Regulation of the dual specificity protein phosphatase, DsPTP1, through interactions with calmodulin.

Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.

In plants and animals, Ca 2ϩ acts as a mediator of stimulus response coupling in the regulation of diverse cellular functions triggered by a variety of biotic and abiotic external stimuli (1)(2)(3)(4)(5). The cytosolic free calcium concentration ([Ca 2ϩ ] cytosol ) is transiently elevated with the complex forms of amplitude, frequency, and duration, in response to specific stimuli (6). The complex Ca 2ϩ signals are decoded by Ca 2ϩ sensor(s), and transduced to downstream elements for cellular processes (7)(8)(9). Calmodulin (CaM), 1 a ubiquitous Ca 2ϩ -binding protein with four EF hands, is a well characterized primary Ca 2ϩ sensor in all eukaryotes. In most cases, the active form of CaM (Ca 2ϩbound) regulates the activity and function of a wide range of CaM-binding proteins (CaMBP), including metabolic enzymes, transcription factors, ion channels, protein kinases/phosphatases, and structural proteins (10,11). Therefore, CaM acts as a multifunctional protein in Ca 2ϩ -mediated signal transduction networks and regulates the activities of proteins that are structurally distinct and opposite proteins, such as protein phosphorylation and dephosphorylation (12)(13)(14).
Reversible phosphorylation of proteins is an essential element of the numerous mechanisms that have evolved to facilitate the communication of external stimuli across cell surfaces, subsequently leading to alterations in the activities and functions of other intracellular proteins. Protein kinase and phosphatase play central roles in diverse cellular mechanisms (15)(16)(17). Protein kinase catalyzes the covalent attachment of a phosphate group to an amino acid side chain with a hydroxyl group. A number of protein kinase types have been widely studied in higher plants. Protein phosphatases are less numerous but more diverse, and can therefore be grouped into Ser/ Thr, Tyr, and dual specificity classes according to substrate specificity (18,19). Ser/Thr phosphatases were originally subdivided into protein phosphatase 1 (PP1) and protein phosphatase 2 (PP2) groups, based on differential sensitivity to the small inhibitors (20). PP2 proteins are further distinguished by metal ion requirements. Specifically, PP2C and protein phosphatase 2B (PP2B) require Mg 2ϩ and Ca 2ϩ , respectively, whereas protein phosphatase 2A (PP2A) types have no ion requirement (21). Proteins of PP1, PP2A, and PP2B groups share high sequence similarity and constitute the protein phosphatase P (PPP) family. In contrast, PP2C proteins display low sequence similarity to the PPP family and compose the protein phosphatase M (PPM) family, together with pyruvate dehydrogenase phosphatase and other Mg 2ϩ -dependent Ser/Thr phosphatases (22,23). Interestingly, members of the PPP and PPM families share similar structural folding characteristics (24), suggesting a common mechanism of catalysis. Several conserved acidic residues generate complex metal ions essential to their activities (25)(26)(27). A metal-bound water molecule acts as a nucleophile to directly displace phosphate from a specific amino acid of the substrate in an acid-base catalytic mechanism (28). Tyr phosphatases and Ser/Thr phosphatases have distinct evolutionary origins and catalytic mechanisms. Conventional Tyr phosphatases (PTP) are specific for phosphorylated Tyr residues. However, dual specificity phosphatases (DsPTP) act on both phosphorylated Tyr and phosphorylated Ser/Thr residues (29). PTP and DsPTP contain a catalytic core motif with a conserved Cys residue, which acts as a nucleophile, displacing the phosphate group from the substrate and forming a phosphoryl-cysteinyl intermediate (30). This subgroup of Tyr phosphatases regulates mitogen-activated protein kinase (MAPK) in a variety of signal transduction pathways in both animals and yeast (31). In fact, both AtPTP1 and AtDsPTP1 dephosphorylate and deactivate a MAPK protein (AtMPK4) in vitro (32,33). A putative DsPTP gene, AtMKP1, is essential for UV resistance in Arabidopsis (34).
The Ca 2ϩ /CaM-dependent protein phosphatase, calcineurin (PP2B), is ubiquitous in animals and yeast, and mediates a variety of intracellular signaling pathways (35). Studies reporting the in vitro and in vivo effects of introducing heterologous calcineurin in plants indirectly support the existence of functional calcineurin in plants (36 -38). A number of potential calcineurin partners have been identified in plants, including immunophilins (39 -41), and "calcineurin B-like proteins" (42). However, the calcineurin catalytic subunit (calcineurin A) is not encoded by the Arabidopsis genome, and to date, no calcineurin A-related sequences have been reported from other plant species in the public sequence data bases.
