Catalytic Activation of the Plant MAPK Phosphatase NtMKP1 by Its Physiological Substrate Salicylic Acid-induced Protein Kinase but Not by Calmodulins*

MAPK phosphatases (MKPs) are negative regulators of MAPKs. Previously, we identified NtMKP1 as a novel calmodulin (CaM)-binding protein (Yamakawa, H., Katou, S., Seo, S., Mitsuhara, I., Kamada, H., and Ohashi, Y. (2004) J. Biol. Chem. 279, 928-936). In this study, we characterized the interaction of NtMKP1 with substrate MAPKs and CaM. NtMKP1 (produced by in vitro transcription/translation) inactivated salicylic acid-induced protein kinase (SIPK) through dephosphorylation of the TEY motif of SIPK. CaM bound but unexpectedly did not activate the phosphatase activity of NtMKP1. NtMKP1 has four characteristic domains, viz. a dual-specificity phosphatase catalytic domain, a gelsolin homology domain, a CaM-binding domain, and C-terminal domain. Deletion analysis revealed that the N-terminal non-catalytic region of NtMKP1 bound SIPK and was essential for inactivating SIPK, whereas the CaM-binding and C-terminal domains were dispensable. Moreover, the phosphatase activity of NtMKP1 was increased strongly by the binding of SIPK, but weakly by another MAPK, wound-induced protein kinase. Swapping and site-directed mutagenesis of SIPK and wound-induced protein kinase revealed that the strong activation of NtMKP1 phosphatase activity by SIPK partially depended on the putative common docking domain of SIPK. On the other hand, conversion of Lys41 and Arg43 of NtMKP1 to Ala (K41A/R43A) abolished the interaction with SIPK. Expression of constitutively active MAPK kinase in Nicotiana benthamiana induced activation of SIPK and cell death. Simultaneous expression of either NtMKP1 or NtMKP1 L443R, which was unable to bind CaM, compromised the constitutively active MAPK kinase-induced responses, whereas that of NtMKP1 K41A/R43A did not. These results indicate that the regulation of NtMKP1 activity by SIPK binding, but not by CaM binding, is important for the function of NtMKP1.

The MAPKs 3 are key molecules of signal transduction responding to various extracellular stimuli in eukaryotes. The activity of MAPKs is strictly regulated via phosphorylation of the conserved TXY motif by an upstream MAPK kinase (MAPKK) (1,2). Conversely, MAPKs are dephosphorylated and inactivated by protein phosphatases, including tyrosine-specific phosphatases; serine/threonine-specific phosphatases; and dual-specificity MAPK phosphatases (MKPs), which are highly specific to MAPKs (3)(4)(5). In mammals, the 14 members of the MAPK family represent three major subfamilies, viz. ERK, JNK, and p38 (1,2). On the other hand, the mammalian MKP family has 10 members, which share sequence homology, but differ in substrate specificity and subcellular localization (3)(4)(5). Mammalian MKPs interact with their substrate MAPKs via a substrate-binding domain at their N termini, and such an interaction increases the catalytic activity of some MKPs. This "substrate-triggered activation" of MKP phosphatase activity has been shown to play an important role in determining substrate specificity at the subfamily level (6 -9).
Plant MAPKs also constitute a large family. However, no obvious JNK or p38 type of MAPK has been identified in plants. The Arabidopsis genome contains 20 MAPKs, 12 of which are the ERK type, and the others are the plant-specific type (10). In contrast to many members of the MAPK family, plant MKPs likely form a small gene family. Only five MKPs are predicted in the Arabidopsis genome, including the previously reported AtMKP1, IBR5, PHS1, and DsPTP1 (11). This disproportionate ratio of MAPK to MKP may suggest that one MKP regulates multiple MAPKs in plants and that other types of protein phosphatases such as protein phosphatase 2C (12) and tyrosine-specific phosphatase (13) also play an important role in the regulation of plant MAPKs. Mutation of the AtMKP1, IBR5, and PHS1 genes causes sensitivity to genotoxic stress, reduced responses to the phytohormones auxin and abscisic acid, and compromised cortical microtubule functions, respectively (14 -16). This genetic evidence clearly reveals the important biological role of plant MKPs. However, the biochemical characteristics of plant MKPs are not well understood. Although yeast two-hybrid analysis showed that AtMKP1 specifically interacts with AtMPK3, AtMPK4, and AtMPK6 among AtMPK1-9 (17), the phosphatase activity of AtMKP1 and IBR5 has not been proven. PHS1 has general phosphatase activity as shown by the hydrolysis of an artificial substrate, but its target MAPK is unclear (16). DsPTP1 dephosphorylates and inactivates AtMPK4 in vitro; however, it has been suggested that DsPTP1 is unlikely to inactivate AtMPK4 in vivo (13,18).
Previously, we identified a putative tobacco ortholog of AtMKP1 (NtMKP1) as a calmodulin (CaM)-binding protein (19). It has also been reported that CaM binds two different sites of DsPTP1 (20). To date, no MKP from organisms other than plants has been shown to bind CaM. Therefore, interaction with CaM seems to be a unique feature of plant MKPs. Compared with mammalian MKPs, NtMKP1 and AtMKP1 are large proteins carrying several characteristic domains, viz. a dual-specificity phosphatase domain, a gelsolin homology (GH) domain, a CaMbinding domain, and a long C-terminal domain containing a serine-rich region (see Fig. 4A). In tobacco, two MAPKs, salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), are activated by a variety of extracellular stimuli, including wounding (21,22). We showed previously that overexpression of NtMKP1 compromises wound-induced activation of SIPK and WIPK, suggesting a role for NtMKP1 in regulating these MAPKs in vivo (19). In fact, the activity of AtMPK6, the Arabidopsis ortholog of SIPK, is stronger in an atmkp1 loss-of-function mutant than in the wild type (17). These results indicate that SIPK is a physiological substrate of NtMKP1.
