Biochimie 108 (2015) 13e19
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Research paper
The activity of the wheat MAP kinase phosphatase 1 is regulated by manganese and by calmodulin nie Robe c, d, Benoit Ranty c, d, Christian Mazars c, d, Mouna Ghorbel a, c, Ikram Zaidi a, Euge Jean-Philippe Galaud c, d, **, Moez Hanin a, b, * a
Laboratory of Plant Protection and Improvement, Center of Biotechnology of Sfax, BP1177, 3018 Sfax, Tunisia University of Sfax, Institute of Biotechnology, BP “1175”, 3038 Sfax, Tunisia Universit e de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences V eg etales, BP 42617, F-31326 Castanet-Tolosan, France d CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 October 2014 Accepted 15 October 2014 Available online 30 October 2014
MAPK phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes and play key roles in the regulation of different cellular processes. However in plants, little is known about the regulation of these Dual Specific Phosphatases (DSPs) by Ca2þ and calmodulin (CaM). Here, we showed that the wheat MKP (TMKP1) harboring a calmodulin (CaM) binding domain, binds to CaM in a Ca2þ-dependent manner. In addition, TMKP1 exhibited a phosphatase activity in vitro that is specifically enhanced by Mn2þ and to a lesser extent by Mg2þ, but without any synergistic effect between the two bivalent cations. Most interestingly, CaM/Ca2þ complex inhibits the catalytic activity of TMKP1 in a CaM-dose dependent manner. However, in the presence of Mn2þ this activity is enhanced by CaM/Ca2þ complex. These dual regulatory effects seem to be mediated via interaction of CaM/Ca2þ to the CaM binding domain in the Cterminal part of TMKP1. Such effects were not reported so far, and raise a possible role for CaM and Mn2þ in the regulation of plant MKPs during cellular response to external signals. te française de biochimie et biologie Mole culaire (SFBBM). All rights © 2014 Elsevier B.V. and Socie reserved.
Keywords: Calmodulin Ca2þ Enzyme regulation MAP kinase phosphatase Mn2þ
1. Introduction In all eukaryotes, mitogen-activated protein kinases (MAPKs) belong to a conserved family of proteins that regulate a large number of physiological processes, including cell proliferation, differentiation, development, and stress responses. MAPKs are activated by phosphorylation cascades relayed on three interlinked components, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a terminal MAP kinase (MAPK). MAPKs are activated by dual phosphorylation of Thr and Tyr residues within their T-X-Y consensus sequence by the dual-specificity MAPKKs.
Abbreviations: DSP, dual specificity phosphatase; MKP, MAP kinase phosphatase; CaM, calmodulin; OMFP, 3-O-methylfluorescein phosphate. * Corresponding author. Laboratory of Plant Protection and Improvement, Center of Biotechnology of Sfax, BP1177, 3018 Sfax, Tunisia. Tel./fax: þ216 74 875 818. de Toulouse, UPS, UMR 5546, Laboratoire de ** Corresponding author. Universite ge tales, BP 42617, F-31326, Castanet-Tolosan, France. Recherche en Sciences Ve Tel.: þ33 562 34323828; fax: þ33 562 34323802. E-mail addresses: [email protected] (J.-P. Galaud), [email protected] (M. Hanin).
However, this phosphorylation needs to be tightly regulated [1,2] and protein phosphatases are known to act as important negative regulators of MAPK signaling pathways as they control the magnitude and duration of MAPK activities. They can be ranged into three major groups: tyrosine phosphatases (PTPS), serineethreonine phosphatases (PSTPs) and DSP (Ser/Thr and Tyr) phosphatases that include MAPK phosphatases (MKPs). Given that full inactivation of MAPKs requires dual dephosphorylation, MKPs are an important group of phosphatases dedicated to the regulation of MAPK signaling [1e3]. It has been previously reported that defects in such MKPs have a broad range of detrimental effects in multicellular eukaryotes including mammals [4,5] and plants [6e8]. In plants, whereas MAPKs constitute a large family, the number of MKP encoding genes per genome is very limited [9,10]. The Arabidopsis genome encodes only five MKPs namely DUALSPECIFICITY PROTEIN TYROSINE PHOSPHATASE 1 (DsPTP1), MAP KINASE PHOSPHATASE 2 (MKP2), INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5), PROPYZAMIDE HYPERSENSITIVE 1 (PHS1) and MAP KINASE PHOSPHATASE 1 (MKP1) [10]. Experimental evidence confirmed that all these phosphatases are active since they interact with and dephosphorylate MAPKs [11e15].