In the present study, we identify a CaM-binding DsPTP1 (33) by screening with HRP-conjugated CaM as a probe. DsPTP1 has distinct biochemical characteristics from calcineurin. Dephosphorylation of pNPP, a common synthetic protein phosphatase substrate, by DsPTP1 was increased in the presence of CaM. However, the tyrosine dephosphorylation activity of DsPTP1 on phosphorylated MBP was inhibited by CaM. Our results demonstrate that Ca 2ϩ signaling mediated by CaM contributes to the protein dephosphorylation mechanism in plants via DsPTP1 regulation.

EXPERIMENTAL PROCEDURES
Screening of the Arabidopsis cDNA Expression Library-A cDNA expression library was constructed from Arabidopsis thaliana (ecotype Columbia) in a ZAPII vector (Stratagene, La Jolla, CA). Arabidopsis calmodulin 2 (AtCaM2) was conjugated to maleimide-activated HRP using the EZ-Link maleimide-activated HRP conjugation kit (Pierce), as described in a previous report (43). We screened the Arabidopsis cDNA expression library using HRP-conjugated AtCaM2 (AtCaM2::HRP) as a probe. Approximately 5 ϫ 10 4 pfu cells were plated per 15-cm LB plate, using Escherichia coli XL1-blue MRF (Stratagene) as the host strain. Plates were incubated at 42°C until plaques appeared and overlaid with nitrocellulose filters previously soaked in 10 mM isopropyl-1-thio-␤-D-galactopyranoside. Incubation was continued at 37°C for 6-8 h, and plates were cooled to 4°C. Filters were removed and rinsed twice in a large volume of TBS-T (Tris-based saline containing 0.1% (v/v) Tween 20). Next, filters were blocked by incubation in 7% (w/v) nonfat dry milk/TBS-T overnight. Blocked filters were washed three times with TBS-T for 5 min and equilibrated in overlay buffer (50 mM imidazole-HCl, pH 7.5, 150 mM NaCl) for 1 h. Membranes were blocked a second time by incubating filters in overlay buffer containing 9% (v/v) gelatin (Sigma), 0.5% (v/v) Tween 20, and 1 mM CaCl 2 for 3.5 h. AtCaM2::HRP was added to gelatin-containing buffer at a final concentration of 0.2 g/ml, and filters were incubated for 1 h. The final washing was performed in three steps, with each step consisting of 5 repeats of a 5-min wash; first, in TBS-T, 50 mM imidazole-HCl (pH 7.5), and 1 mM CaCl 2 ; second, in 20 mM Tris-HCl (pH 7.5), 0.5% Tween 20, 50 mM imidazole-HCl, 0.5 M KCl, and 1 mM CaCl 2 ; and third, in 20 mM Tris-HCl (pH 7.5), 0.1% Tween 20, and 1 mM MgCl 2 . Bound CaM::HRP was visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences). Recombinants (5 ϫ 10 5 ) were screened, and 50 positive clones were isolated after three rounds of screening. cDNA inserts were recovered by in vivo excision with helper phage (ExAssist, Stratagene, La Jolla, CA). To confirm binding to CaM, we expressed positive clones as ␤-galactosidase fusion proteins in E. coli. Clones were examined for CaM binding by a CaM::HRP overlay assay, as described above. In brief, we transformed positive clones into XL1-blue MRF (Stratagene) and induced the expression of ␤-galactosidase fusion proteins by 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside treatment. Isopropyl-1-thio-␤-D-galactopyranoside-induced E. coli crude proteins (20 g) were separated on a 10% SDS-polyacrylamide gel and transferred onto an Immobilon-MP membrane (polyvinylidene difluoride, Millipore). The membrane was rinsed in TBS-T, blocked by incubation in 7% (w/v) nonfat dry milk/TBS-T overnight, and processed as described above. For determination of Ca 2ϩ independent binding of CaM, 5 mM EGTA was substituted for 1 mM CaCl 2 in all overlay buffers. The cDNA sequences of the resulting positive clones were determined from both strands by dideoxynucleotide chain termination, using an automatic DNA sequencer (ABI 373A, Applied Biosystems Inc.).