Previously, we failed to detect the phosphatase activity of NtMKP1 (19). To demonstrate that NtMKP1 is an active MAPK phosphatase, we produced the NtMKP1 protein by in vitro transcription/translation. We also prepared phosphorylated SIPK as a substrate of NtMKP1. Here, we show that NtMKP1 is an active phosphatase that dephosphorylates the conserved TEY motif of SIPK in vitro. The N-terminal non-catalytic region of NtMKP1 interacted with SIPK and was essential for inactivating SIPK. Moreover, the phosphatase activity of NtMKP1 was significantly increased by the binding of SIPK. We show that the catalytic activation of NtMKP1 by SIPK, but not by CaM, is important for the function of NtMKP1 both in vitro and in vivo.

EXPERIMENTAL PROCEDURES
Plant Materials-Nicotiana benthamiana was grown in a temperature-controlled growth room maintained at 25°C with a 16-h light/8-h dark cycle. Four-to five-week-old plants were used for experiments.

Production of Recombinant Proteins of MAPKs and Constitutively Active MAPKKs-
The open reading frames of SIPK and WIPK were amplified by PCR with MscI and XhoI sites attached to the 5Ј-and 3Ј-ends, respectively, and cloned into the corresponding sites of the pET32a vector (Novagen), allowing the production of each MAPK with a thioredoxin tag at the N terminus and a His 6 tag at the C terminus. The open reading frame of MEK DD , a constitutively active form of StMEK1 (23), was amplified by PCR with NcoI and HindIII sites attached to the 5Ј-and 3Ј-ends, respectively, and cloned into the corresponding sites of pET32a (Novagen), allowing the production of MEK DD with a thioredoxin tag, a His 6 tag, and an S⅐tag at the N terminus. The resulting constructs were used to transform Escherichia coli strain BL21(DE3) (Novagen). The conditions used to induce expression of recombinant proteins were as follows: SIPK, (S/W)IPK, SIPK N , and SIPK NN , 0.1 mM isopropyl ␤-D-thiogalactopyranoside (IPTG) at 26°C for 3 h; WIPK, 0.05 mM IPTG at 20°C for 20 h; (W/S)IPK, 0.01 mM IPTG at 20°C for 20 h; and MEK DD , 0.5 mM IPTG at 26°C for 3 h. Purification of His 6tagged proteins with a nickel-Sepharose column (Amersham Biosciences) was performed according to the manufacturer's recommendations. Purified proteins were desalted on a PD-10 desalting column (Amersham Biosciences) equilibrated with 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl and concentrated using a Microcon YM-30 concentrator (Amicon, Inc.).
The autophosphorylation and myelin basic protein (MBP) phosphorylation activities of SIPK and WIPK were assayed as follows. Purified recombinant SIPK and WIPK (0.4 g each) were incubated at 25°C for 15 min in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 , 0.25 mg/ml MBP, 50 M ATP, and 50 Ci/ml [␥-32 P]ATP. The reactions were terminated by the addition of SDS sample buffer. Phosphorylated SIPK, WIPK, and MBP were visualized by autoradiography after separation on a 15% SDS-polyacrylamide gel.
Phosphorylation of SIPK by MEK DD -Purified SIPK (50 g/ml) and MEK DD (10 g/ml) were incubated at 30°C for 30 min in buffer containing 20 mM HEPES-KOH (pH 7.5), 10 mM MgCl 2 , 1 mM dithiothreitol, and 500 M ATP. Two-hundred microliters of 50% S protein-agarose (Novagen) was added to the reaction mixture to remove MEK DD . The mixture was rotated at room temperature for 30 min, and the agarose beads were removed by brief centrifugation. The resulting supernatant containing phosphorylated SIPK was concentrated using a Microcon YM-30 concentrator, mixed with an equal volume of glycerol, and stored at Ϫ80°C.
Inactivation of SIPK by NtMKP1 Produced by in Vitro Transcription/ Translation-The open reading frame of NtMKP1 was fused at its N terminus to a FLAG epitope (FLAG-MKP1) and cloned into the in vitro translation vector pTNT (Promega). In vitro transcription/translation was performed using a TNT wheat germ extract kit (Promega) at 30°C for 2 h according to the manufacturer's recommendations. In vitro translated products were diluted with buffer A (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Triton X-100) containing 1 mM EGTA and 1ϫ Complete (Roche Applied Science); 2 l of anti-FLAG antibody M2 (Sigma) was added; and the mixture was incubated at 4°C for 1 h. Then, 20 l of protein A-Sepharose was added, and the reaction mixture was rotated at 4°C for 1 h. Note that anti-FLAG antibody M2 is isotype 1 of mouse immunoglobulin G and binds protein G stronger than protein A. However, protein G-Sepharose had greater nonspecific binding in our assay system. Therefore, we used protein A-Sepharose to precipitate anti-FLAG antibody M2. The beads were collected by brief centrifugation and washed three times with 1 ml of buffer A containing 1 mM EGTA and twice with buffer B (20 mM Tris-HCl (pH 7.5) and 1 mM dithiothreitol). Then, the beads were suspended in buffer B containing 5 g/ml phosphorylated SIPK and incubated at ambient temperature with continuous agitation. At specific times, reaction mixtures were flash-centrifuged, and supernatants containing SIPK were removed. The kinase activities of SIPK were assayed at ambient temperature for 10 min with 0.25 mg/ml MBP, 50 M ATP, and 5 Ci/ml [␥-32 P]ATP. The reactions were terminated by the addition of SDS sample buffer. The phosphorylation of MBP was analyzed by autoradiography after separation on a 15% SDS-polyacrylamide gel. To investigate the effect of CaM, 1 M NtCaM1, NtCaM3, or NtCaM13 was added to the reaction mixture in the presence (100 M CaCl 2 ) or absence (5 mM EGTA) of Ca 2ϩ .
