodulin (CaM) was puriWed from rat testis as described previously [18].
cDNA cloning of zebraWsh CaMKP-N SMART RACE cDNA AmpliWcation kit (Clontech) was used to clone a full-length coding sequence for zCaMKP-N using primers based on a GenBank zebraWsh whole genome shotgun (WGS) clones (Accession Nos.: 132560840 and 45566463). 5⬘-RACE Wrst strand cDNA was primed from mRNA of adult zebraWsh with SuperscriptII reverse transcriptase using a SMART II oligonucleotide and a 5⬘-RACE cDNA synthesis primer. The 5⬘-end of the cDNA was ampliWed by PCR with gene-speciWc primer 1 (AS1: 5⬘-GTCTCTGAGGAACACTACGATTACCGT-3⬘) and a univer-
1 Abbreviations used: CaM, calmodulin; CaMK, Ca2+/calmodulin-dependent protein kinase; CaMKK, Ca2+/calmodulin-dependent protein kinase kinase; CaMKP, Ca2+/calmodulin-dependent protein kinase phosphatase; CaMKP-N, nuclear Ca2+/calmodulin-dependent protein kinase phosphatase; NLS, nuclear localization signal; PBS, phosphate-buVered saline; PP2C, protein phosphatase 2C.
sal primer mix and 5⬘-RACE Wrst strand cDNA as a template, using an Advantage 2 PCR kit (Clontech). The 3⬘-RACE Wrst strand cDNA was primed using 3⬘-RACE cDNA synthesis primer. The 3⬘-end of the cDNA was obtained by PCR using a universal primer mix and gene-speciWc primer 2 (S1: 5⬘-CTGGCGCGACTTGTCTTCAATAAG-3⬘), and 3⬘RACE Wrst strand cDNA as a template. The 5⬘- and 3⬘-RACE PCR products were cloned into pGEM-T Easy vector (Promega), and their DNA sequences determined. The nucleotide sequences of the products of 5⬘RACE and 3⬘-RACE were overlapped and aligned using the DNASIS computer program developed by Hitachi Software Engineering. An open reading frame of 1899 nucleotides was generated. A sense primer (5⬘AGACTTCTTGCCCGGCTCCA-3⬘) and an antisense primer (5⬘ACGTCCACCGCAGGAGAGTG-3⬘) were designed from the outside sequences of the open reading frame. A full-length cDNA was prepared by PCR using these primers and 3⬘-RACE ready cDNA library as a template with Pyrobest DNA polymerase (TaKaRa). The PCR product was cloned into a pGEM-T Easy vector, and Wve independent clones were sequenced (pGEMzCaMKPN-1, -2, -3, -4, -5).
Construction of plasmids In the case of zCaMKP-N(WT), the following primers were used for PCR with pGEMzCaMKPN-5 as a template: 5⬘-upstream primer (5⬘GCTAGCATGGCCGGCTCTGCCAACGA-3⬘) and 3⬘-downstream primer (5⬘-CTCGAGGACCCTTTTGCTATGGGTTACATGAGG-3⬘). The NheI (underlined)–XhoI (double underlined) fragment was inserted into the NheI–XhoI sites of pET-23a(+) (Novagen) to generate plasmid pETzCaMKPN(WT). Mutagenesis on Asp-188 was performed by the inverse PCR method [19], with the following primers: D188A-upstream primer, 5⬘-GCCGGTCACGGCGGGGTGGACGCT-3⬘ and D188Adownstream primer, 5⬘-AAACACGGCAAAGTAGGCCTGCTCTTC-3⬘ (underline shows the site of mutation), and pETzCaMKPN(WT) as a template. The 5⬘-ends of the PCR fragment were then phosphorylated by T4 polynucleotide kinase (Nippon Gene) and self-ligated by T4 DNA ligase (Nippon Gene), and the recombinant plasmid obtained was designated as pETzCaMKPN(D188A). To express zCaMKPN and its mutants in mammalian cells, the following plasmids were prepared. pczCaMKPN; the DNA fragment coding C-terminal myc/His6-tagged full-length zCaMKPN was ampliWed with PCR using primers (5⬘-AAGCTTGCGATGGCC GGCTCTGCCAA-3⬘ and 5⬘-CTCGAGACCCTTTTGCTATGGGTTA CATGAGG-3⬘), and pETzCaMKPN(WT) as a template, the HindIII (underlined)–XhoI (double underlined) fragment was inserted into HindIII–XhoI sites of pcDNA3.1(+)/myc-His B (Invitrogen). pczCaMKPN(D188A) was prepared by the same procedure except that pETzCaMKPN(D188A) was used as a template. pczCaMKPN(1–574); the DNA fragment coding residues 1–574 of zCaMKP-N, lacking the putative nuclear localization signal, was ampliWed with PCR using primers (5⬘-AAGCTTGCGATGGCCGGCTCTGCCAA-3⬘ and 5⬘-CTCGAG CCCTCCTGAGAGAAGAGCGCAGC-3⬘) and inserted into HindIII (underlined)–XhoI (double underlined) sites of pcDNA3.1(+)/myc-His