http://dx.doi.org/10.1016/j.biochi.2014.10.021 te française de biochimie et biologie Mole culaire (SFBBM). All rights reserved. 0300-9084/© 2014 Elsevier B.V. and Socie
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Calmodulins (CaMs) are highly conserved calcium binding proteins in eukaryotic cells in both amino acid composition and number (148aa) [16]. Considered as an ubiquitous intracellular Ca2þ sensor involved in the transduction of a variety of extracellular signals in eukaryotes [17], CaM contains four canonical calciumbinding motifs called EF-hands encompassing the well-known helix-loop-helix structure. Each motif can bind a single Ca2þ ion that relays Ca2þ signals into downstream effectors influencing a broad range of cellular processes including Ca2þ homeostasis [18], stress responses such as cold, wind, wounding and pathogenic attacks, and gene regulation [19e21]. Interestingly, AtMKP1 from Arabidopsis, as well as tobacco (NtMKP1) and rice (OsMKP1) orthologs were identified as CaM-binding proteins [22e24]. In addition, it has been reported that the in vitro phosphatase activity of AtMKP1 using the artificial substrate OMFP (3-O-methylfluorescein phosphate) increases in the presence of CaM in the reaction medium [22]. In contrast, the catalytic activity of NtMKP1 did not vary by adding CaM, while it is increased in the presence of its substrates such as WIPK and SIPK, two orthologs of AtMPK3 and AtMPK6 respectively [25]. Collectively, these results indicate that the molecular mechanisms regulating the activity of MKPs may be complex and can vary between plant species. We recently identified the first MAPK phosphatase (TMKP1) in durum wheat. TMKP1 harbors the four characteristic domains of MKPs i.e. a dual specificity protein phosphatase (DSP) catalytic domain, a gelsolin-like domain, a putative CaM-binding domain (CaMBD), and a serine-rich region, indicating that TMKP1 could be the closest ortholog of the Arabidopsis AtMKP1 [26]. TMKP1 accumulates in nuclear/nucleolar compartments and controls the subcellular localization of its MAPK partners TMPK3 and TMPK6. In vitro studies have also demonstrated that TMKP1 is an active phosphatase that physically interacts with TMPK3 and TMPK6. Furthermore, heterologous functional analysis showed that the overexpression of TMKP1 in Saccharomyces cerevisiae, leads to salt stress tolerance (especially LiCl) that is dependent on the phosphatase activity of the protein [27]. In this study, we provide experimental evidence that TMKP1 activity is enhanced by the presence of Mn2þ and to a lesser extent by Mg2þ and that TMKP1 binds to CaM. Most importantly, the CaM/ Ca2þ complex was shown to stimulate the phosphatase activity of TMKP1 in the presence of Mn2þ. These data were never described so far and suggest a possible contribution of calmodulin and Mn2þ in the modulation of wheat MKP phosphatase activity in plant cells. 2. Methods 2.1. Overexpression and purification of recombinant proteins in Escherichia coli To produce the recombinant proteins GST_TMKP1, GST_TMKP1C214G (dead phosphatase mutant form), and GST_AtCaM1, the ORFs of TMKP1 (accession no. EU502843), TMKP1C214G, and AtCaM1 (At5g37780) were amplified by PCR with the Pfu Taq polymerase, digested by the appropriate restriction enzymes, and cloned in-frame with a GST tag either into the pGEX-6P1 or pGEX2TK expression vectors (Amersham Biosciences). For the production of the recombinant proteins His_TMKP1 and His_TMKP1C214G, the corresponding ORFs were cloned in frame with the polyhistidine tag in the pET28a expression vector (Novagen, Madison, WI, USA). A truncated form His_DCTMKP1 that harbors only the first 380 amino acids (where the C-terminal part including the predicted CaM binding domain was deleted) was derived from the recombinant His_TMKP1 by a digestion with HindIII restriction enzyme and following a self religation of the corresponding plasmid pHis_TMKP1. The resulting constructs pGST_TMKP1,