Interaction of FLAG-MKP1 with NtCaM1-FLAG-MKP1 was produced by in vitro transcription/translation and purified. FLAG-MKP1 bound to Sepharose was suspended in 0.5 M NtCaM1 in buffer A containing 100 M CaCl 2 or 1 mM EGTA and rotated at 4°C for 1 h. The beads were collected by brief centrifugation and washed three times with 1 ml of buffer A containing 100 M CaCl 2 or 1 mM EGTA. The beads were suspended in SDS sample buffer and used for immunoblot analyses with anti-NtCaM1 or anti-NtMKP1 antibody, respectively. The CaM overlay assay was performed as described previously (19), but 1 g/ml alkaline phosphatase-labeled NtCaM1 was used.
Production of Recombinant NtMKP1 Proteins in E. coli-The cDNA fragments corresponding to residues 1-114 and 115-361 of NtMKP1 were amplified by PCR with BamHI and XhoI sites attached to the 5Јand 3Ј-ends, respectively, and cloned into the corresponding sites of pGEX-4T-1, resulting in glutathione S-transferase (GST)-MKP1-(1- , and GST-MKP1-(468 -862) were expressed and purified as described previously (19). The inactivation of SIPK by GST-MKP1 derivatives bound to glutathione-Sepharose was performed similarly to the inactivation by FLAG-MKP1.
Production and Purification of Antibody and Immunoblot Analyses-Polyclonal antiserum was raised in rabbits using GST-MKP1-(1-114) as an antigen. Purification of anti-NtMKP1 antibody was performed as follows. The cDNA fragment corresponding to residues 1-114 of NtMKP1 was amplified by PCR with XhoI sites attached to the 5Ј-and 3Ј-ends and cloned into the corresponding sites of the pET15b vector, allowing the production of N-terminally His 6 -tagged NtMKP1-(1-114). The resulting construct was used to transform E. coli strain BL21(DE3) (Novagen). The recombinant protein was induced to express with 1 mM IPTG at 37°C for 3 h and purified with a nickel-Sepharose column according to the manufacturer's recommendations. Purified His 6tagged NtMKP1-(1-114) protein (1-2 mg) was coupled to an N-hydroxysuccinimide-activated Sepharose column (Amersham Biosciences). Anti-NtMKP1 antiserum was applied to the column and washed extensively with 20 mM Tris-HCl (pH 7.5), 1 M NaCl, and 1% Triton X-100. Bound antibodies were eluted with 0.1 M sodium citrate (pH 3) and immediately neutralized.
Phosphatase Assay Using 3-O-Methylfluorescein Phosphate (OMFP) as a Substrate-The phosphatase activity of GST-MKP1 was assayed at 30°C in 100 l of reaction buffer containing 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, and 500 M OMFP. The amount of 3-O-methylfluorescein was determined from the absorbance at 477 nm. The nonenzymatic hydrolysis of OMFP was corrected by measuring the control reaction without enzyme. To investigate the effect of CaM, EDTA was omitted from the reaction, and recombinant NtCaM1, NtCaM3, or NtCaM13 was added to the reaction in the presence (100 M CaCl 2 ) or absence (5 mM EGTA) of Ca 2ϩ .
Agroinfiltration-For agroinfiltration experiments, the open reading frames of SIPK, FLAG-MEK DD , and FLAG-MKP1 derivatives were cloned into the multiple cloning sites of the binary vector pEl2⍀ (26). Various mutations were generated using a Mutan-Super Express Km kit (Takara) and confirmed by sequencing. The constructs were used to transform Agrobacterium tumefaciens strain C58C1 by electroporation. Agrobacterium cells were cultured; harvested by centrifugation; suspended in 10 mM MES-NaOH (pH 5.6), 10 mM MgCl 2 , and 150 M acetosyringone to A 600 ϭ 1.0; incubated at room temperature for 2 h; and infiltrated into leaves of N. benthamiana (4 -5 weeks old) using a syringe without a needle.
For simultaneous expression, Agrobacterium cells carrying the vector control or FLAG-MKP1 derivatives were mixed with 0.0526 volume of Agrobacterium cells carrying FLAG-MEK DD immediately before infil-tration. Under this condition, accumulation of MEK DD was still three to four times higher than that of MKP1 as judged by immunoblot analysis with anti-FLAG antibody. The activity of SIPK was measured 2 days after infiltration by immunocomplex kinase assay as described previously (21).
Precipitation of SIPK by GST-fused NtMKP1 Derivatives-Total protein was extracted from N. benthamiana leaves infiltrated with Agrobacterium cells carrying SIPK 2 days after infiltration as described previously (21). Total protein was mixed with GST alone or GST-MKP1 derivatives bound to glutathione-Sepharose and rotated overnight at 4°C. The beads were washed three times with 1 ml of buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, and 0.5% Triton X-100. Precipitated proteins were denatured with SDS sample buffer and used for immunoblotting.
Construction of (S/W)IPK, (W/S)IPK, SIPK N , and SIPK NN -(S/W)IPK and (W/S)IPK were constructed as reported by Zhang and Liu (27). A PstI site introduced into the WIPK gene does not change the amino acid sequence. Various mutations were generated using the Mutan-Super Express Km kit and confirmed by sequencing.