B. zCaMKP-N(448–633) was ampliWed with PCR using EGFP-zPN-U1342/ ERI primer (5⬘-GAATTCTGATGGAGGTGCGGAGAATGG-3⬘) and LHis6/SalI primer (5⬘-TTTGTCGACTCAGTGGTGGTGGTGGTGG TG-3⬘), and pETzCaMKPN(WT) as a template. zCaMKP-N(448–574) was ampliWed with PCR using EGFP-zPN-U1342/ERI primer and LHis6/ SalI primer and pETzCaMKPN(1–574) as a template. zCaMKP-N(489– 633) was ampliWed with PCR using EGFP-zPN-U1465/ERI primer (5⬘and LHis6/SalI GAATTCTATCACAGGGCCGGATGTGCA-3⬘) primer and pETzCaMKPN(WT) as a template. EcoRI (underlined)–SalI (double underlined) fragment was inserted into the EcoRI–SalI sites of pEGFP-C1 (Clontech) to generate plasmid pEGFP-zCaMKPN(448–633), (448–574), and (489–633), respectively. In the case of rat CaMKIV(WT), the following primers were used for PCR: CaMKIV(WT)-5⬘ upstream primer (5⬘-GATATCGTTATGCTCA AAGTCACGGTGCC-3⬘) and CaMKIV(WT)-3⬘ downstream primer (5⬘The EcoRV CTCGAGTACTCTGGCAGAATAGCATCCTG-3⬘). (underlined)–XhoI (double underlined) fragment was inserted into the EcoRV–XhoI sites of pcDNA3.1(+)/myc-His B to generate plasmid
T. Nimura et al. / Archives of Biochemistry and Biophysics 457 (2007) 205–216 pcrCaMKIV(WT). Mutagenesis on Lys-71 was performed by the inverse PCR method [19], with the following primers: CaMKIV(K71R)-5⬘ upstream primer, 5⬘-CGCGTGTTAAAGAAAACAGTGGACAAGA-3⬘ and CaMKIV(K71R)-3⬘ downstream primer, 5⬘-GAGAGCATAGGGC TTCTGGG-3⬘ (underline shows the site of mutation) and pETrCaMKIV(WT) as a template. The 5⬘-ends of the PCR fragment were phosphorylated by T4 polynucleotide kinase and self-ligated by T4 DNA ligase, and the recombinant plasmid thus obtained was designated as pETrCaMKIV(K71R).
Expression of proteins in Escherichia coli and puriWcation In the case of zCaMKP-N, pETzCaMKPN(WT or D188A) was introduced into E. coli strain Rosetta(DE3) (Novagen). The transformed bacteria were grown at 37 °C to an A600 of 0.5, and then isopropyl--Dthiogalactopyranoside was added to a Wnal concentration of 0.25 mM. After 6 h at 25 °C, the bacteria were harvested by centrifugation and suspended in buVer A (20 mM Tris–HCl (pH 7.5) containing 150 mM NaCl, and 0.05% Tween 40). After sonication, cell debris was removed by centrifugation (20,000g) at 4 °C for 10 min, and supernatant obtained was loaded on a HiTrap Chelating HP column (Amersham Biosciences) preequilibrated with buVer A. The column was washed with buVer A, buVer A containing 20 mM imidazole, buVer A containing 50 mM imidazole, and then eluted with buVer A containing 200 mM imidazole. The active fraction was dialyzed against buVer B (20 mM Tris–HCl (pH 7.5) containing 0.05% Tween 40 and 14.2 mM 2-mercaptoethanol) and used for phosphatase assay. In the case of CaMKIV(K71R), the pETrCaMKIV(K71R) construct was introduced into E. coli strain BL21(DE3) (Novagen). The transformed bacteria were grown at 37 °C for 12 h, harvested by centrifugation, and suspended in buVer A. CaMKIV(K71R) expressed in E. coli was extracted and puriWed with a HiTrap Chelating HP column as described above. The active fraction was dialyzed against buVer A and stored in aliquots at ¡80 °C until use.
Production of antibodies An antibody against zCaMKP-N was produced by immunizing rabbits with the antigenic peptide, CYEDRMDSFTDRTSLS, corresponding to the amino acid residues 474–488 of zCaMKP-N. Immunization was carried out essentially according to a previously described procedure [20]. Monoclonal antibody to detect phospho-CaMKIV at Thr-196 was prepared as described [21].
SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting SDS–PAGE was performed essentially according to the method of Laemmli [22] on slab gels consisting of a 10% acrylamide separation gel and a 3% stacking gel. The resolved proteins were electrophoretically transferred to nitrocellulose membranes (Protran BA85, Schleicher and Schuell) and immuno-reactive protein bands were detected essentially according to the method described previously [23].
