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Characterization of a Novel Plant PP2C-like Protein Ser/Thr Phosphatase as a Calmodulin-binding Protein

2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês

10.1074/jbc.m301369200

1083-351X

Daisuke Takezawa,

Enzyme Structure and Function

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or β-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324–346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three α-helices in the C-terminal domain of human PP2Cα, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants. Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or β-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324–346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three α-helices in the C-terminal domain of human PP2Cα, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants. Plants can sense changes in environmental conditions such as light, temperature, and water status as signals and show specific responses. Cytosolic free Ca2+, the level of which is increased by the extracellular signals, has been demonstrated to be an intracellular messenger that regulates cellular processes necessary for the plant responses. Physiological processes such as responses to light, wounding, low temperature, and pathogen attacks have been suggested to be regulated by Ca2+ in plants (1Knight M.R. Campbell A.K. Smith S.M. Trewavas A.J. Nature. 1991; 352: 524-526Crossref PubMed Scopus (927) Google Scholar, 2Knight M.R. Smith S.M. Trewavas A.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4967-4971Crossref PubMed Scopus (273) Google Scholar, 3Poovaiah B.W. Reddy A.S.N. CRC Crit. Rev. Plant Sci. 1993; 12: 185-211Crossref PubMed Scopus (328) Google Scholar, 4Bush D.S. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1995; 46: 95-122Crossref Scopus (909) Google Scholar). Calmodulin (CaM), 1The abbreviations used are: CaM, calmodulin; ABI, abscisic acid-insensitive; CKII, casein kinase II; GST, glutathione S-transferase; MBP, myelin basic protein; MsCaM, M. sativa calmodulin isoform; PCaMPP, Physcomitrella CaM-binding protein phosphatase; PCM1 and PCM6, potato calmodulin isoforms 1 and 6; PKA, cyclic AMP-dependent protein kinase; PP1, PP2A, PP2B, and PP2C, types 1, 2A, 2B, and 2C phosphoprotein phosphatases; PVDF, polyvinylidene difluoride; DTT, dithiothreitol.1The abbreviations used are: CaM, calmodulin; ABI, abscisic acid-insensitive; CKII, casein kinase II; GST, glutathione S-transferase; MBP, myelin basic protein; MsCaM, M. sativa calmodulin isoform; PCaMPP, Physcomitrella CaM-binding protein phosphatase; PCM1 and PCM6, potato calmodulin isoforms 1 and 6; PKA, cyclic AMP-dependent protein kinase; PP1, PP2A, PP2B, and PP2C, types 1, 2A, 2B, and 2C phosphoprotein phosphatases; PVDF, polyvinylidene difluoride; DTT, dithiothreitol. a multifunctional Ca2+-binding protein, has been postulated to be a primary sensor of cytosolic Ca2+ (5Klee C.B. Crouch T.H. Richman P.G. Annu. Rev. Biochem. 1980; 49: 489-515Crossref PubMed Scopus (824) Google Scholar). In the presence of Ca2+, CaM binds to a number of intracellular target proteins and modulates their functions. These target CaM-binding proteins are involved in the regulation of diverse cellular functions, including metabolism, ion transport, transcription, and signal transduction (6Snedden W.A. Fromm H. New Phytol. 