pGST_TMKP1C214G, pGST_AtCaM1, pHis_TMKP1, pHis_TMKP1C214G and pHis_DCTMKP1 were introduced into the Rosetta E. coli strain (DE3) (Novagen). The production of these different recombinant proteins was induced by 0.8 mM isopropyl b-D-thiogalactopyranoside (IPTG) for 4 h at 37 C. Recombinant proteins were extracted and purified by affinity chromatography either on Glutathione Sepharose 4B beads (Amersham Biosciences) as previously described [28] or on nickel columns (GE Healthcare) according to described procedures. Protein quantification was performed using the Bradford method [29] and the correct size of recombinant proteins was checked by SDS-PAGE electrophoresis. 2.2. Phosphatase activity The phosphatase assays were performed as previously described [22,25e27] using 1 mg of purified recombinant GST_TMKP1, GST_TMKP1C214G, His_TMKP1C214G His_TMKP1, or His_DCTMKP1 with 3-O-methylfluorescein phosphate (OMFP, Sigma) as a substrate. Phosphatase activities were assayed by measuring the amount of OMF released by absorbance at l ¼ 477 nm in the presence or absence of calmodulin as well as bivalent cations such as Mn2þ, Mg2þ, Ca2þ, Fe2þ, Zn2þ and Cu2þ. 2.3. GST pull down assay Prior to binding, Gluthatione Sepharose 4B beads were washed with the appropriate buffer (TriseHCl 20 mM; pH 7.4, EDTA 1 mM, DTT 0.5 mM, NaCl 150 mM, 0.5% Triton, PMSF 1 mM), and equilibrated with the same buffer. Then, the beads were incubated with 10 mg of GST_AtCaM1 or GST for 2 h at 4 C and washed to eliminate the non fixed proteins. The His_TMKP1 or His_DCTMKP1 proteins (15 mg) were then incubated with the immobilized proteins overnight at 4 C. After extensive washes, proteins were dissociated from the beads by boiling them in TriseHCl 50 mM, pH 6.8, DTT 1 mM, SDS 2%, glycerol 10%, bromophenol blue 0.1% and separated by SDSePAGE. The His_TMKP1 and His_DCTMKP1 proteins were finally detected by western blot using the anti-TMKP1 antibody as previously described [26]. As controls, fractions the His_TMKP1 and of His_DCTMKP1 used in the pull-down assays, were also analyzed by western blot. 3. Results 3.1. Phosphatase activity of TMKP1 is enhanced by Mn2þ Our previous phosphatase assays using OMFP as a substrate have shown that the catalytic activity of GST_TMKP1 is weak [26] as it was also reported for the activity of AtMKP1 [22] and AtMKP2 [8].
Table 1 Phosphatase activity of various forms of the recombinant TMKP1 protein fused either to GST or to His tags. Assays were performed as already described [27] in reaction buffers containing either the OMFP (500 mM) as a substrate alone or with 1 mg of GST, GST_TMKP1, His_TMKP1 (native or heat denatured), or dead phosphatase mutated form GST_TMKP1C214G. Data presented are mean values ± S.E of initial rate (103 mmol of OMF/ min) from three independent assays. Enzyme
OMF release (103 mmol/min)
e GST Native His_TMKP1 Native GST_TMKP1 Heat-denatured GST_TMKP1 GST_TMKP1C214G
0.02 0.02 1.10 1.10 0.02 0.03
± ± ± ± ± ±
0.02 0.01 0.01 0.01 0.02 0.02
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For this reason, we have first determined the initial reaction rate (Vo) by measuring enzyme's kinetics of the purified recombinant proteins GST_TMKP1 and His_TMKP1 during the first 10 min. As shown in Table 1, the Vo for both proteins is as expected, fairly the same and relatively low (103 mmoles of OMF released per min) indicating that with His or GST tags added to TMKP1, the activity remains constant. We also confirmed that the phosphatase activity depends on the tertiary structure of the protein since protein denaturation by heat treatment completely abolishes the phosphatase activity as it was the case with the dead phosphatase mutated form GST_TMKP1C214G [27]. Because it is known that phosphatase activities can be modulated by bivalent cations [30,31], different enzyme assays were performed with TMKP1 in the presence of 2 mM of Mn2þ, Mg2þ, Ca2þ, Fe2þ, Zn2þ or Cu2þ. We noticed that the catalytic activity is not significantly modified by bivalent cations such as Ca2þ, Fe2þ, Zn2þ, Cu2þ (Suppl. Fig. 1). In contrast, TMKP1 activity is significantly stimulated in the presence of 2 mM Mn2þ or Mg2þ. Thus, a doseeresponse assay was performed with these two cations separately and the results showed that the activity of TMKP1 is enhanced by increasing Mn2þ (Fig. 1A) or Mg2þ (Fig. 1B) concentrations. The maximal activity of TMKP1 (5-fold higher than in control