Protein phosphatase assay Protein phosphatase assay was carried out using a phosphopeptide pp10 (YGGMHRQETpVDC), which contains the amino acid sequence around the autophosphorylation site of CaMKII, as a substrate [24]. The reaction mixture (50 l) contained 50 mM Tris–HCl (pH 8.0), 2 mM MgCl2 (or MnCl2), 0.1 mM EGTA, 0.01% Tween 20, 40 M pp10, and an appropriate amount of zCaMKP-N. The reaction was started by adding zCaMKP-N and incubated at 30 °C for 10 min. Phosphate released in the mixture was determined by malachite green assay [25]. Protein phosphatase activity of CaMKP-N was also determined using phosphorylated CaMKIV mutant. Recombinant CaMKIV(K71R) (40 g/ ml) was phosphorylated by CaMKK (5 g/ml) at 30 °C for 30 min in a
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reaction mixture comprising 40 mM Hepes–NaOH (pH 8.0), 5 mM Mg(CH3COO)2, 2 mM dithiothreitol, 0.1 mM EGTA, 0.1 mM ATP, 1 mM CaCl2, and 1 M CaM, and the reaction stopped by adding 2 mM EGTA. Dephosphorylation of a phosphoprotein substrate was carried out at 30 °C for 30 min in a reaction mixture containing 20 mM Tris–HCl (pH 7.5), 2 mM MnCl2, 0.05% Tween 40, 20 g/ml phospho-CaMKIV(K71R), and an appropriate amount of zCaMKP-N. The reaction was started by the addition of zCaMKP-N and terminated by the addition of an equal amount of SDS sample buVer. The sample was then subjected to SDS– PAGE and analyzed by Western blotting analysis using phospho-CaMKIV antibody.
Cell culture and transfection Mouse neuroblastoma, Neuro2a, was cultured in Dulbecco’s modiWed Eagle’s medium (DMEM, Sigma) containing 10% heat inactivated fetal calf serum. Cells were grown at 37 °C in a humidiWed incubator with a 5% CO2/95% air atmosphere. Transfection of Neuro2a cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Neuro2a cells were plated at 4 £ 104 in a 35-mm dish in 2 ml of DMEM containing 10% fetal calf serum. After 48 h of culture, cells were incubated for 24 h in 1 ml of DMEM containing 5% fetal calf serum, 6 l of Lipofectamine 2000, and 5 g 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 M ionomycin in DMEM at 37 °C. After stimulation, the medium was removed and 150 l of SDS sample buVer added to stop the reaction. Samples were boiled for 5 min, electrophoresed on SDS–polyacrylamide gel, and analyzed by Western blotting.
Immunocytochemistry of zCaMKP-N Transfected cells were cultured on cover glass and treated with 3.7% formaldehyde in phosphate-buVered saline (PBS) for 20 min. After being rinsed with PBS, cells were permeabilized with 0.1% Triton X-100 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% bovine serum albumin in PBS at 4 °C for 16 h followed by incubation with Cy3-labeled anti-mouse IgG at room temperature for 2 h. The nuclear chromatin region was stained with DAPI [4⬘,6-diamidino-2-phenylindole] (DOJINDO, Japan) at 2 g/ml. Stained cells were observed under a Xuorescence microscope (model BX51, Olympus, Japan) and images were obtained using a Cool SNAP CCD camera (Roper ScientiWc).
Fish maintenance ZebraWsh, Danio rerio, were maintained at 26 °C and embryos collected from natural crosses of wild-type Wsh. Collected embryos were maintained in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4) at 26 °C. Embryos were staged according to hours post-fertilization (hpf) at 26 °C and morphological criteria [26].
Whole mount in situ hybridization analysis Whole mount in situ hybridization analysis was carried out as described in [27]. Embryos were incubated in E3 medium containing 0.1 mM phenylthiourea to block melanin production. The sense (as negative control) and the antisense RNA probes labeled with digoxigenin(DIG)-UTP were synthesized using the DIG RNA Labeling mix (Roche) from zCaMKP-N cDNA (346–1215) subcloned into pGEM-T Easy. The hybridization was detected by anti-DIG antibody conjugated to alkaline phosphatase and NBT/BCIP as a substrate.
Morpholino oligo injections Morpholino-based oligonucleotides (GENE TOOLS) were solubilized in sterilized water at a concentration of 5 g/l, and diluted to
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working concentrations in sterilized water before injection. ZebraWsh embryos were injected at one- to four-cell stage with morpholino oligonucleotides using a micromanipulator and microinjector (Narishige) according to the method of Nasevicius and Ekker [28]. Injected embryos were cultured in E3 medium at 26 °C. The sequences of mor-
pholino oligonucleotides used in the present study were; zCaMKP-N antisense morpholino oligo (AS-MO), 5⬘-GCCATCGCTTCTC ATTCCTGGAGAG-3⬘ and zCaMKP-N control morpholino oligo (5 mis-MO), and 5⬘-GCGATGGCTTCTGATTCCTCGACAG -3⬘ (underlines show the sites of mismatch).
Fig. 1. Nucleotide and deduced amino acid sequences of zebraWsh CaMKP-N. The amino acid residues are numbered beginning with the Wrst Met and are shown with a one-letter code below the nucleotide sequence. The termination codon is denoted by an asterisk. The solid underline and double underline indicate an acidic amino acid cluster and a PP2C motif (PROSITE entry No. PS01032), respectively. A predicted NLS is boxed.
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Acridine orange staining Dechorionated embryos were stained with the vital dye, acridine orange (Sigma) to examine the occurrence of apoptosis. After treatment
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with 2 g/ml of the dye in E3 medium for 30 min, embryos were washed with E3 medium. The visualization and photography was performed using Xuorescence microscope, SMZ1500 (Nikon, Japan), and COOLPIX4500 (Nikon, Japan).