NtMKP1 Inactivates SIPK via Dephosphorylation of the Conserved TEY Motif in Vitro-
To determine the phosphatase activity of NtMKP1, we prepared phosphorylated SIPK as a substrate of NtMKP1. Recombinant SIPK and MEK DD , a constitutively active form of StMEK1 MAPKK that activates SIPK in N. benthamiana (23,28,29), were expressed in E. coli and purified by nickel affinity column chromatography. SDS-PAGE analysis followed by Coomassie Brilliant Blue staining of purified fractions detected a single band on the gels (Fig. 1A). Phosphorylation of SIPK by MEK DD enhanced the activity of SIPK for MBP by ϳ50 -100fold. After the reaction, MEK DD was removed by S protein-Sepharose. Recombinant NtMKP1 was produced as an N-terminal FLAG fusion protein (FLAG-MKP1) by in vitro transcription/translation using wheat germ extract. The production of FLAG-MKP1 was confirmed by immunoblot analyses using antibodies against NtMKP1 and FLAG. Both antibodies recognized a protein of ϳ100 kDa, which corresponds to the predicted molecular mass of FLAG-MKP1 (96.6 kDa) (Fig. 1B). FLAG-MKP1 was purified from wheat germ extract by immunoprecipitation using anti-FLAG antibody and protein A-Sepharose, and the beads were washed with buffer containing EGTA to remove wheat germ CaM. The Sepharose beads attached to FLAG-MKP1 were suspended in buffer containing phosphorylated SIPK and incubated with gentle agitation. At specific times, FLAG-MKP1 bound to the beads was removed by brief centrifugation, and the kinase activity of the supernatant containing SIPK was measured using MBP as a substrate. As shown in Fig. 1C, SIPK activity was decreased by incubation with FLAG-MKP1 within 15 min, whereas the amount of SIPK protein was constant, demonstrating that NtMKP1 inactivates SIPK in vitro.
The activity of MAPK is strictly correlated with the phosphorylation of the conserved TXY motif. SIPK activity is also regulated by the phosphorylation of the TEY motif (27). We investigated whether NtMKP1 inactivates SIPK via dephosphorylation of the conserved TEY motif by conducting an immunoblot analysis using anti-phospho-p44/42 MAPK antibody, which specifically recognizes the phosphorylated form of the TEY motif of mammalian ERK MAPK. Although this antibody has been used to monitor the phosphorylation of SIPK (30 -32), we first confirmed the specificity of the antibody in an immunoblot analysis. As shown in Fig. 2A, anti-phospho-p44/42 MAPK antibody recognized SIPK phosphorylated by MEK DD , but not non-phosphorylated SIPK. We then investigated whether FLAG-MKP1 dephosphorylates the TEY motif of SIPK in vitro. The phosphorylation of the conserved TEY motif of SIPK decreased upon incubation with FLAG-MKP1, whereas the amount of SIPK protein was constant (Fig. 2B). Collectively, these results indicate that NtMKP1 inactivates SIPK via dephosphorylation of its TEY motif.
Calmodulin Binds but Does Not Activate FLAG-MKP1-In a previous study, we identified NtMKP1 as a CaM-binding protein (19). We first confirmed the interaction of FLAG-MKP1 with NtCaM1. NtCaM1 was mixed with FLAG-MKP1 bound to Sepharose beads in the presence or absence of Ca 2ϩ . Precipitated NtCaM1 was detected by immunoblot analysis using antibody against NtCaM1. As shown in Fig. 3A, NtCaM1 was precipitated by FLAG-MKP1 in a Ca 2ϩ -dependent manner. Previously, we showed that Trp 440 and Leu 443 , but not Gly 450 , are essential for the GST-fused CaM-binding domain of NtMKP1 to bind CaM (19). We investigated whether these amino acids are also critical for full-length NtMKP1 to bind CaM. The FLAG-MKP1 W440R, L443R, and G450R recombinant proteins, in which Trp 440 , Leu 443 , and Gly 450 were converted to Arg, respectively, were produced by in vitro transcription/ translation and purified by immunoprecipitation. Ligand binding assay using alkaline phosphatase-labeled NtCaM1 showed that FLAG-tagged A, recombinant proteins of SIPK and MEK DD were produced in E. coli and purified by nickel affinity chromatography. The purified proteins (0.5 g) were separated on an SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue (CBB). The positions of molecular mass markers in kilodaltons are indicated on the left. B, FLAG-MKP1 was produced by in vitro transcription/translation using wheat germ extracts. The production of FLAG-MKP1 was confirmed by immunoblot analyses using anti-FLAG antibody (␣-FLAG) or anti-Nt-MKP1 antibody (␣-MKP1). C, FLAG-MKP1 was purified by immunoprecipitation with anti-FLAG antibody and protein A-Sepharose. The precipitated beads were suspended in buffer containing 5 g/ml phosphorylated SIPK and incubated at ambient temperature with continuous agitation. At the indicated times, the beads were removed by flash centrifugation. The kinase activity of SIPK was assayed with MBP as a substrate, and SIPK protein was detected by immunoblot analysis with anti-SIPK antibody (␣-SIPK). The production of FLAG-MKP1 protein was confirmed by immunoblot analysis with anti-NtMKP1 antibody.

FIGURE 2. NtMKP1 dephosphorylates the conserved TEY motif of SIPK in vitro.
A, the specificity of anti-phospho-p44/42 MAPK antibody was confirmed by immunoblot analyses with non-phosphorylated and phosphorylated SIPK (p-SIPK). B, FLAG-MKP1 was purified by immunoprecipitation with anti-FLAG antibody and protein A-Sepharose. The precipitated beads were suspended in buffer containing 5 g/ml phosphorylated SIPK and incubated at ambient temperature with continuous agitation. At the indicated times, the beads were removed by flash centrifugation. The amounts of phosphorylated SIPK and total SIPK protein were determined by immunoblot analyses with anti-phospho-p44/42 MAPK antibody (␣-pTEpY) and anti-SIPK antibody (␣-SIPK), respectively. The production of FLAG-MKP1 protein was confirmed by immunoblot analysis with anti-NtMKP1 antibody (␣-MKP1).
As one well known and characterized effect of CaM binding to targeted proteins is activation of the enzymatic activity of the proteins (33,34), we investigated the effect of NtCaM1 binding on the phosphatase activity of FLAG-MKP1. However, we found no significant activation of FLAG-MKP1 upon the addition of not only NtCaM1, but also NtCaM3 and NtCaM13 (up to 1 M), 4 which are different types of tobacco CaMs from NtCaM1 (35). As most target proteins are significantly activated by CaM at nanomolar levels (33,35), and 1 M NtCaM1 was sufficient to detect interaction between NtCaM1 and NtMKP1 (Fig. 3A), CaM binding might have function other than enzymatic activation of NtMKP1.