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conditions) is reached using 2 mM Mn2þ (Fig. 1A) which corresponds in our experimental conditions to ~1 mM of free Mn2þ as estimated using Maxchelator program (http://maxchelator. stanford.edu/webmaxc/webmaxcS.htm) and according to Bers et al. [32]. The catalytic activity of TMKP1 starts to increase only with 1.5 mM of Mn2þ (4 fold increase) Fig. 1A. Therefore, we performed additional series of phosphatase assays with intermediate Mn2þ concentrations ranging from 1 to 1.5 mM. As shown in Fig. 1A (inset), no significant increase in the TMKP1 activity was registered with up to 1.4 mM of Mn2þ, and an increase was only observed in the presence of 1.5 mM of Mn2þ. This result suggests that the phosphatase activity of TMKP1 does not increase in a gradual manner by increasing Mn concentrations but rather suddenly above a threshold level of this cation (1.5 mM). It is worth to note that Mn2þ stimulation is specific and not artifactual since no activity could be detected when similar assays are performed using the dead phosphatase mutated forms GST_TMKP1C214G or His_TMKP1C214G, in the presence of increasing Mn2þ concentrations (Suppl. Table 1). In the presence of Mg2þ (Fig. 1B), the activity of TMKP1 also increases in a dose-dependent manner, but to a lesser extent than with Mn2þ (with a maximum of 2.5 fold increase using 3 mM Mg2þ compared to the basal activity). Therefore, Mn2þ appears more efficient than Mg2þ on TMKP1 activity in vitro and was used in the further experiments. In order to explore any additive, synergistic or competitive effects between the two cations, TMKP1 activity was measured after adding a mixture of 2 mM Mn2þ and 3 mM Mg2þ. As shown in Table 2, the TMKP1 activity increases about 4 fold similar to what was obtained with 2 mM Mn2þ alone indicating that there is no synergistic effect between the two cations. This result suggests that Mn2þ and Mg2þ might share a similar mechanism of action on TMKP1 activity but with a different efficiency. 3.2. TMKP1 binds to the Arabidopsis calmodulin AtCaM1 in Ca2þ dependent manner Similarly to other plant MKPs, TMKP1 protein sequence analysis revealed the presence of two putative CaM binding motifs in the Cterminus of TMKP1. The first motif is highly conserved and located at the position 398-449, whereas the second one is less conserved and positioned from residues 618 to 669. In order to investigate whether TMKP1 binds CaM, in vitro CaM binding assays were performed using AtCaM1, a CaM isoform almost identical to known wheat CaM sequences sharing 97% identity (Suppl. Fig. 2). To perform a GST pull down assay, the recombinant proteins His_TMKP1, His_DCTMKP1 and GST_AtCaM1 were first purified (Fig. 2A). Then, the purified His_TMKP1 was mixed with the Sepharose bound GST_AtCaM1 and the interaction between the two proteins was investigated by immunoblotting the membrane using the TMKP1 antibody. As shown in Fig. 2B, the
Table 2 Stimulatory effects of Mn2þ and Mg2þ on the phosphatase activity of the recombinant protein GST_TMKP1. Assays were performed in a reaction buffer containing the OMFP (500 mM) as a substrate in the absence or presence of various cation compositions. Data presented are mean values ± S.E of initial rate (103 mmol of OMF/min) from three independent assays.
Fig. 1. Stimulatory effects of Mn2þ and Mg2þ on the in vitro phosphatase activity of the recombinant GST_TMKP1. TMKP1 activity was assayed with 1 mg of recombinant GST_TMKP1 and 500 mM of OMFP as a substrate, in the presence of increasing concentrations of Mn2þ (A and inset), or Mg2þ (B). Values are means of initial rates (103 mmol of OMF/min) ± S.E from three independent experiments.
Cation concentration
OMF release (103 mmol/min)
0 5 mM Mn2þ 5 mM Mg2þ 2 mM Mn2þ/3 mM Mg2þ
1.10 4.70 2.60 4.16
± ± ± ±
0.01 0.10 0.30 0.20
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Fig. 2. AtCaM1 binds to TMKP1 in vitro. (A) SDS-PAGE analyses of the purified recombinant proteins His_TMKP1 (left panel), His_DCTMKP1 (middle panel) and GST_AtCaM1 (right panel). Protein extracts from non-induced (lane1) and IPTGinduced (lane2) E. coli cells expressing pHis_TMKP1 are also presented. Positions of the purified proteins are indicated by arrows. The size of protein standards is given in kDa on the left side. (B) Pulled down proteins were analyzed by immunoblotting with anti-TMKP1 antibody (left panel). Eluates from GST-bound sepharose beads incubated with His_TMKP1 and GST (lane 1); eluates from ungrafted beads incubated with purified His_TMKP1 (lane 2); eluates from GST_AtCaM1-bound sepharose beads alone (lane 3); eluates from GST_AtCaM1 bound sepharose beads incubated with His_TMKP1 without (lane 4) or with 2 mM Ca2þ (lane 5), and in the presence of 2 mM Ca2þ and 5 mM EGTA (lane 6); eluates from GST_AtCaM1-bound sepharose beads incubated with His_DCTMKP1 in the presence of 2 mM Ca2þ (lane 7). Inputs of His_TMKP1 and His_DCTMKP1 were also run as controls (right panel). Positions of molecular weight markers are indicated on the left side of each panel. Experiments were repeated three times with identical results.