CaM Mobility Shift Assay with a Synthetic Peptide-Peptides corresponding to a stretch of 20 amino acids within CaMBDI ( 26 KVKN-QIQALVRVIKVARTYR 47 ) and 28 amino acids ( 151 KKHGMT-LAQALQHVKSKRPVASPNAGFI 180 ) in CaMBDII of DsPTP1 were generated at a peptide synthesis facility (Peptron and Peptipharm). The CaM binding abilities of the synthetic peptides were determined from the relative mobility shift of CaM in the presence of the peptide (44). CaM (303 pmol) was incubated with increasing concentrations of peptide (molar ratios: 0, 0.25, 0.5, 1.0, 2.0, 2.5, 3.0, and 3.5) in binding buffer (100 mM Tris-HCl, pH 7.2, plus 0.1 mM CaCl 2 or 2 mM EGTA) at room temperature for 1 h. Half the volume of 50% glycerol plus the tracer, bromphenol blue, was added, and mixtures were electrophoresed on 15% non-denaturing polyacrylamide gels containing 0.375 M Tris-HCl (pH 8.8), and either 0.1 mM CaCl 2 or 2 mM EGTA. Gels were run at a constant voltage of 100 V in electrode buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, and either 0.1 mM CaCl 2 or 2 mM EGTA) and stained with Coomassie Brilliant Blue for visualization.
Phosphodiesterase Competition Assay-Cyclic nucleotide PDE (phosphodiesterase) assays were performed using commercially available bovine heart CaM-deficient PDE (Sigma). The initial reaction mixture (100 l) contained buffer (100 mM imidazole-HCl, 2.56 mM cAMP, 5.13 mM MgSO 4 , 1.28 mM CaCl 2 ) with varying amounts of AtCaM2 (1 to 200 nM) and a fixed concentration of synthetic peptide (100 nM). The reaction was initiated by the addition of PDE (0.5 milliunits/l). The basal level of enzyme activity was determined in the absence of AtCaM2, and stimulated activity was determined in the presence of AtCaM2 and CaCl 2 . After incubation at 37°C for 30 min, the reaction was terminated by placing the reaction tubes into a boiling water bath for 5 min and then on ice for 2 min. Following brief centrifugation, 50 l of alkaline phosphatase (10 units) was added and the mixture incubated at 37°C for 10 min. The reaction was stopped by adding 500 l of 10% trichloroacetic acid. After vortexing, precipitates were spun down and the supernatant (400 l) was transferred to a new tube. After adding 1 ml of phosphate reagent (45), the supernatant was incubated at 37°C for 30 min and assayed for P i at A 660 . The dissociation constant (K d ) of AtCaM2 for the peptide was calculated from the concentration of AtCaM2 (nM) required to obtain half-maximal (50%) PDE activity, either in the presence (100 nM) or absence of peptide. The following equation was used to calculate dissociation constants (44): is the total concentration of peptide added, and [CaM] and K represent the concentrations of CaM required to obtain half-maximal activation of PDE in the presence or absence of peptides, respectively.
Yeast Split Ubiquitin Assay-The yeast split ubiquitin assay was performed as described previously (46). Saccharomyces cerevisiae strain JD53 was used for all the experiments. AtCaM2 and DsPTP1 (WT and mutant) cDNA were cloned into pMet-Ste14-C ub -RUra3, replacing yeast Ste14. AtCaM2 and DsPTP1 (WT and mutant) cDNA were cloned into modified versions of the pCup-N ub -Sec62 vector, replacing yeast Sec62 (47). Interactions between each pair of proteins were tested on selective medium containing 1 mg/ml 5-FOA and selective medium lacking uracil. Plates were incubated at 30°C for 3-5 days, unless specified otherwise.
For phosphatase assays with another protein substrate, MBP was labeled either at tyrosine ([ 32 P]Tyr MBP) or serine/threonine ([ 32 P]Ser/ Thr MBP) residues, using specific protein kinase. Experiments were performed according to the manufacturer's instructions. For tyrosine labeling, 25 units of human c-Src tyrosine kinase (Upstate Biotechnology Inc., Lake Placid, NY) were incubated with 1 mg of substrate and 50 Ci [ 32 P]ATP (PerkinElmer Life Sciences) in 50 l of reaction buffer (25 mM Tris-HCl, pH 7.2, 5 mM MnCl 2 , 0.5 mM EGTA, 0.05 mM Na 3 VO 4 , 25 mM Mg acetate) for 4 h at 30°C. Radiolabeled MBP was precipitated with 15% trichloroacetic acid, washed three times with cold 20% trichloroacetic acid and twice with cold acetone, air-dried, dissolved in 0.2 M Tris-HCl (pH 8.0), and stored at 4°C. For 32 P labeling of serine/ threonine residues in MBP, the procedure described for Src kinase was applied to 50 units of bovine heart protein kinase A (Sigma) in a different reaction buffer (25 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 100 mM NaCl, 12 mM MgCl 2 ). Labeled protein was purified as described for the Src-labeled substrate.