2001; 151: 35-66Crossref PubMed Scopus (384) Google Scholar, 7Zielinski R.E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 697-725Crossref PubMed Scopus (394) Google Scholar). Among the CaM-binding proteins, CaM-dependent protein kinases and phosphatases play major roles in diversifying the signals triggered by Ca2+, because these enzymes modify functions of multiple cellular proteins by phosphorylation and dephosphorylation. CaM-regulated protein phosphorylation plays pivotal roles in the regulation of signal perception and gene activation, as has been well documented in mammalian neuronal cells (8Klee C.B. Neurochem. Res. 1991; 16: 1059-1065Crossref PubMed Scopus (119) Google Scholar). In animals, several classes of CaM-dependent protein kinases, such as phosphorylase kinase, myosin light chain kinase, CaM kinases I, II, and VI, and CaM kinase kinase, are known to exist, whereas only one class of CaM-dependent protein phosphatase, calcineurin, has been reported. Calcineurin, which was identified as a major CaM-binding protein in the brain, is known as type 2B protein Ser/Thr phosphatase (PP2B) and belongs to the PPP family of phosphatases, having a moderate degree of sequence similarity to PP1 and PP2A (9Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2151) Google Scholar, 10Kincaid R. Adv. Second Messenger Phosphoprotein Res. 1993; 27: 1-23PubMed Google Scholar, 11Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (600) Google Scholar). In plant systems, protein phosphatases that bind CaM or those regulated by CaM have not been characterized. However, physiological evidence has suggested that some of the Ca2+-dependent cellular processes are regulated by PP2B-like phosphatases. Bovine calcineurin has been shown to modulate the current of the slowly activating vacuolar channel of the stomata guard cell, positively at low concentrations and negatively at higher concentrations (12Allen G.J. Sanders D. Plant Cell. 1995; 7: 1473-1483Crossref PubMed Google Scholar). The PP2B inhibitor cyclosporin A has been shown to inhibit Ca2+-stimulated phosphatase activity (13Kinoshita T. Shimazaki K. Plant Cell Physiol. 1999; 40: 53-59Crossref PubMed Scopus (19) Google Scholar) and enhance phosphorylation of proteins in guard cells (14Li J. Lee Y.R.J. Assmann S.M. Plant Physiol. 1998; 116: 785-795Crossref PubMed Scopus (156) Google Scholar). Possible involvement of PP2B in the regulation of salt stress in plants has also been suggested. Overexpression of catalytic and regulatory subunits of yeast PP2B in transgenic tobacco resulted in an enhancement of salt tolerance (15Pardo J.M. Reddy M.P. Yang S. Maggio A. Huh G.H. Matsumoto T. Coca M.A. Paino-D'Urzo M. Koiwa H. Yun D.J. Watad A.A. Bressan R.A. Hasegawa P.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9681-9686Crossref PubMed Scopus (185) Google Scholar). Despite this physiological evidence, no PP2B-like genes in plants have been characterized. Complete genome sequencing of the model plant Arabidopsis thaliana revealed that there are no genes encoding Ser/Thr phosphatase with sequence similarity to the catalytic subunit of PP2B. These facts raise the possibility that plants might possess CaM-regulated protein phosphatases that belong to a different class from that of PP2B. Screening of cDNA expression libraries with labeled CaM as a ligand has been successfully used for isolation of plant genes encoding CaM-binding proteins (6Snedden W.A. Fromm H. New Phytol. 2001; 151: 35-66Crossref PubMed Scopus (384) Google Scholar, 16Fromm H. Chua N.-H. Plant Mol. Biol. Rep. 1993; 10: 199-206Crossref Scopus (60) Google Scholar, 17Reddy A.S.N. Takezawa D. Fromm H. Poovaiah B.W. Plant Sci. 1993; 94: 109-117Crossref Scopus (36) Google Scholar). However, genes of protein phosphatases that bind CaM have not been isolated from plants by this method. This could possibly be due to low abundance of mRNA expressed in specific cell types in complex tissue organization. In this study, screening with 35S-labeled CaM was carried out using a library made from protonema tissues of the moss Physcomitrella patens, which has relatively uniform cells with simple structures. One of the isolated clones, designated PCaMPP, encoded a protein with similarity to the type 2C protein Ser/Thr phosphatases (PP2Cs). PP2Cs belong to the PPM family