Fig. 2. Alignment of deduced amino acid sequence of zCaMKP-N with other mammalian phosphatases. (A) Amino acid sequence of zCaMKP-N was aligned with rat CaMKP-N and human CaMKP-N using CLUSTAL W. Identical amino acids are shaded, and gaps inserted into the sequences are indicated by dots. The acidic amino acid clusters are underlined. A predicted nuclear localization signal is double underlined. (B) Alignment of catalytic domains of zCaMKP-N (Accession No.: AB113302), rat CaMKP (Accession No.: AB023634), and rat PP2C (Accession No.: J04503). Catalytic domains of zCaMKP-N(residues 156–405), rCaMKP(residues 162–411), and rPP2C(residues 33–293) are aligned using CLUSTAL W. The seven amino acid residues shown by the arrows indicate the critical residues for the binding of metal ions. (C) Schematic illustration of the primary structures of rat PP2C, rat CaMKP, rat CaMKP-N, and zebraWsh CaMKP-N. Catalytic domains and acidic amino acid clusters are shown by gray and black boxes, respectively. The dark gray with asterisk indicates the position of the PP2C motif. Amino acid identity between indicated catalytic domains are also shown.
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Other methods Protein concentrations were determined by the method of Bensadoun and Weinstein using bovine serum albumin as a standard [29]. Nucleotide sequences were determined on both strands by the dideoxynucleotide chain termination method with a BigDye Terminator Cycle Sequencing Ready Reaction Kit Ver. 3.1 (Applied Biosystems) and a DNA Sequencer (model 3100, Applied Biosystems).
Results cDNA cloning of zebraWsh CaMKP-N In earlier papers, cDNA clones for human CaMKP-N [16] and rat CaMKP-N [17] were isolated and recombinant enzymes characterized in vitro. However, the physiological functions of these mammalian CaMKP-Ns are not well understood. To investigate the physiological signiWcance of CaMKP-N in vivo, we used zebraWsh as a model animal in this study. To isolate a cDNA clone for zCaMKP-N, BLAST search against the zebraWsh whole genome shotgun sequence database was carried out using the tblastn program. By using the amino acid sequence of human CaMKP-N as a probe, two DNA sequences (Accession Nos.: 132560840 and 45566463) were found to contain the central part and the C-terminal parts, respectively, of zCaMKP-N. To obtain the 5⬘- and 3⬘-terminal segments of the cDNA, 5⬘- and 3⬘-RACE were performed using two gene speciWc primers derived from the sequences of the whole genome shotgun clones. The nucleotide sequences of the products of 5⬘-RACE and 3⬘-RACE were overlapped, and a set of primers was designed from the outside sequences of the open reading frame. A full-length cDNA was ampliWed by PCR using these primers and a 3⬘-RACE ready cDNA library as a template, and then subcloned into pGEM-T Easy vector. The nucleotide sequence of the cDNA clone for zCaMKP-N was determined by sequencing Wve independent clones. The nucleotide sequence and the deduced amino acid sequence of zCaMKP-N are shown in Fig. 1. The open reading frame of 1899 nucleotides encoded a polypeptide of 633 amino acids containing a protein phosphatase 2C (PP2C) motif (PROSITE entry No. PS01032) (Fig. 1, double underlined). From the deduced amino acid sequences, the molecular mass, and pI of zCaMKP-N were calculated to be 71,036 and 4.97, respectively. The similarity in the sequence of zCaMKP-N to those of other mammalian homologues was analyzed using CLUSTAL W software [30]. An alignment of the deduced amino acid sequence of zCaMKP-N with those of rat and human enzymes is shown in Fig. 2A. The number of amino acid residues of zCaMKP-N(633 amino acids) is much smaller than those of rat CaMKP-N (750 amino acids) or human CaMKP-N(757 amino acids); zCaMKP-N exhibited 49% and 48% identities with those of rat and human enzymes, respectively. An alignment of the catalytic domain of zCaMKP-N with those of rat CaMKP (Accession No.: AB023634) and rat PP2C (Accession No.: J04503) is
shown in Fig. 2B. Seven amino acid residues in zCaMKPN, corresponding to residues Arg-33, Glu-37, Asp-38, Asp60, His-62, Asp-239, and Asp-282 of PP2C, were found to be conserved in PPM family phosphatases. These highly conserved amino acids have been reported to be essential residues for catalytic functions such as metal ion binding in the PP2C family [31]. Structures of zCaMKP-N, rat CaMKP-N, rat CaMKP, and rat PP2C are schematically illustrated in Fig. 2C. A characteristic feature of rat CaMKP and rat CaMKP-N is an acidic amino acid cluster, which is essential for stimulation by poly L-lysine [17,32], at the N-terminal region of the molecule. Although an acidic amino acid cluster is also found in zCaMKP-N, it is localized in the C-terminal region of the catalytic domain (Fig. 2C). Characterization of zCaMKP-N To determine whether zCaMKP-N possessed protein phosphatase activity, His6-tagged recombinant zCaMKPN was expressed in E. coli and puriWed using a HiTrap Chelating HP column. The puriWed zCaMKP-N showed a major protein band of approximately 80 kDa on SDS–
Fig. 3. PuriWcation of recombinant zCaMKP-N and determination of its activity. (A) Electrophoretic analysis of puriWed zCaMKP-N. His6-tagged zCaMKP-N expressed in E. coli was puriWed using a HiTrap Chelating HP column. PuriWed zCaMKP-N (0.5 g protein) was subjected to 10% SDS–PAGE and stained with Coomassie brilliant blue (left panel), and detected by Western blotting analysis with anti-His6 antibody (right panel). (B) PuriWed zCaMKP-N (8 g/ml) was assayed using 40 M pp10 as a substrate in the presence of 2 mM MgCl2 or 2 mM MnCl2, or in its absence, in the standard reaction mixture. Phosphate released in the reaction mixture was determined as described in “Materials and methods.” Data are means § SD values from three independent determinations.