Binding to SIPK Activates the Phosphatase Activity of NtMKP1-In addition to a catalytic domain, mammalian MKPs have a substratebinding domain at their N termini (5). In Fig. 4C, deletion of the N-terminal non-catalytic region (residues 1-114) or the GH domain (residues 277-361) of GST-MKP1-(1-361) reduced the phosphatase activity despite the presence of an intact catalytic domain. This result raised the possibility that the N-terminal non-catalytic region or the GH domain binds SIPK. Therefore, the SIPK-binding domain of NtMKP1 was mapped by coprecipitation assay with total protein extracts prepared from tobacco N. benthamiana leaves overexpressing the SIPK gene. SIPK was precipitated by GST-MKP1-(1-361), but not by GST-MKP1-(362-467), GST-MKP1-(468 -862), or GST alone (Fig. 5A, left  panel). Further deletion analysis confirmed that the N-terminal 114 amino acids of NtMKP1 were required and sufficient to bind and precipitate SIPK (Fig. 5A, right panel). These results indicate that the N-terminal non-catalytic region of NtMKP1 binds SIPK. However, the involvement of the C-terminal domain in the binding to SIPK cannot be ruled out because GST-MKP1-(468 -862) was highly unstable in the extracts despite the presence of a protease inhibitor mixture (compare before (Fig. 5A, left panel, lane 5) and after (lane 4) incubation with the extracts).
We tested whether NtMKP1 is activated by the binding of its substrate SIPK using an artificial substrate. Although p-nitrophenyl phosphate is the most commonly used artificial substrate of MKPs, we found no significant dephosphorylation of p-nitrophenyl phosphate by NtMKP1 (19). It has been reported that MKPs prefer bulky polycyclic aryl phosphates such as OMFP (36). Naoi and Hashimoto (16) reported that PHS1, an Arabidopsis MKP involved in microtubule organization, dephosphorylates OMFP, but not p-nitrophenyl phosphate. Therefore, the activity of NtMKP1 was assayed using OMFP as a substrate. We found no significant activity of FLAG-MKP1, again probably because only a small amount of FLAG-MKP1 protein was produced by in vitro transcription/translation. Then, we used partially purified GST-MKP1 as another source of the phosphatase. As shown in Fig. 5B, the partially purified fraction of GST-MKP1 contains a major band of 125 kDa (indicated by the arrowhead), which corresponds to the predicted molecular mass of GST-MKP1 (122.7 kDa). As expected, GST-MKP1 dephosphorylated OMFP in a time-and concentration-dependent manner (Fig.  5B). Moreover, the dephosphorylation of OMFP by GST-MKP1 was significantly increased by the addition of recombinant SIPK in a dosedependent manner (Fig. 5C). This effect was independent of the kinase activity of SIPK because the reaction mixture contained EDTA, which chelates Mg 2ϩ and inhibits kinase activity. We analyzed the effect of CaM binding on the OMFP phosphatase activity of GST-MKP1 in the presence or absence of SIPK, but found no significant change in the dephosphorylation activity of GST-MKP1 (Fig. 5D). As purified GST-MKP1-(1-361) also dephosphorylated OMFP (Fig. 5E) in a similar manner, possible dephosphorylation by other proteins can be ruled out. The OMFP phosphatase activity of GST-MKP1-(115-361), which could not inactivate SIPK (Fig. 4B), was also analyzed. Interestingly, purified GST-MKP1-(115-361) dephosphorylated OMFP more effectively than did purified GST-MKP1-(1-361) (Fig. 5E). This result further indicates that the N-terminal non-catalytic region of NtMKP1 is involved in the binding to SIPK, but is not essential for phosphatase activity.
Differential Activation of NtMKP1 by SIPK and WIPK-WIPK is another stress-inducible tobacco MAPK, and some forms of stress, including pathogen-induced cell death, induce activation of both WIPK and SIPK (22). WIPK is highly similar to SIPK in primary structure, and 4 S. Katou and Y. Ohashi, unpublished data.  MKP1). B, recombinant proteins of FLAGtagged WT MKP1 and mutants W440R, L443R, and G450R were produced by in vitro transcription/ translation. They were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with alkaline phosphatase-labeled NtCaM1 (AP-NtCaM1). A parallel membrane blot was subjected to immunoblot analysis with anti-NtMKP1 antibody to verify that similar amounts of FLAG-MKP1 proteins were used. they share the same upstream MAPKK, NtMEK2 (37). However, their activities are regulated differentially at both the transcriptional and post-translational levels (27,38). We tested whether WIPK also activates OMFP hydrolysis of GST-MKP1. The ability of recombinant SIPK and WIPK to activate the OMFP phosphatase activity of GST-MKP1 was compared (Fig. 6A). WIPK also stimulated the dephosphorylation of OMFP by GST-MKP1 in a dose-dependent manner, but its effect was weaker than that of SIPK (Fig. 6C). Because WIPK could phosphorylate more WIPK itself and MBP than did SIPK (Fig. 6B), the possibility that the WIPK protein is folded improperly and is unable to activate NtMKP1 was ruled out. To investigate the role of the CaM-binding and C-terminal domains of NtMKP1 in the catalytic activation of NtMKP1 by SIPK and WIPK, the effect of SIPK and WIPK on the OMFP phosphatase activity of GST-MKP1-(1-361) was analyzed. Like GST-MKP1, the OMFP phosphatase activity of GST-MKP1-(1-361) was activated strongly by SIPK and weakly by WIPK in a dose-dependent manner (Fig.  6D). These results indicate that the CaM-binding and C-terminal domains are dispensable for the catalytic activation of NtMKP1 by SIPK and WIPK. As the expression of GST-MKP1 in E. coli is very weak, we used GST-MKP1-(1-361) in the subsequent experiments.