Protein phosphatase activity was determined by measuring the release of free 32 P from labeled substrates. Assays were performed as described previously (32,33), with some modifications. For calmodulin analysis, 2 g of wild-type DsPTP1 or mutant protein (L35R, K166E, or L35R/K166E) was mixed with 2 ϫ 10 4 cpm of 32 P-labeled substrate in phosphatase buffer (50 mM Tris-HCl, pH 7.0, 2 mM dithiothreitol) in a total volume of 150 l. Following incubation of the reaction mixture at 30°C for 1 h, the reaction was terminated by the addition of 2 volumes of 25% trichloroacetic acid. Next, 20 g of bovine serum albumin (Amresco, Burnsville, MN) was added as carrier protein to facilitate precipitation. Proteins were pelleted by centrifugation at 12,000 rpm for 10 min, and the supernatant was subjected to liquid scintillation counting (Beckman LS 6000TA). Blank incubations were performed in the absence of DsPTP1. The phosphatase activity of DsPTP1 protein was calculated as percentage dephosphorylation of the substrate after subtraction of blank readings.
Furthermore, protein phosphatase activity was confirmed by measuring 32 P-labeled substrates. 32 P-Labeled substrate (2 ϫ 10 6 cpm) was added to a 20 l reaction mixture containing 50 mM Tris-HCl, pH 7.0, 2 mM dithiothreitol and DsPTP1 protein, and incubated at 30°C for 1 h. The reaction conditions were similar, except for reaction volume and substrate concentration (2 ϫ 10 6 cpm). The mixture was separated on 13% SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. To visualize dephosphorylation activity, the gel was exposed to x-ray films after drying.

RESULTS
The Arabidopsis CaM-binding Protein, DsPTP1-To identify the molecular components of Ca 2ϩ /CaM-mediated signaling pathways, we screened an Arabidopsis cDNA expression library using HRP-conjugated CaM as a probe (43). Fifty positive clones were obtained from about 5 ϫ 10 5 recombinant phages. DNA sequencing of the clones and comparisons with known sequences in the GenBank TM data base revealed known plant CaMBP, such as kinesin-like proteins (48), CaM-binding heatshock proteins (49), and two CaMBP of unknown function. These additionally included several novel CaMBP with high homology to known proteins, and others with no significant homology to any reported proteins.
One of isolated clones that bound CaM was DsPTP1, which is already reported in the literature. Gupta et al. (33) showed that DsPTP1 encodes an active protein phosphatase that hydrolyzes the phosphate group on both phosphoserine/threonine and phosphotyrosine residues of substrates. Moreover, the catalytic mechanism of DsPTP1 involves a similar to phosphoenzyme intermediate of PTP, as evident from data showing that the replacement of Cys 135 with serine eliminates phosphatase activity (33).
Mapping of CaMBD within DsPTP1-Comparative analysis of the CaM-binding regions of the numerous reported CaMBP proteins has led to the identification of multiple sequence motifs required for CaM complex formation (50). Based on the structural characteristics of known CaMBD, two putative CaMBD are predicted in DsPTP1. One is located in the N terminus from Lys 26 to Arg 47 (CaMBDI), whereas the other is present in the C terminus from Lys 151 to Ile 180 (CaMBD II) (Fig. 1A).
To confirm the presence of the putative CaMBD of DsPTP1, we generated GST-fused constructs containing full-length cDNA (designated D0) and four serial fragment constructs (D1, D2, D3, and D4) (Fig. 1A). Recombinant fusion proteins were produced in E. coli, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes for Western blotting and CaM overlay assays. Expression of the GST fusion proteins was verified by probing the blot with an anti-GST antibody. Three recombinant proteins (D0, D1, and D4) that contained the putative CaMBD interacted with HRP-conjugated CaM (CaM::HRP), whereas GST only (GST) and GST fusion proteins lacking the predicted CaM binding region (D2 and D3) did not interact with CaM::HRP. CaM bound to DsPTP1 in the presence, but not the absence of Ca 2ϩ (Fig. 1B). These results indicate that CaM binds DsPTP1 at two distinct regions in a Ca 2ϩ -dependent manner, specifically, CaMBDI in the N terminus and CaMBD II in the C terminus.