of Ser/Thr phosphatases, which are distinct from PP1, PP2A, or PP2B and are present in a wide variety of eukaryotes, including animals, yeast, and plants (9Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2151) Google Scholar). PP2Cs are postulated to be monomeric enzymes, and their regulation by other protein molecules is not well understood. Thus, characterization of PCaMPP may lead to understanding of unidentified regulatory mechanisms for PP2C-related protein phosphatases by CaM. In this study, recombinant proteins of PCaMPP were produced, and interaction with CaM and regulation of the phosphatase activity were examined. Plant Material—Protonema tissues of the moss P. patens were grown at 25 °C under continuous light (60 μmol m-2 s-1) on agar plates as described by Minami et al. (18Minami A. Nagao M. Arakawa K. Fujikawa S. Takezawa D. J. Plant Physiol. 2003; 160: 475-483Crossref PubMed Scopus (66) Google Scholar). CaMs—Bovine CaM was purchased from Sigma. Potato CaMs PCM1 and -6 (19Takezawa D. Liu Z.H. An G. Poovaiah B.W. Plant Mol. Biol. 1995; 27: 693-703Crossref PubMed Scopus (92) Google Scholar) were expressed in Escherichia coli cells and purified by phenyl-Sepharose column chromatography as described by Poovaiah et al. (20Poovaiah B.W. Takezawa D. An G. Han T.-J. J. Plant Physiol. 1996; 149: 553-558Crossref PubMed Scopus (31) Google Scholar). The MsCaM gene was isolated from 8-day-old seedlings of alfalfa (Medicago sativa L.) by reverse transcriptase-PCR using 5′-ATGGCNGAYATHCTNTC-3′ and 5′-CGAACTGTCATCATCATCTTGAC-3′. The MsCaM protein was expressed in E. coli by using a TOPO101 expression system (catalog number 45-1072, Invitrogen) and purified by phenyl-Sepharose chromatography. Screening with Labeled CaMs—Potato calmodulins PCM1 and PCM6 were labeled with [35S]methionine as described by Fromm and Chua (16Fromm H. Chua N.-H. Plant Mol. Biol. Rep. 1993; 10: 199-206Crossref Scopus (60) Google Scholar). The λZAP cDNA library of P. patens protonemata was used for screening by using 35S-labeled PCM1 and PCM6 as ligands. Positive clones detected by autoradiography were selected and subjected to a second or third round of screening. Plasmids were prepared from phage clones by an in vivo excision protocol, and nucleotide sequences were determined. The genomic DNA corresponding to the PCaMPP cDNA was obtained by PCR using oligonucleotides, 5′-GTTGCAACGCAAGTGAGCCGTCAG-3′ and 5′-CGTATTCACTGACGGAAATTTAAC-3′, comprising 5′- and 3′-end sequences of the PCaMPP cDNA, respectively. The nucleotide sequence was determined by direct sequencing using the amplified DNA as a template. Constructs for Recombinant Proteins—GST fusion proteins of PCaMPP were prepared by using pGEX-2TK vectors (Amersham Biosciences). Constructs for making deletion proteins were made by PCR using oligonucleotide primers with a BglII (AGATCT) or BamHI (GGATCC) site for N-terminal deletions and an EcoRI (GAATTC) site for C-terminal deletions. The amplified fragments were fused in-frame to the BamHI/EcoRI sites of the pGEX-2TK plasmid. The oligonucleotide pairs used for making deletion constructs were 5′-GAGGGATCCGCAAAGAACATCGTCAAG-3′/5′-GCAAGGAATTCATCACCATCATTTGCTTTC-3′ for GST-PCaMPP-(291–336), 5′-GAGGGATCCGCAAAGAACATCGTCAAG-3′/5′-CCAGAACGAATTCCTTTTTTGGACCGTGTG-3′ for GST-PCaMPP-(291–348), 5′-GTCGAGATCTTTCGCACCAGCAGTC-3′/5′-GATCCAGAATTCGTTGTCCATC-3′ for GST-PCaMPP-(324–373), and 5′-GTCGGATCCCAAATGATGGTGATGAGATTC-3′/5′-GATCCAGAATTCGTTGTCCATC-3′ for GST-PCaMPP-(332–373). A histidine tag (His tag) was added to the fusion constructs by ligation of phosphorylated oligonucleotides, 5′-GTCAGCCATCATCACCATCACCATTG-3′ and 5′-AATTCAATGGTGATGGTGATGATGGCTAGC-3′, to the C-terminal end of the PCaMPP coding region to make the construct for GST-PCaMPPh. Site-directed mutagenesis of the PCaMPP active site was carried out by PCR mutagenesis