The Putative Common Docking Domain of SIPK Is Partially Responsible for Activation of NtMKP1 by SIPK-SIPK and WIPK share 72%
identity in their amino acid sequences. SIPK is slightly larger than WIPK because of its N-terminal extension (Fig. 7A), and a major difference between SIPK and WIPK exists in the N terminus. To determine whether the N-terminal extension of SIPK is responsible for the strong activation of GST-MKP1-(1-361), two chimeras, (S/W)IPK and (W/S)IPK, were constructed as reported by Zhang and Liu (27). The (S/W)IPK construct has the SIPK N terminus fused to the WIPK C terminus, whereas (W/S)IPK construct has the WIPK N terminus fused to the SIPK C terminus (Fig. 7A). The ability of the recombinant proteins produced to activate the OMFP phosphatase activity of GST-MKP1-(1-361) was compared (Fig. 7B). As shown in Fig. 7C, (W/S)IPK stimulated the dephosphorylation of OMFP by GST-MKP1-(1-361) like SIPK, whereas (S/W)IPK acted like WIPK. This result suggests that the C terminus of SIPK determines the strong activation of NtMKP1 phosphatase activity by SIPK.
It has been demonstrated that the C-terminal acidic motif of mammalian MAPKs, known as the common docking (CD) domain, is responsible for the interaction with the upstream MAPKK and sub- strates as well as negative regulator MKPs (39). The CD domain has two invariable acidic amino acids that are also conserved in plant MAPKs. A Drosophila melanogaster sevenmaker gain-of-function mutant, in which the second invariable acidic amino acid (Asp) of the CD domain is converted to Asn, shows significantly reduced sensitivity to MKP (40,41). SIPK also has a putative CD domain, in which Asp 350 and Asp 352 correspond to invariable acidic amino acids. To investigate the importance of these acidic amino acids, either the second or both Asp residues of SIPK were replaced with Asn, resulting in SIPK N and SIPK NN , respectively (Fig. 7A). Recombinant proteins were then produced (Fig. 7D), and their ability to activate the OMFP phosphatase activity of GST-MKP1-(1-361) was compared. Both SIPK N and SIPK NN stimulated the dephosphorylationofOMFPbyGST-MKP1-(1-361)inaconcentrationdependent manner (Fig. 7E), although their efficiency was about a half that of SIPK.
The N-terminal Non-catalytic Region, but Not CaM Binding, Is Required for NtMKP1 to Inactivate SIPK in Vivo-Transient expression of MEK DD , a constitutively active form of StMEK1 MAPKK, in tobacco N. benthamiana induces cell death probably through activation of endogenous SIPK (23,(27)(28)(29). Because NtMKP1 inactivates SIPK in vitro (Fig. 1C) and its overexpression compromises wound-induced activation of SIPK (19), we expected the simultaneous expression of NtMKP1 to compromise MEK DD -induced cell death. Agrobacterium cells carrying FLAG-MEK DD were mixed with Agrobacterium cells carrying either the vector control or FLAG-MKP1 and then infiltrated into N. benthamiana leaves (agroinfiltration). As expected, expression of WT MKP1 compromised both the cell death (Fig. 8C) and SIPK activation (Fig. 8B, lane 3) induced by MEK DD . In contrast, expression of MKP1 C201S, in which the putative catalytically essential Cys residue was replaced with Ser, failed to suppress either response (Fig. 8, B, lane  13; and F), suggesting the significance of the phosphatase activity of NtMKP1. To investigate the significance of the N-terminal non-catalytic region of NtMKP1 and CaM binding in vivo, we first compared protein accumulation of NtMKP1 derivatives in N. benthamiana. FLAG-MKP1 derivatives were transiently expressed in N. benthamiana by agroinfiltration, and their accumulation was detected by immunoblot analysis with anti-NtMKP1 antibody. Compared with WT MKP1, the level of MKP1 L443R was slightly but reproducibly low (Fig. 8A, lane

5)
, whereas the level of MKP1 W440R was quite low. 4 Therefore, we used MKP1 L443R to investigate the importance of CaM binding. In addition to bands of the predicted sizes, both anti-FLAG and anti-Nt-MKP1 antibodies detected multiple bands in extracts from leaves expressing WT MKP1, MKP1 L443R, MKP1 C201S, and MKP1⌬N (Fig.   8A, asterisks), suggesting that NtMKP1 is an unstable protein. In contrast, the level of MKP1-(1-361) was much higher than that of WT MKP1, and a single band of the predicted size was detected by immunoblot analysis (Fig. 8A, lane 3). Because anti-NtMKP1 antibody was raised against the N-terminal 114 amino acids of NtMKP1, a parallel blot was subjected to immunoblot analysis with anti-FLAG antibody to detect MKP1⌬N, which lacks the N-terminal 114-amino acid non-catalytic region. As reported previously (42), anti-FLAG antibody detected a nonspecific band, which could be used as a reference for the loading of each lane, but migrated to the same position as full-length NtMKP1 (Fig.  8A, black arrowhead). MKP1⌬N accumulated to a similar level compared with WT MKP1 (Fig. 8A, lane 10).