To ascertain whether other DsPTP bind CaM, DsPTP1e (At2g04550) and DsPTP1b (At3g06110) in Arabidopsis (29) were cloned into the GST fusion vector. Recombinant protein phosphatases were produced in E. coli, and subjected to Western blotting and a CaM::HRP overlay assay. Although DsPTP1e and DsPTP1b display significant sequence homology to DsPTP1, CaM did not bind these proteins (Fig. 1B). This finding indicates that interactions between CaM and DsPTP1 are highly specific.
Binding of a Synthetic Peptide to CaM-To confirm CaM binding to CaMBDI (N terminus) and CaMBDII (C terminus) of DsPTP1, peptides corresponding to the two domains were employed for a CaM mobility shift assay on non-denaturing polyacrylamide gels (44).
As shown in Fig. 2A, the amount of peptide-CaM complex increased with increasing concentrations of the synthetic peptide in the presence of 0.1 mM Ca 2ϩ , whereas the complex was undetectable in the presence of 2 mM EGTA. This finding is consistent with data from a previous CaM overlay assay under similar Ca 2ϩ and EGTA conditions. In the case of CaMBDI, about 60% CaM was shifted at a molar ratio of 1:1 (peptide: CaM), and all the CaM was shifted at molar ratios of 3:1 and 3.5:1 (peptide:CaM). For CaMBDII, about 40% CaM was shifted at a molar ratio of 1:1 (peptide:CaM), and all CaM was shifted at a ratio of 2.5:1 (peptide:CaM) (Fig. 2A). These results indicate that the 20-mer corresponding to CaMBDI (Lys 26 to Arg 47 ) and 28-mer corresponding to CaMBDII (Lys 151 to Ile 180 ) are sufficient for CaM binding in a Ca 2ϩ -dependent manner.
To confirm binding of the synthetic peptides to CaM, we performed a competition assay with PDE, a Ca 2ϩ /CaM-dependent enzyme, in the presence of the two CaMBD peptides. To determine K d values, dose-dependent activation of PDE by CaM was monitored either in the presence (100 nM) or absence of peptides (Fig. 2B). The activation curves shifted to the right in the presence of the peptide, indicating competition between PDE and the peptide for binding to CaM. The concentration required to achieve half-maximal activation of PDE was 8.4 nM in the absence of peptides, whereas 73.5 (9-fold) and 16.8 nM (2-fold) in the presence of the each of two peptides (CaMBDI and CaMBDII, respectively). The K d values of the peptide for PDE activation by ACaM2 were determined as 4.50 (CaMBDI) and 11.82 nM (CaMBDII). Our data imply that the binding affinity of CaMBDI for CaM is higher than that of CaMBDII.
Identification of Critical Residues of the CaM-binding Motifs-CaM-binding proteins possess a region comprising a basic amphipathic helix consisting of ϳ16 -34 amino acids (51,52). The sequence of CaM is highly conserved and identical among vertebrates (50,53). In contrast, the Ca 2ϩ /CaM-binding regions of targets exhibit low sequence homology (54). To identify the critical residues of CaMBDI and CaMBDII for CaM binding, we performed site-directed mutagenesis. Several mutant proteins were produced in E. coli and analyzed for CaM binding with a CaM overlay assay.
Within CaMBDI, hydrophobic residues important for CaM binding (specifically, Val 28 , Ile 32 , Leu 35 , and Val 36 ) were separately replaced with Arg and denoted V28R, I32R, L35R, and V36R, respectively (Fig. 3A). Because CaMBDI may comprise an IQ-like motif, Gln 33 was replaced with the acidic residue, Asp, and the resulting construct denoted Q33D (Fig. 3A). Each mutation was introduced into the GST::D1 fusion construct containing CaMBDI. Fusion proteins were expressed in E. coli and subjected to a CaM binding overlay assay (Fig. 3B).
To determine the effect of CaM binding to full-length DsPTP1, we constructed a CaM-binding negative mutant, GST::DsPTP1. Based on previous mutagenesis results, we substituted Ile 32 and Leu 35 with Arg in CaMBDI, and Lys 166 with Glu in CaMBDII, resulting in CaM-binding negative mutants (denoted I32R/K166E and L35R/ K166E, respectively). GST fusion proteins were produced and subjected to a CaM binding overlay assay. As shown in Fig. 5, CaM bound to DsPTP1 mutants with a single amino acid substitution (I32R, L35R, and K166E) because of the presence of the remaining CaMBD, but not to the double mutants (I32R/K166E and L35R/ K166E) of DsPTP1.