using 5′-CTGCGGATCCATTATGACTGGCCAAAACCG-3′ for PCaMPP(G92A/D-93S)h and 5′-CTGCGGATCCATCATGACCGTC-3′ for PCaMPP(N95D)h. Expression of Recombinant Proteins in E. coli—E. coli cells harboring the plasmids of various constructs of PCaMPP were grown at 37 °C in the LB medium until the A 600 reached 0.6. Isopropyl-β-d-galactopyranoside was added to a final concentration of 0.1 mm to the culture, and the proteins were expressed by further incubation at 16 °C for 10–16 h. The cells were collected by centrifugation, suspended in TE (10 mm Tris-Cl (pH 8.0), and 1 mm EDTA), and stored at -80 °C. Column Chromatography—Ni2+ affinity chromatography was carried out using a His-bind purification kit (Novagen, Madison, WI) according to the protocol provided by the manufacturer. CaM-agarose chromatography was carried out essentially as described previously (21Takezawa D. Ramachandiran S. Paranjape V. Poovaiah B.W. J. Biol. Chem. 1996; 271: 8126-8132Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) with slight modifications. The proteins in a column-loading buffer containing 25 mm Tris-HCl (pH 7.5), 50 mm NaCl, 0.05 mm EDTA, 0.05% (w/v) Triton X-100, 10% (v/v) ethylene glycol, and 1 mm CaCl2 were loaded onto a column of CaM-agarose (P4385, Sigma). After washing with the same buffer twice, the column was further washed twice with the same buffer as that described above but containing 500 mm NaCl. The column was again equilibrated with the loading buffer and then developed with elution buffer containing 2.5 mm EGTA instead of CaCl2. The purified proteins were collected and concentrated by using Ultrafree microfiltration units (Millipore, Bedford, MA). Glycerol was added to 25% (w/v), and the proteins were stored at -80 °C. Biotinylated CaM Binding Assays—Proteins produced by plaques on agar plates were transferred onto a nitrocellulose membrane by capillary action. The bacterially expressed recombinant proteins were electrophoresed on 10% SDS-polyacrylamide gel and electrophoretically transferred onto a PVDF membrane (Immobilon-P, Millipore, Bedford, MA). The membranes were incubated for 1 h in a binding buffer (25 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm CaCl2) with 1% (w/v) bovine serum albumin and then incubated with biotinylated CaM (catalog number 208697, Calbiochem) in the same solution for 2.5 h. The membranes were washed in the binding buffer three times for 5 min each time and then incubated with alkaline phosphatase-conjugated streptavidin. After washing three times using the binding buffer, the bound biotinylated calmodulin was detected by nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate reagents in alkaline phosphate buffer (100 mm Tris-Cl (pH 9.5), 10 mm MgCl2 and NaCl, 1 mm CaCl2). Peptide Binding Assay on Non-denaturing Polyacrylamide Gel—CaM (50 pmol) and various amounts of a synthetic peptide were incubated for 5 min at room temperature in 50 mm Hepes (pH 7.5) and 1 mm CaCl2. The mixture was loaded on a 15% non-denaturing polyacrylamide gel. The lower gel contained 0.375 m Tris-HCl (pH 8.8), and the upper gel contained 0.125 m Tris-HCl (pH 6.8) as buffers, and both of the gels were supplemented with either 1 mm CaCl2 or 2 mm EGTA. Electrophoresis was carried out at 120 V for 2.5 h in a buffer containing 0.02 m Tris, 0.192 m glycine, and either 1 mm CaCl2 or 2 mm EGTA. CaMs and CaM-peptide complexes were visualized by staining with Coomassie Brilliant Blue R-250. Protein Phosphatase Assays—Protein phosphatase activity was determined by using myelin basic protein (MBP) phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) or Abl tyrosine kinase and casein-phosphorylated by casein kinase II (CKII). To prepare Ser/Thr-phosphorylated MBP, 500 μg of MBP (Upstate Biotechnology, Inc., Lake Placid, NY) was phosphorylated using 25 units of PKA (P2645, Sigma) in a buffer containing 