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PAGE (Fig. 3A, left panel) and Western blotting analysis using anti-His6 antibody (Fig. 3A, right panel). Maximal enzyme activity was observed when Mn2+ was added in the reaction mixture, but about two-third enzyme activity was also observed with Mg2+ (Fig. 3B). The phosphatase activity was not aVected by additions of 10 M okadaic acid or 100 nM calyculin A (data not shown), suggesting that zCaMKP-N is also a member of the PPM family phosphatases. Subcellular localization of zCaMKP-N In a previous paper, we reported that the human CaMKP-N was exclusively localized in the cell nucleus [16]. To examine subcellular localization of zCaMKP-N, myctagged zCaMKP-N was transiently expressed in Neuro2a cells and detected by indirect immunoXuorescence. As revealed by Xuorescence microscopic analysis, zCaMKPN(WT) was exclusively detected in the nucleus (Fig. 4B, upper panels), as in case of human enzyme. A predicted nuclear localization signal (NLS), RKKRRLDVLPLRR, was found in the C-terminal region (residues 575–587) of zCaMKP-N (Fig. 4A). To examine whether this region is responsible for nuclear localization, a C-terminal truncated mutant, zCaMKP-N(1–574), lacking the putative NLS was also transiently expressed in Neuro2a cells. In contrast to zCaMKP-N(WT), Xuorescence microscopic analysis
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indicated that zCaMKP-N(1–574) was localized in nuclei and cytoplasm (Fig. 4B, lower panels). Then, to investigate further that RKKRRLDVLPLRR (residues 575–587) sequence is NLS, EGFP-fused C-terminal region of zCaMKP-N mutants were expressed in Neuro2a cells (Fig. 4C). EGFP-zCaMKP-N(448–633 and 489–633) with 575–587 residues localized in nuclei, while EGFPzCaMKP-N(448–574) without the sequence localized in nuclei and cytosol (Fig. 4D). These results suggest that zCaMKP-N is clearly localized in the nucleus as in cases of rat and human enzymes, and that the NLS of this enzyme exists at the C-terminal region (575–587) of the enzyme. Dephosphorylation of CaMKIV by zCaMKP-N First we examined whether zCaMKP-N dephosphorylates phospho-CaMKIV in vitro. In this experiment, we constructed an expression vector for a point mutant of zCaMKP-N that showed no phosphatase activity. This mutant, zCaMKP-N(D188A), was prepared by changing Asp-188 that corresponds to Asp-60 of PP2C (essential for metal binding, see Fig. 2B) to Ala. The His6-tagged zCaMKP-N(D188A) mutant expressed in E. coli was detected at the same position (approximately 80 kDa) as that of zCaMKP-N(WT) by Western blotting analysis using anti-His6 antibody (Fig. 5A). Since the zCaMKPN(D188A) mutant showed essentially no phosphatase
Fig. 4. Nuclear localization of zCaMKP-N. (A) Schematic illustration of wild type (WT) zCaMKP-N and its C-terminal deletion mutant, zCaMKP-N(1– 574). The phosphatase domain is shaded in gray and the predicted NLS is shaded in black (red letters are basic amino acids). (B) Subcellular localization of zCaMKP-N(WT) and zCaMKP-N(1–574). Neuro2a cells were transfected with myc-tagged zCaMKP-N(WT) or myc-tagged zCaMKP-N(1–574) and transiently expressed myc-tagged proteins were stained by means of indirect immunoXuorescence with anti-myc antibody (left panels), and visualized by Xuorescence microscopy. Staining images with DAPI are shown in the central panels. Merged images are also shown in the right panels. (C) Schematic illustration of EGFP-fused C-terminal region of zCaMKP-N(448–633), (448–574), and (489–633). Black boxes indicate predicted NLS(575–587, see A). (D) Subcellular localization of EGFP- zCaMKP-N(448–633), (448–574), and (489–633). Neuro2a cells were transfected with above mutants and the expressed proteins were visualized by Xuorescence microscopy (left panels). Staining images with DAPI are shown in the central panels. Merged images are also shown in the right panels. (For interpretation of the references to color in this Wgure legend, the reader is referred to the web version of this paper.)