Determining in Vivo Interactions between DsPTP1 and CaM with a Split Ubiquitin Assay in Yeast-To examine the direct in vivo interactions between DsPTP1 and CaM, we used the yeast split ubiquitin assay system, based on the reassembly of the N-and C-terminal halves (N ub and C ub ) of ubiquitin (Ub) (46,47). DsPTP1 and CaM were fused to the C terminus of N ub and the N terminus of C ub . The C ub of ubiquitin was linked to an N-terminal modified Ura3p reporter containing Arg at position 1 (RUra3p). If CaM interacts with the DsPTP1 protein, N ub and C ub should reassemble into native-like Ub, followed by cleavage of RUra3p by ubiquitin-specific proteases. Released RUra3p is then rapidly degraded through the N-end rule pathway of protein degradation (55). Consequently, cells containing CaM-C ub -RUra3p and N ub -DsPTP1 or DsPTP1-C ub -RUra3p and N ub -CaM are unable to grow on plates lacking uracil, but grow on plates containing 5-FOA, which is converted into toxic 5-fluorouracil by RUra3p. In the opposite cases, yeast cells are uracil prototrophs and 5-FOA sensitive.
As shown in Fig. 6, cells co-expressing CaM-C ub -RUra3p and N ub -DsPTP1 or DsPTP1-C ub -RUra3p and N ub -CaM were unable to grow on plates lacking uracil, but grew on plates containing 5-FOA, indicating that DsPTP1 effectively forms stable complexes with CaM in vivo. In negative controls, no interactions were observed between CaM-C ub -RUra3p and N ub or C ub -  (Fig. 5).
Effect of CaM on DsPTP1 Phosphatase Activity-A phosphatase cleaves the phosphate group of pNPP, resulting in a yellow nitrophenol product that may be conveniently quantified by absorbance at 405 nm. Wild-type and mutant proteins (I32R, L35R, K166E, L35R/K166E, and I32R/K166E) were purified and subjected to a phosphatase assay using pNPP as a substrate. DsPTP1 displayed very low specific activity against pNPP, compared with well known phosphatases such as AtPTP1 (33).
To measure the CaM-dependent activity of DsPTP1, we used a constant amount of DsPTP1 (5 g) with increasing CaM concentrations. Activity was increased, proportional to the level of CaM. To determine the CaMBD critical for CaM-dependent activation of DsPTP1, we performed similar experiments with mutant phosphatases. The phosphatase activity of the CaMBD I mutant (L35R) was increased with the concentration of CaM, similar to wild-type DsPTP1 (Fig. 7, A and B). However, phosphatase activities of CaMBDII mutants (K166E and L35R/K166E) were not affected upon the addition of CaM (Fig. 7, C and D). These results indicate that CaMBDII, rather than CaMBDI, is important for CaM-dependent activation of DsPTP1. In the absence of Ca 2ϩ (5 mM EGTA), the phosphatase activity of DsPTP1 was not affected by CaM (Fig. 7).
Because DsPTP1 is a dual specificity protein phosphatase, we performed a phosphatase assay using phospho-Ser/Thr and phospho-Tyr-labeled MBP as a substrate. Phospho-Ser/Thr and phospho-Tyr-labeled MBP were prepared with Ser/Thr kinase and tyrosine kinase, respectively (33). DsPTP1 wild-type and mutant proteins, including L35R, K166E, and L35R/K166E, dephosphorylated both phospho-Tyr and phospho-Ser/Thr in an enzyme concentration-dependent manner. However, CaMbinding mutants (I32R and I32R/K166E) of DsPTP1 displayed no phosphatase activity (Supplemental Materials Fig. 2). Notably, DsPTP1 proteins displayed higher activity on phosphotyrosine-labeled MBP than on phospho-Ser/Thr-labeled MBP. This finding strongly indicates that the DsPTP1 gene encodes a functional dual specificity protein phosphatase. Similar to the experiment performed with the pNPP substrate, to identify the effect of CaM on the phosphatase activity of DsPTP1, we performed the phosphatase assay with CaM in the presence of Ca 2ϩ . As described under "Experimental Procedures," the phosphatase assay was performed using phospho-Ser/Thr and phospho-Tyr-labeled MBP as substrates.