Like WT MKP1, MKP1 L443R compromised the MEK DD -induced cell death and activation of SIPK (Fig. 8, B, lane 7; and E). Moreover, MKP1-(1-361) also suppressed both (Fig. 8, B, lane 5; and D), suggesting that CaM binding is dispensable for the function of NtMKP1 in vivo. In contrast, no significant difference in the timing and extent of MEK DD -induced cell death was observed between MKP1⌬N and the vector control (Fig. 8, G-I). SIPK activity in leaves expressing MKP1⌬N or MKP1 C201S was reproducibly higher than that in leaves expressing the vector control (Fig. 8B, lanes 9 and 13), probably due to some dominant-negative effects of MKP1⌬N and MKP1 C201S against endogenous NtMKP1. These results indicate that the N-terminal non-catalytic region, but not CaM binding, is required for the function of NtMKP1 in vivo. Expression of MEK DD was confirmed by immunoblot analysis with anti-FLAG antibody (Fig. 8B, lower panels). We found a change in the mobility of MEK DD upon SDS-PAGE that shows a strict correlation with the activation of SIPK; when SIPK was activated, the mobility of MEK DD was decreased. We observed phosphorylation of MEK DD by SIPK when SIPK was phosphorylated and activated by MEK DD . 4 Therefore, the mobility shift of MEK DD was likely caused by phosphorylation by activated SIPK.
Identification of Amino Acids in the N-terminal Non-catalytic Region of NtMKP1 Essential for Interaction with SIPK-In mammalian MKPs, a cluster of three or four basic amino acids (Arg and Lys) at the N terminus is involved in the interaction with substrate MAPKs (5). PHS1, an Arabidopsis MKP involved in microtubule organization, also has such a cluster at its N terminus (16). However, NtMKP1 has no such cluster in its N-terminal non-catalytic region, suggesting that a novel MAPKinteracting motif exists in NtMKP1. To identify the SIPK-interacting motif of NtMKP1, we first prepared deletion constructs of NtMKP1 (Fig. 9A) and tested their ability to suppress MEK DD -induced cell death. MKP1-(39 -862) suppressed cell death, whereas like MKP1⌬N, MKP1-(82-862) did not (Fig. 9A), indicating that the SIPK-interacting motif of NtMKP1 exists between amino acids 39 and 82. Alignment of amino acid sequences of the N-terminal non-catalytic region of NtMKP1 and its putative orthologs from other plant species revealed conservation of three positively charged amino acids: Lys 41 , Arg 43 , and Arg 56 . To determine whether these amino acids are required for interaction with SIPK, we prepared NtMKP1 mutants in which one or two of them were converted to Ala (Fig. 9A). We also converted Arg 15 , Lys 16 , Lys 105 , and Lys 106 , which are also conserved positively charged amino acids, to Ala as a negative control (Fig. 9A). As shown in Fig. 9 (C-G), the K41A/ R43A mutation specifically abolished the ability of NtMKP1 to suppress the MEK DD -induced cell death and activation of SIPK, although mutations did not change the stability of NtMKP1 proteins (Fig. 9B). In addition to the K41A/R43A mutation, the R56A mutation slightly decreased the ability of NtMKP1 to suppress MEK DD -induced responses (Fig. 9, C and F). However, MKP1 R56A clearly repressed MEK DD -induced cell death more effectively than the vector control, whereas we found no significant difference between the vector control and MKP1 K41A/R43A, 4 suggesting that Lys 41 and Arg 43 are major and essential SIPK-interacting sites of NtMKP1. Immunoblot analysis with anti-FLAG antibody confirmed the expression of MEK DD (Fig. 9C, lower  panel). The decreased mobility of MEK DD upon SDS-PAGE again correlated with the activation of SIPK (Fig. 9C, lower panel). The effect of mutations on the interaction of the N-terminal non-catalytic region of NtMKP1 with SIPK was investigated by coprecipitation analysis with total protein extracts prepared from N. benthamiana leaves overexpressing the SIPK gene. Consistent with the effect on the cell death-suppressing activity of FLAG-MKP1 (Fig. 9, D-G), the K41A/R43A mutation abolished the interaction of GST-MKP1-(1-114) with SIPK, whereas the R56A mutation decreased it (Fig. 9H, upper panel), indicating that Lys 41 and Arg 43 are essential for NtMKP1 to interact with SIPK. The direct interaction of GST-MKP1-(1-114) derivatives with purified recombinant SIPK (Fig. 1A) was investigated. Although the interaction of GST-MKP1-(1-114) with recombinant SIPK was greatly diminished by the K41A/R43A mutation, it was still detected (Fig. 9H, middle  panel). These results indicate that Lys 41 and Arg 43 of NtMKP1 are major SIPK-interacting sites and are required for high affinity binding to SIPK.

DISCUSSION
Catalytic Activation of NtMKP1 by SIPK but Not by CaM-In this study, we have demonstrated that NtMKP1 is an active phosphatase that inactivates its physiological substrate SIPK by dephosphorylating the conserved TEY motif of SIPK ( Figs. 1 and 2). We further showed that the catalytic activity of NtMKP1 was increased by the binding of SIPK, but not by CaM (Fig. 5, C and D), showing for the first time that substrate-triggered activation is an evolutionarily conserved mechanism that regulates the activity of mammalian and plant MKPs. The N-terminal non-catalytic region of NtMKP1 was found to interact with SIPK and to be essential for the inactivation of SIPK both in vitro and in vivo (Figs. 4, 5, and 8). The interaction between mammalian MKPs and their substrate MAPKs is mediated via the interaction between a cluster of basic amino acids at the N terminus of MKP and two invariable acidic amino acids within the CD domain located at the C terminus of MAPK (5). However, NtMKP1 has no such cluster in its N-terminal non-catalytic region. Alternatively, we identified Lys 41 and Arg 43 as important amino acids of NtMKP1 for interaction with and inactivation of SIPK ( Fig. 9, C-H). These amino acids are well conserved among putative NtMKP1 orthologs of other plant species and therefore represent a novel MAPK-docking site of MKP. On the other hand, the CD domain and two invariable acidic amino acids are conserved in SIPK. However, mutation of both invariable acidic amino acids of SIPK only partially compromised the activation of MKP1 by SIPK (Fig. 7E). Zhang and Liu (27) showed by transient expression that such a mutation of SIPK does not enhance its activity, but rather weakens it. It is therefore likely that a novel mechanism underlies the interaction of SIPK with NtMKP1.