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 1 mm DTT, 1 mm unlabeled ATP, and 1.85 MBq [α-32P]ATP. To prepare Tyr-phosphorylated MBP, a protein phosphatase assay system (catalog number P0785S, New England Biolabs) was used. Casein (C8032, Sigma) was phosphorylated using 2500 units of CKII (catalog number P6010S, New England Biolabs) in a buffer containing 20 mm Tris-HCl (pH 7.5), 350 mm NaCl, 1 mm EDTA, 10 mm 2-mercaptoethanol, 0.1% (w/v) Triton X-100, 1 mm unlabeled ATP, and 1.85 MBq [γ-32P]ATP. All the phosphorylation reactions were carried out at 30 °C overnight. The phosphorylated MBP and casein were precipitated by trichloroacetic acid at 10% (w/v) and collected by centrifugation at 12,000 × g for 30 min at 4 °C. The precipitated proteins were solubilized in a buffer containing 50 mm Tris-HCl (pH 8.5), 0.1 mm EDTA, 2 mm DTT, and 0.01%(w/v) Brij35. The proteins were dialyzed against 25 mm Tris-HCl (pH 7.5), 0.1 mm EDTA, 2 mm DTT, and 0.01% (w/v) Brij35 and used as substrates. Phosphatase assays were carried out using 20–50 ng of the purified recombinant enzyme and 5 μg of 32P-labeled protein substrates in a buffer containing 50 mm Tris-HCl (pH 7.0), 0.1 mm EDTA, 5 mm DTT, and 0.01% Brij35 in a 25-μl reaction volume with or without various effectors. The enzyme reactions were carried out at 30 °C and terminated by addition of 100 μl of 20% (w/v) trichloroacetic acid. Precipitated proteins were removed by centrifugation at 12,000 × g for 30 min. The supernatants were subjected to scintillation counting to determine radioactive phosphate (32Pi) released during the phosphatase reaction. Characterization of the CaM-binding Protein Similar to PP2C—A cDNA library of P. patens was used for screening with 35S-CaM to isolate genes encoding CaM-binding proteins. The screening resulted in identification of a clone that showed strong binding to 35S-labeled plant CaMs, which was further confirmed by CaM binding assays using biotinylated bovine CaM. Sequence analysis of the isolated clone revealed that it encoded a protein of 373 amino acids with a predicted molecular mass of 40.2 kDa. The amino acid sequence of the protein was found to have the highest similarity to a gene product of A. thaliana registered as phosphatase 2C-like protein (GenBank™ accession number AF419561). The sequence also had moderate sequence similarity to other plant, yeast, and animal PP2Cs. Thus, the protein was designated Physcomitrella CaM-binding protein phosphatase (PCaMPP). Alignment of amino acid sequences of PCaMPP and human PP2Cα revealed moderate degrees of similarity (Fig. 1). Eukaryotic PP2Cs are known to possess conserved amino acid residues associating with divalent metals such as Mg2+ and Mn2+ as revealed by analysis of the crystal structure of human PP2Cα (22Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (384) Google Scholar). Although amino acid identity of the presumed catalytic domains of human PP2Cα (amino acids 2–290) and the corresponding region of PCaMPP (amino acids 29–316) was only 22%, all of the metal-associating residues corresponding to Glu-37, Asp-38, Asp-60, Gly-61, Asp-239, and Asp-282 of human PP2Cα were conserved in PCaMPP (Fig. 1, open arrowheads). PCaMPP also possessed other highly conserved amino acids such as the residues corresponding to Thr-128, Gly-145, Gly-198, Val-221, and Gly-240 of human PP2Cα. CaM Binding Analyses of PCaMPP—In order to examine CaM-binding properties of PCaMPP, a GST fusion protein with the PCaMPP coding region (GST-PCaMPP-(373)) was expressed in E. coli and used for CaM binding assays. Fig. 3 shows that the expressed protein binds to CaM in the presence of Ca2+ but not in the presence of EGTA. The Ca2+-dependent binding of CaM to GST-PCaMPP-(373) was dramatically reduced when a 50-fold excess amount of unlabeled CaM was present. In order to identify