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Fig. 5. Dephosphorylation of CaMKIV by zCaMKP-N. (A) Wild type (WT) and point mutant (D188A) of zCaMKP-Ns (0.5 g protein) were expressed in E. coli, subjected to SDS–PAGE, and detected by Western blotting analysis using anti-His6 antibody. (B) Phosphatase activities of WT and D188A of CaMKP-Ns (8 g/ml) were determined by the standard malachite green assay as described in “Materials and methods.” Data are means § values from three independent experiments. (C) Phosphatase activities of zCaMKP-N(WT) and zCaMKP-N(D188A) were determined using phospho-CaMKIV(K71R) as a substrate. PhosphoCaMKIV(K71R) (20 g/ml) was incubated in a standard reaction mixture (20 l) containing varying amounts of WT or D188A mutant of zCaMKP-N (0.625, 1.25, 2.5, 5, and 10 g/ml) at 30 °C for 30 min, stopped the reaction by the addition of SDS sample buVer, and subjected to SDS– PAGE. Phospho-CaMKIV(K71R) was detected by Western blotting analysis using phospho-CaMKIV monoclonal antibody as described in “Materials and methods.”
activity (Fig. 5B), this point mutant can be used as a phosphatase-dead zCaMKP-N. When CaMKIV(K71R) phosphorylated by CaMKK was incubated with varying amounts of zCaMKP-N, CaMKIV(K71R) was clearly dephosphorylated in a dose-dependent manner (Fig. 5C). In contrast, the CaMKP-N(D188A) mutant did not dephosphorylate CaMKIV(K71R) at all, even at the highest dose (Fig. 5C). These results indicated that zCaMKP-N(WT) but not zCaMKP-N(D188A) eYciently dephosphorylates phospho-CaMKIV(K71R) in vitro. Next, we examined whether zCaMKP-N dephosphorylates phospho-CaMKIV in vivo. Neuro2a cells were transiently transfected with CaMKIV or cotransfected with CaMKIV and zCaMKP-N, and stimulated by ionomycin, a calcium ionophore. The phosphorylation level of CaMKIV was then analyzed by Western blotting analysis using a phospho-CaMKIV speciWc antibody. The time course of phosphorylation of CaMKIV after ionomycin treatment is shown in Fig. 6A. CaMKIV was phosphorylated by ionomycin treatment, while CaMKIV remained unphosphorylated in cells cotransfected with zCaMKP-N (Fig. 6A). The phosphorylation level of CaMKIV in cells transfected with only CaMKIV was markedly increased, while in cells coex-
Fig. 6. Dephosphorylation of CaMKIV by zCaMKP-N in Neuro2a cells. (A) pcrCaMKIV(WT) (2.5 g) was cotransfected with or without pczCaMKPN (2.5 g) to Neuro2a cells and the total amount of DNA was adjusted to 5 g with the empty vector. Cells were stimulated by 1 M ionomycin, and lysed with SDS sample buVer at the indicated times. The cell lysates (10 g protein) were subjected to SDS–PAGE and then analysed by Western blotting analysis using both CaMKIV and phospho-CaMKIV antibodies. (B) Neuro2a cells were transfected with pcrCaMKIV in the absence or presence of pczCaMKPN(WT) or pczCaMKPN(D188A) as in (A); cells were then stimulated with 1 M ionomycin for 10 min. After stimulation, the cells were lysed with SDS sample buVer and the cell lysates (10 g protein) subjected to SDS–PAGE and analyzed by Western blotting using CaMKIV, phospho-CaMKIV, and zCaMKP-N antibodies. An asterisk indicates a possible proteolytic product of zCaMKP-N. (C) Neuro2a cells were transiently transfected with myc-tagged zCaMKPN(D188A). These cells were stained by means of indirect immunoXuorescence with anti-myc antibody (left panel) or DAPI staining (central panel), and visualized by Xuorescence microscopy. A merged image is also shown in the right panel. These data are representative of at least three independent experiments with similar results.
pressed with zCaMKP-N(WT) it was signiWcantly suppressed (Fig. 6B, lanes 2 and 3). In contrast, the phosphorylation level of CaMKIV in cells coexpressed with zCaMKP-N(D188A) mutant was not suppressed at all (Fig. 6B, lane 4). The zCaMKP-N(D188A) mutant was found to be localized in the nucleus in transformed Neuro2a cells, as in case of zCaMKP-N(WT) (Fig. 6C). Therefore, inability of zCaMKP-N(D188A) to dephosphorylate phospho-CaMKIV was not due to mislocalization of the mutant. These results indicated that phospho-CaMKIV