Interestingly, the activity of DsPTP1 on phospho-Tyr was inhibited by the addition of CaM, whereas activity on phospho-Ser/Thr was not affected (Fig. 8A). The effect on DsPTP1 phosphatase activity was dependent upon the concentration of CaM. Additionally, activity of the CaMBDI mutant (L35R) was inhibited (Fig. 8B), whereas CaMBDII mutants (K166E and L35R/K166E) were not affected by CaM (Fig. 8, C and D). These results are consistent with the ability of CaM to bind DsPTP1, and are similar to data obtained with the pNPP substrate (Fig.  7). To confirm the inhibitory effect of DsPTP1 on phospho-Tyr in the presence of CaM, regulation of DsPTP1 activity by CaM was additionally monitored by SDS-PAGE and autoradiography. As shown in Fig. 9, the addition of increasing amounts of DsPTP1 to phospho-Ser/Thr and phospho-Tyr substrates re-FIG. 6. The use of split ubiquitin to monitor interactions between CaM and DsPTP1 in vivo. Shown are serial dilutions of cells co-expressing N ub or Nub-DsPTP1 (WT and CaMBD mutant) fusion, together with CaM (AtCaM2-C ub -RUra3) and C ub or DsPTP1-C ub -RUra3 (WT and CaMBD mutant) fusion together with CaM (N ub -AtCaM2) on plates lacking tryptophan and histidine (control) additionally lacking uracil (ϪUra) or containing 5-FOA (ϩFOA). All proteins were expressed from single copy vectors. duced the amount of 32 P labeling in the MBP protein, indicating dephosphorylation of phosphorylated MBP by DsPTP1. In a parallel experiment, we measured the regulation of DsPTP1 phosphatase activity by CaM. High dephosphorylation activity of DsPTP1 on phospho-Tyr was detected in the absence of CaM (Fig. 9A). Following the addition of CaM, the activity of DsPTP1 on phospho-Tyr was significantly decreased. However, the dephosphorylation of phospho-Ser/Thr activity by DsPTP1 was not influenced by CaM (Fig. 9B). Moreover, the inhibition of dephosphorylation of phospho-Tyr by DsPTP1 was dependent upon the concentration of CaM (Fig. 9C). These results are consistent with data on the phosphorylated MBP substrate from a previous phosphatase assay of DsPTP1 (Fig. 8A). DISCUSSION Plant and animal cells generate Ca 2ϩ signals with different amplitudes, frequencies, and durations in response to a variety of external stimuli, and use these signals to control numerous cellular processes (2)(3)(4)(5)(6). CaM, a small acidic protein, plays a vital role in transducing Ca 2ϩ signals by modulating the activities of numerous target proteins (48, 49, 56 -58). One of the important mechanisms in post-translational modification for signal transduction is phosphorylation by protein kinase and dephosphorylation by protein phosphatase. Ca 2ϩ -mediated protein phosphorylation and dephosphorylation constitute one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses (17, 59, 60). Calcineurin (protein phosphatase 2B), the only serine/threonine phosphatase under control of Ca 2ϩ /CaM, is an important mediator in signal transmission, and connects Ca 2ϩ -dependent signaling to a wide variety of cellular responses (61).
Recently, Kutuzor et al. (62) suggested that protein phosphatase 7 (PP7) is a candidate plant calcineurin, based on data on the activity of PP7 interactions with CaM and its resistance to okadaic acid. However, calcineurin is activated, whereas PP7 is inhibited by CaM. The investigators propose that this discrepancy may be because of the necessity for factors other than CaM for PP7 activation or substrate specificity, as stimulation of plant calcineurin by CaM is observed with peptide or protein substrates (62).
To elucidate the role(s) of CaM in phosphorylation/dephosphorylation signal transduction, we isolated potential CaMbinding proteins by screening an Arabidopsis cDNA expression library using HRP-conjugated CaM as a probe. We characterized a CaM-binding protein encoded by DsPTP1, a dual specificity protein phosphatase from Arabidopsis (33).