the CaM-binding region of PCaMPP, various PCaMPP deletion proteins were expressed in E. coli and used for CaM binding assays. CaM binding assays of the C-terminal deletion proteins revealed that GST-PCaMPP-(347) bound to CaM but that GST-PCaMPP-(336) did not, indicating that the CaM-binding region is present in the C-terminal domain of PCaMPP (Fig. 2).Fig. 2CaM binding assays of GST fusion proteins of PCaMPP. A, schematic representation of the constructs of GST-PCaMPP-(373), GST-PCaMPP-(348), and GST-PCaMPP-(336). The PCaMPP catalytic domain (c) and the C-terminal domain (hatched box) are indicated. B, results of CaM binding assays of GST-PCaMPP proteins. Lysates of control E. coli cells (lane 1) and cells carrying GST-PCaMPP-(373) (lane 2), GST-PCaMPP-(348) (lane 3), and GST-PCaMPP-(336) (lane 4) were either stained with Coomassie Brilliant Blue to visualize total proteins (left) or transferred onto PVDF membranes (right panels). The membranes were incubated with 35S-labeled CaM (1 μg ml-1) in the presence of either 2.5 mm EGTA, 0.5 mm CaCl2 (Ca2 +), or 0.5 mm CaCl2 plus 50 μg ml-1 of unlabeled bovine CaM (Ca2 + + CaM). Positions of molecular mass markers are shown in kDa on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Further deletion analysis of the PCaMPP C-terminal domain revealed that GST-PCaMPP-(324–373) and GST-PCaMPP-(291–348) bound to CaM, whereas GST-PCaMPP-(332–373) and GST-PCaMPP-(291–336) did not (Fig. 3, A and B). The CaM binding of GST-PCaMPP-(324–373) and GST-PCaMPP-(291–348) was Ca2+-dependent and was dramatically reduced in the presence of an excess amount of unlabeled CaM. These results indicated that the CaM-binding region of PCaMPP is present between amino acids 324 and 348. The results of CaM-agarose binding tests indicated that both GST-PCaMPP-(324–373) and GST-PCaMPP-(291–348) bound CaM-agarose in the presence of Ca2+, which were eluted out by a buffer containing 2.5 mm EGTA (Fig. 3C). CaM Binding Assays Using a Synthetic Peptide—To confirm CaM binding to the PCaMPP C-terminal domain identified by deletion analyses, a synthetic peptide, CBPep-1, having the same amino acid sequence as that of PCaMPP-(320–346) was used for a CaM-mobility shift assay on a non-denaturing polyacrylamide gel (Fig. 4). CBPep-1 has a basic and hydrophobic character commonly found in a number of CaM-binding sequences of other proteins (Fig. 4A). In a non-denaturing gel with pH 8.8, bovine CaM that has acidic charges was mobilized toward the anode. Electrophoresis in the presence of Ca2+ showed that the mobility of CaM in the non-denaturing gel was shifted toward the cathode by the addition of increasing amounts of CBPep-1 (Fig. 4B). The mobility shift by CBPep-1 was not observed when the electrophoresis was carried out in the presence of 2 mm EGTA instead of Ca2+. The mobility shift assays using different plant CaM isoforms, PCM6, PCM1, and MsCaM, with CBPep-1 indicated that all of the CaM isoforms tested bound CBPep-1 in a similar manner to binding by bovine CaM (Fig. 4C). Phosphatase Activity of PCaMPP—Recombinant GST fusion proteins of PCaMPP were used for protein phosphatase assays in order to determine enzymatic properties of PCaMPP. MBP phosphorylated by cyclic AMP-dependent protein kinase (PKA) or Abl tyrosine kinase (AblK) and casein phosphorylated by casein kinase II (CKII) were used as substrates. GST-PCaMPPh, with an extra C-terminal His tag, was used for phosphatase assays, because the His-tagged enzyme was apparently less vulnerable to degradation during the expression in bacteria and the protein purification was further facilitated by Ni2+-affinity chromatography (Fig. 5A). The phosphatase assays using different substrates revealed that GST-PCaMPPh has activity toward PKA-phosphorylated MBP. However, it had very little activity toward CKII-phosphorylated casein or