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could be eYciently dephosphorylated by zCaMKP-N in the nucleus of living cells. Localization of zCaMKP-N gene transcript In order to elucidate the role of zCaMKP-N in the early development of zebraWsh, we examined the temporal and spatial patterns of zCaMKP-N gene expression using whole mount in situ hybridization. Positive signals were seen in brain and neural tube after 48 hpf, especially in telencephalon, diencephalon, epiphysis, and cerebellum but not mesencephalon (Figs. 7B–D). These results were in good agreements with tissue localization of mammalian CaMKP-N, which is highly expressed in central nervous system (CNS) [16,17]. Gene knockdown of zCaMKP-N Morpholino-mediated knockdown of genes in zebraWsh embryos has become a routine and eYcient method to provide information about gene function in vivo [28]. To investigate the biological signiWcance of CaMKP-N in zebraWsh embryogenesis, we carried out a gene knockdown experiment using an antisense morpholino oligonucleotide designed on the basis of the sequence at the 5⬘-untranslated region that included the initiation Met of zCaMKP-N mRNA. ZebraWsh embryos at 1–4 cell stage were injected with an antisense morpholino oligo (AS-MO) or a 5-bases mismatch morpholino oligo (5mis-MO) (Fig. 8A). These embryos were cultured at 26 °C and observed by stereoscopic microscopy. Injection of the AS-MO in a concentration of 1 g/l (approximately 0.8 ng injection per egg) led to an increase in the number of embryos with morphological and cellular abnormalities. On the other hand, embryos injected with the 5mis-MO at the same concentration developed normally. Acridine orange is known to stain apoptotic cells, but not necrotic cells [33]. Therefore, we can examine
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whether apoptosis occurs in the embryos with morphological abnormalities by acridine orange staining. The cells in the brain and spinal cord of the embryos injected AS-MO exhibited typical apoptotic images by acridine orange staining analysis (Fig. 8B, lower panels), but not embryos injected 5mis-MO (Fig. 8B, upper panels). In good agreement with the results of in situ hybridization, apoptotic cells were observed in telencephalon, diencephalon, epiphysis, cerebellum, and spinal cord but not mesencephalon (Fig. 8B, lower panel). At 72 hpf, a signiWcant increase of round-shaped embryos was observed (Fig. 8C). Among about 100 embryos tested, less than 2% and 7% were abnormal in embryos with no injection and 5mis-MO-injected embryos, respectively. In contrast, more than 80% and 95% of embryos were abnormal when 0.5 g/l (0.4 ng/embryo) and 1 g/l (0.8 ng/embryo) of AS-MO were injected, respectively (Fig. 8D). These results suggest that zCaMKPN is indispensable for normal development, especially in CNS, of zebraWsh embryos. Discussion We previously found a protein phosphatase having a certain homology with CaMKP in the human database and named it CaMKP-N [16]. CaMKP-N has similar biochemical properties to CaMKP, but in contrast to CaMKP it localizes exclusively in cell nuclei. To clarify the physiological signiWcance of CaMKP-N, we cloned a CaMKP-N homologue of zebraWsh and analyzed its biological functions in this study. The cDNA of zCaMKP-N possessed an open reading frame of 1899 bp encoding 633 amino acids, and exhibited identities of 49% and 48% with rat CaMKPN and human CaMKP-N, respectively. The catalytic domain of zCaMKP-N was highly homologous to mammalian CaMKP-Ns, and the PP2C motif, which is highly conserved among PPM family phosphatases, was found in this region (Fig. 2).
Fig. 7. Localization of zCaMKP-N gene transcript in zebraWsh embryos. ZebraWsh embryos (48 hpf) were analyzed by whole mount in situ hybridization with DIG-labeled RNA probes. The hybridization was detected by anti-DIG antibody conjugated to alkaline phosphatase. (A) Sense RNA probe, (B) antisense RNA probe. (C) Represents an enlarged view of (B). (D) Dorsal view of (C). Red arrows indicate forebrain, in which strong signal was detected.
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Fig. 8. Gene knockdown of zCaMKP-N during zebraWsh embryogenesis. (A) Sequences of zCaMKP-N mRNA around the 5⬘ 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) Acridine orange staining of zebraWsh embryos injected with morpholino oligos. AS-MO (lower panels) or 5mis-MO (upper panels) was injected into 1–4 cell stage embryos at a concentration of 1 g/l (about 0.8 ng/ embryo). At 48 hpf, embryos were stained with acridine orange and observed by stereoscopic microscopy as described in “Materials and methods”. (C) Phenotypes of zebraWsh embryos injected with AS-MO (right panel) or 5mis-MO (left panel) at 72 hpf. (D) Frequency of abnormal phenotypes in embryos injected with 5mis-MO (1 g/l) or AS-MO (0.5 or 1 g/l), and control embryos without injection.