The activation of SIPK, as well as the accumulation of NtMKP1 mRNA, is induced by both wounding and a hypersensitive reaction against tobacco mosaic virus infection (19,43). However, the accumulation of NtMKP1 mRNA is much delayed compared with the activation of SIPK (19,43). Similarly, activation of AtMPK6 (an Arabidopsis SIPK ortholog) is rapidly induced by UVC treatment, whereas the AtMKP1 mRNA level remains constant (17). These results suggest the importance of post-transcriptional regulation of plant MKPs. The catalytic activation of NtMKP1 by its substrate MAPK shown in this study may represent such a regulatory mechanism of NtMKP1.
The catalytic activity of NtMKP1 was increased strongly by the binding of SIPK, but weakly by the binding of WIPK (Fig. 6). AtMKP1 was also shown to interact strongly with AtMPK6 (an Arabidopsis SIPK ortholog), but weakly with AtMPK3 (an Arabidopsis WIPK ortholog) (17). In mammalian MKPs, it has been shown that substrate-triggered activation plays an important role in determining substrate specificity at the subfamily level (6 -9). Previously, we found that wound-induced activation of SIPK is more frequently compromised than that of WIPK in NtMKP1-overexpressing plants, although wounding induces much stronger activity of SIPK compared with WIPK (19). This correlation suggests that substrate-triggered activation seems to play an important role in determining the substrate specificity of NtMKP1. However, WIPK activity was suppressed to a similar extent as SIPK activity in one line of our NtMKP1-overexpressing plants (19). Several lines of evidence show that SIPK and WIPK activities are not independent. SIPK and WIPK share the same upstream MAPKK, NtMEK2, and silencing of the SIPK gene by RNA interference leads to enhanced activation of WIPK by ozone (32,37). Therefore, SIPK and WIPK activities should be carefully compared. The correlation of substrate-triggered activation with substrate specificity of mammalian MKPs has been shown at the subfamily level (ERK, JNK, and p38). In contrast, most plant MAPKs, including SIPK and WIPK, are the ERK type. It would be interesting to investigate whether substrate-triggered activation plays a role in deter-mining the substrate specificity of MKP within the same MAPK subfamily. We will investigate the correlation between the substrate-triggered activation and substrate specificity of NtMKP1 using tobacco MAPKs, including SIPK and WIPK.
Role of the CaM-binding and C-terminal Domains of NtMKP1-Nt-MKP1 was identified as a CaM-binding protein, but our results demonstrated that CaM binding was not essential for NtMKP1 to inactivate SIPK in vitro or in vivo (Figs. 3, 4, and 8). Moreover, neither the SIPKinactivating activity nor the OMFP phosphatase activity of NtMKP1 was stimulated by the addition of CaM (Fig. 5). 4 The cell death-suppressing activity of NtMKP1 derivatives seems to correlate with their protein expression level. The level of NtMKP1 L443R protein was slightly but reproducibly lower than that of NtMKP1, and its cell deathsuppressing activity was weaker than that of NtMKP1 (Fig. 8). These results suggest that CaM binding might not stimulate NtMKP1 activity. Nevertheless, we cannot rule out the possibility that CaM binding activates NtMKP1 because it has been reported that the native forms of catalase and NADK2 are activated by CaM, but the recombinant forms are not (44,45). In contrast to NtMKP1, a recent report showed that the phosphatase activity of DsPTP1 is regulated by CaM binding (20). Interestingly, DsPTP1 activity for p-nitrophenyl phosphate is stimulated by CaM, whereas that for tyrosine-phosphorylated MBP is inhibited by CaM (20). This result suggests that the effect of CaM binding may depend on the type of substrate. It is also of interest whether the phosphatase activity of DsPTP1 is activated not only by CaM, but also by its substrate MAPK(s).
NtMKP1 and its putative orthologs from other plant species have a long C-terminal domain, which is not present in most mammalian MKPs. The C-terminal domain is less conserved compared with the N-terminal domain, but the serine-rich region is a conserved feature of the C-terminal domain. In human MKPs, only VH5 and MKP7 have a C-terminal stretch containing PEST sequences (5). PEST sequences are abundant in Pro, Glu, Ser, and Thr residues and are frequently found in unstable proteins (46). In addition to PEST sequences, MKP7 has a serine-rich region in its C-terminal stretch, and deletion of the C-terminal stretch stabilizes MKP7 (9). These results suggest that the C-terminal stretch of mammalian MKPs is involved in protein stability. Although no apparent PEST sequence was found in the C-terminal domain of NtMKP1, our results suggest that the C-terminal domain also contributes to the stability of the NtMKP1 protein. Immunoblot analysis of N-terminally FLAG-tagged NtMKP1 expressed in N. benthamiana detected multiple truncated proteins (Fig. 8A). Like MKP7, the level of NtMKP1-(1-361), which lacks the C-terminal domain, was much higher than that of full-length NtMKP1 (Fig. 8A). The GST-fused C-terminal domain was unstable in the protein extracts from N. benthamiana (Fig. 5A). Some mammalian MKPs are phosphorylated by MAPK, and such phosphorylation regulates degradation of MKPs by the proteasome (47,48). In addition, one effect of CaM binding to targeted proteins is stabilization of the proteins (49). Therefore, the degradation mechanism of NtMKP1 in relation to phosphorylation and CaM binding should be a subject of further analysis.
In this study, we have demonstrated that NtMKP1 is an active phosphatase that inactivates SIPK. Although NtMKP1 is quite different from mammalian MKPs in primary structure, the catalytic activity of NtMKP1 was significantly increased by substrate MAPKs, similar to mammalian MKPs. Our results indicate that the activities of mammalian and plant MKPs are regulated, at least in part, by an evolutionarily conserved mechanism. On the other hand, CaM binding is a unique feature of plant MKPs, although the role of CaM binding to NtMKP1 is still unclear. By elucidating the regulatory mechanism of MKP, an important negative regulator of MAPK, we will understand how the activity of MAPK is regulated.