Most of the 50 or more CaMBD isolated so far comprise stretches of 16 -35 residues that display segregation of basic and polar residues on one side and hydrophobic amino acids on the other in a helical wheel representation (51)(52)(53)(54). Ca 2ϩ -dependent CaM-binding motifs have been classified into two major groups, specifically, 1- (5) ping of expression serial fragments (Fig. 1), and confirmed by CaM mobility shift and PDE enzyme competition assays with a synthetic peptide corresponding to a 20-amino acid stretch (from Lys 26 to Arg-47) and a 28-amino acid stretch (from Lys 151 to Ile 180 ) (Fig. 2). The hydrophobic and basic residues in CaMBD play a critical role in binding (54). We further confirmed the predicted CaM-binding motif by showing that the substitution of a single amino acid (Ile 32 3 Arg or Leu 35 3 Arg (Fig. 3) and Lys 166 3 Glu (Fig. 4)) resulted in the loss of the Ca 2ϩ -dependent CaM binding ability of CaMBDI and CaMB-DII of DsPTP1, respectively. In vivo interactions between CaM and DsPTP1 were confirmed by the split ubiquitin assay in yeast, which was developed to monitor protein-protein interactions in a living cell (64) and is widely used for membrane proteins (58,65) and transcriptional regulators (46). DsPTP1 has several salient differences, compared with calcineurin. Although both are CaM-binding protein phosphatases regulated by CaM, calcineurin hydrolyzes phosphoserine/ threonine, whereas DsPTP1 is capable of using both phosphotyrosine and phosphoserine/threonine as substrates. An additional difference is that calcineurin consists of two subunits, whereas DsPTP1 appears to be a monomer in the absence of calmodulin. However, the possibility of the existence of another regulatory protein of DsPTP1 cannot be excluded in view of low phosphatase activity in vitro. Therefore, we speculate that in animals, DsPTP1 binds CaM, such as rhodopsin protein phosphatase (RDGC) (63) and protein-tyrosine phosphatase ␣ (64).
In this report, we show that DsPTP1 activity on pNPP and phosphorylated MBP is regulated by CaM. As shown in Figs. 7 and 8, DsPTP1 activity on pNPP is stimulated about 5-fold and that on the phosphotyrosine residue of phosphorylated MBP is inhibited about 3-fold in the presence of CaM. This regulation of phosphatase activity is distinct from that reported for previously known CaM-regulated protein phosphatases. Most CaM-binding phosphatases are activated or inhibited by CaM. However, to date, this type of differential regulation of phosphatase activity of substrates by CaM has not been observed in either plants or mammals. The dephosphorylation of pNPP provides an easy, rapid, and accurate method for the quantification of protein phosphatase assay and permits an insight into reaction kinetics. A number of investigators have reported protein phosphatase activities of dephosphorylating pNPP. However, in some cases, it is unclear whether this represents broad substrate specificity of these phosphatases or extrinsic alkaline phosphatase activity. We have investigated the properties of DsPTP1 in this respect. Our data show that DsPTP1 dephosphorylates both pNPP and phosphorylated MBP, and is differentially regulated by CaM. In plants, it is unclear whether DsPTP1 activity is enhanced or inhibited by CaM. Therefore, the real substrate of DsPTP1 must be identified to confirm the in vivo effect of CaM on the phosphatase. Gupta et al. (33) recently suggested that DsPTP1 specifically dephosphorylates and inactivates Arabidopsis MAPK4, using an in vitro kinase assay. However, no direct evidence has been presented on whether MAPK4 is the real substrate of DsPTP1. In addition to DsPTP1, other protein phosphatases have been implicated in MAPK inactivation. These include tyrosine-specific protein phosphatase (65) and serine/threonine protein phosphatases that have been identified in plants and yeast (66 -68). To further explore the possibility of MAPK4 binding to DsPTP1, we examined binding in an in vivo system. We additionally screened proteins interacting with DsPTP1 to identify other regulatory proteins or substrates. Further functional analyses using overexpression transgenic and mutant plants of DsPTP1 (including wild-type and CaM-binding nega-tive mutants) should provide exciting novel information on the role of protein dephosphorylation in CaM-mediated Ca 2ϩ signaling. FIG. 9. Inhibition of DsPTP1 phosphatase activity by CaM using phosphorylated MBP in gel. A, dephosphorylation of phosphorylated MBP in the presence of increasing concentrations of DsPTP1. B, tyrosine dephosphorylation in the presence of Ca 2ϩ /CaM. C, CaM inhibited tyrosine dephosphorylation activity of DsPTP1 in a concentrationdependent manner. Using isotope-labeled phosphorylated MBP (2 ϫ 10 6 cpm) and DsPTP1, proteins (WT, L35R, K166E, and L35R/K166E) were reacted and separated by SDS-PAGE. Gels were dried, stained with Coomassie Brilliant Blue, and developed with x-ray films for visualization.