When human GFP-CaMKP-N fusion protein was transfected in COS-7 cells, it clearly localizes in the nucleus [16]. Although typical NLS was not found in human CaMKPN, two NLSs, NLS1 and NLS2, were identiWed in its C-terminal region by mutagenesis analysis [34]. In the case of zCaMKP-N, typical NLS was found in the C-terminal region of zCaMKP-N(residues 575–587) (Fig. 4A). When zCaMKP-N was transiently expressed in Neuro2a cells, zCaMKP-N was exclusively localized in the nucleus (Fig. 4B). In contrast to wild type zCaMKP-N, its truncation mutant, zCaMKP-N(1–574), devoid of the C-terminal region including the putative NLS localized in nuclei and cytoplasm (Fig. 4B). In addition, EGFP fusion proteins with residues 448–633 and with residues 489–633, both of which contained the putative NLS, 575–587, translocated
to nuclei, whereas the fusion protein with residues 448–574, which was devoid of the putative NLS, showed homogenous distribution throughout the cytoplasm and nuclei (Fig. 4D). These results suggest that the region 575–587 actually functions as NLS for zCaMKP-N. To examine the enzymatic properties of zCaMKP-N, the recombinant protein was expressed in E. coli and puriWed. PuriWed enzyme exhibited not only Mn2+-dependent but also Mg 2+-dependent protein phosphatase activity (Fig. 3B), and the activity was not aVected by 10 M okadaic acid or 100 nM calyculin A (data not shown). These characteristic properties are in good agreement with those of the PPM family phosphatases including mammalian CaMKP and CaMKP-N [12,13,16]. However, in contrast to zCaMKP-N, human CaMKP-N, as well as rat CaMKP, appeared to be much less active in the presence of Mg2+ than in the presence of Mn2+ [13,16,35]. As shown in Fig. 5C, zCaMKP-N could eYciently dephosphorylate CaMKIV in vitro, as well as could human or rat CaMKP-N [16,17]. Based on the structural features, the subcellular distribution, and the enzymatic properties of zCaMKP-N as described above, we concluded that zCaMKP-N is a zebraWsh homologue of mammalian CaMKP-N. Since both CaMKP-N and CaMKIV are expressed in brain and are speciWcally localized in the nucleus [12,16,17], and since CaMKP-N eYciently dephosphorylates CaMKIV in vitro [16,17], it is highly likely that one of the potential physiological substrates for CaMKP-N is CaMKIV that is phosphorylated by CaMKK. To conWrm this, we examined whether CaMKIV could be dephosphorylated by zCaMKP-N in living cells. The cDNA of CaMKIV was transfected in Neuro2a cells with cDNA of zCaMKPN(WT) or CaMKP-N(D188A), and the phosphorylation level of the transiently expressed CaMKIV was assessed using a phospho-CaMKIV speciWc antibody (Fig. 6). As shown in Fig. 6, CaMKIV was markedly phosphorylated by endogenous CaMKK in response to the increase in the intracellular Ca2+. However, coexpression of wild type zCaMKP-N, but not the inactive mutant zCaMKPN(D188A), signiWcantly attenuated the ionomycin-stimulated phosphorylation of CaMKIV. These results provide the Wrst demonstration that CaMKP-N dephosphorylates CaMKIV in living cells. Thus, zCaMKP-N is suggested to be a potential downregulator for CaMKIV in vivo. To further explore the biological functions of CaMKPN, gene expression of zCaMKP-N in zebraWsh embryos was investigated by whole mount in situ hybridization analysis. In good agreement with the tissue localization of mammalian CaMKP-N [16,17], zCaMKP-N mRNA is predominantly expressed in brain, especially in telencephalon, diencephalon, epiphysis, and cerebellum but not mesencephalon as shown in Fig. 7. During development, zCaMKP-N mRNA was detected in embryos at 24 hpf and gradually increased after 29 hpf (data not shown), suggesting that zCaMKP-N may be involved in the control of neuronal development in the CNS of zebraWsh embryos. We
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further provided evidence about the function of zCaMKPN by morpholino-knockdown experiment in zebraWsh embryos. The injection of the antisense morpholino oligonucleotide, which was designed to functionally knockdown zCaMKP-N, into zebraWsh embryos led to a marked increase of embryos with morphological abnormalities (Fig. 8D). Within the injection of AS-MO, abnormality was also detected at the cellular level when embryos were stained with acridine orange, which was used to stain apoptotic cells, but not necrotic cells, in Drosophila [33]. Dead cells, possibly apoptotic cells, were detected in telencephalon, diencephalon, epiphysis, cerebellum, and spinal cord but not mesencephalon (Fig. 8B, lower panels). On the other hand, few dead cells were detected in 5mis-MOinjected embryos (Fig. 8B, upper panels). Whole mount in situ hybridization using the zCaMKP-N gene suggests that expression of zCaMKP-N mRNA corresponded roughly to the region where dead cells were detected (Fig. 7). These results indicated that zCaMKP-N is related to apoptotic events, especially in the CNS of zebraWsh embryos. It is reported that overexpression of CaMKP results in apoptotic cell death [36], but the mechanism of this phenomenon remains to be elucidated. Although it is also currently unclear why the antisense knockdown of zCaMKP-N caused cellular apoptosis leading to morphological abnormalities of the brain and spinal cord, there have been several reports that CaMKIV is important for regulating apoptotic events [37–40]. Since at least one of the potential targets of CaMKP-N is CaMKIV, as discussed above, it is likely that CaMKP-N also plays a pivotal role in the regulation of cell death and survival through regulating CaMKIV activity. Two opposite eVects of CaMKIV on apoptosis are reported; some reports reveal that activation of CaMKIV prevents cells from apoptosis [38,41], while others show that activation of CaMKIV leads to the promotion of apoptotic cell death [39,40]. Understanding the molecular mechanisms whereby knockdown of zCaMKPN results in apoptosis may shed light on a novel pathway for regulation of apoptotic events. The results presented in this study clearly indicate that CaMKP-N is involved in the regulation of the phosphorylation level of CaMKIV in cells, and that it has important functions in zebraWsh embryogenesis. Further studies will be necessary to further explore the role of CaMKP-N in embryogenesis, including the molecular mechanisms regulating cell death and survival. Acknowledgments We sincerely thank Dr. Naotaka Tanaka and Dr. Kaoru Takegawa for helpful advises on microscopic observation. This work was supported by Grant-in-Aid for ScientiWc Research from the Ministry of Education, Science, Sports and Culture of Japan, and by grants from the Kato Memorial Bioscience Foundation, the Smoking Research Foundation, the Akiyama Foundation, and AIST Research Grant.
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