AblK-phosphorylated MBP (data not shown). Thus, the PKA-phosphorylated MBP was used for the subsequent experiments. To determine divalent cation dependence of GST-PCaMPPh, the effects of Mg2+ and Mn2+ on the phosphatase activity were examined. Fig. 5B shows Mn2+-dependent activation of the GST-PCaMPPh. The activation occurred at an Mn2+ concentration as low as 0.1 mm and was nearly saturated at concentrations above 1 mm. In contrast, Mg2+ up to 5 mm had no effect on GST-PCaMPP phosphatase activity (data not shown). Eukaryotic protein phosphatases can be distinguished by their sensitivities to various organic and inorganic inhibitors. PPM family phosphatases (i.e. PP2Cs) that are insensitive to okadaic acid can be distinguished from the PPP family phosphatases (i.e. PP1, PP2A, and PP2B) that are sensitive. Thus, possible inhibitors of protein phosphatases, okadaic acid (1 and 5 μm), orthovanadate (1 mm), β-glycerophosphate (12.5 mm), and pyrophosphate (5 mm), were used for phosphatase assays to determine enzymatic properties of GST-PCaMPPh. The results of the assays revealed that pyrophosphate had an inhibitory effect on the Mn2+-dependent phosphatase activity (Fig. 5C). The activity was also inhibited by a high concentration (1 mm) of Ca2+, which antagonizes the effect of Mn2+. Effect of Mutations in the Putative Active Site—To examine the catalytic function of PCaMPP, two constructs for mutations in the putative active site were prepared by site-directed mutagenesis (Fig. 6A). In the mutant GST-PCaMPP(D92A/G93S)h, the residues Asp-92 and Gly-93 were replaced by Ala and Ser, respectively. Both Asp and Gly in these positions are highly conserved in known eukaryotic PP2Cs, and the replacement of these residues was expected to cause a great disturbance in the active site (22Das A.K. Helps N.R. Cohen P.T.W. Barford D. EMBO J. 1996; 15: 6798-6809Crossref PubMed Scopus (384) Google Scholar, 23Sheen J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 975-980Crossref PubMed Scopus (228) Google Scholar). In another mutant GST-PCaMPP(N95D)h, Asn-95 was replaced with Asp-95. This N95D mutation was thought to have a milder effect because other PP2Cs do not possess strictly conserved residues in the corresponding position; for example, it is typically Ala in mammalian PP2Cs and Gly in plant PP2Cs. However, it has been shown that replacement with Asp in this position in ABI1 and ABI2 PP2Cs in Arabidopsis mutants causes abscisic acid-insensitive phenotypes (24Leung J. Bouvier-Durand M. Morris P.-C. Guerrier D. Chefdor F. Giraudat J. Science. 1994; 246: 1448-1452Crossref Scopus (659) Google Scholar, 25Meyer K. Leube M.P. Grill E. Science. 1994; 246: 1452-1455Crossref Scopus (602) Google Scholar), indicating that the residue is biologically critical. These mutant proteins, as well as wild-type GST-PCaMPPh, were expressed in E. coli and purified by CaM-affinity chromatography (Fig. 6B). Protein phosphatase assays using these enzymes revealed that the mutants had reduced activity compared with that of the wild-type enzyme (Fig. 6C), indicating that PCaMPP has an active site similar to those of other eukaryotic PP2Cs. D92A/G93S double mutation appeared to have a much more severe effect than an N95D single mutation. Effect of CaM on the Phosphatase Activity—The effect of CaM on the GST-PCaMPPh activity was examined using different concentrations of bovine CaM. The reaction was carried out in the presence of 0.1 mm Ca2+, which alone did not have any effect on the activity. As shown in Fig. 7A, CaM at nanomolar concentrations significantly increased the phosphatase activity of GST-PCaMPPh. This increase was as much as 1.7-fold by saturating CaM concentrations (>0.4 μm), and the CaM concentration needed for half-maximal activation was ∼0.1 μm. The enhancement of the phosphatase activity by CaM was reversed by addition of