His_TMKP1 was pulled down by the GST_AtCaM1 (but not with GST alone). This interaction appears to be Ca2þ dependent since the signal corresponding to TMKP1 is higher when the GST_AtCaM1 was supplemented with 2 mM Ca2þ (Fig. 2B lane 5) while it disappears when the chelating agent EGTA was added to the complex AtCaM1/Ca2þ (Fig. 2B lane 6). Moreover, a truncated form His_DCTMKP1 which has lost the C-terminal part including the CaM binding domain, couldn't be pulled down as expected by GST_AtCaM1/Ca2þ (Fig. 2B lane 7), confirming that TMKP1 interaction to CaM is specific and requires the conserved CaM-binding motifs. Therefore TMKP1 interacts in vitro with AtCaM1 in a Ca2þdependent manner as already reported for other plant MKPs such as AtMKP1, OsMKP1 and NtMKP1 [22e24].
Fig. 3. Comparative analysis of the phosphatase activities of His_TMKP1 and His_DCTMKP1. Phosphatase assays were performed as indicated in Fig. 1, with 1 mg of His_TMKP1 or His_DCTMKP1 in the presence of increasing concentrations of Mn2þ (A) or Mg2þ (B). Values are means of initial rates (103 mmol of OMF/min) ± S.E from three independent experiments.
We then investigated whether the deletion of the C-terminal part containing the CaM binding domain, affects the activity of TMKP1. Interestingly, His_DCTMKP has an activity 4 fold higher than the wild type one in normal assay conditions (without Mn2þ or Mg2þ) (Fig. 3). This finding indicates that the C-terminal part of the enzyme exerts a still unknown and constitutive inhibitory effect on enzyme activity. When mixed with increasing concentrations of Mn2þ or Mg2þ (0.5 mMe5 mM), the activity was also stimulated but to different extents in comparison to the full length protein. Indeed, the activity of His_DCTMKP1 reached its maximum with only 0.5 mM of Mn2þ (versus 2 mM with His_TMKP1) (Fig. 3A). While in the presence of Mg2þ, the activity of the truncated protein is stimulated by adding 1 mM (instead of 1.5 mM for His_TMKP1) and reaches the maximum (2.5-fold) with 2 mM (Fig. 3B).
3.3. Effects of AtCaM1 on TMKP1 activity It is well established that CaM is able to interact with target proteins and to modulate their activities [16]. Consequently, we investigated the effect of AtCaM1 binding on the phosphatase activity of TMKP1, using in vitro assays in the absence of Mn2þ as reported in the previous studies [22,25e27]. Moreover and in order to avoid any possible in vitro dimerization between the two
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recombinant proteins through their GST tags, we used in our phosphatase assays GST_AtCaM1 and His_TMKP1 forms. AtCaM1 alone did not modify TMKP1 activity but surprisingly the addition of calcium results in a significant reduction of the phosphatase activity of TMKP1 using a TMKP1/AtCaM1 molar ratio of 1:4 (Fig. 4A). This activity started to decrease with Ca2þ concentrations as low as 100 mM and inhibition reaches its maximum level using 2 mM of Ca2þ corresponding to a ~70% decrease of the Vo. This inhibition was also shown to be AtCaM1 dose-dependent using
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molar ratios ranging from 1:2 to 1:7 (Suppl. Fig. 3). Moreover, the addition of EGTA a well known Ca2þ chelator, restores the activity of TMKP1 in presence of AtCaM1/Ca2þ whereas EGTA alone does not alter the TMKP1 activity (Fig. 4). This result indicates that Ca2þ is necessary for the calmodulin inhibition of TMKP1. As Mn2þ was shown to enhance the TMKP1 phosphatase activity (Fig. 1A), we evaluated also the effects of AtCaM1/Ca2þ on the TMKP1 activity in the presence of Mn2þ. Remarkably, in a buffer containing 2 mM of Mn2þ, addition of AtCaM1 and calcium rather increases TMKP1 activity by about 2-fold comparatively to the activity measured only in the presence of Mn2þ (Fig. 4B). This stimulatory effect occurs albeit with lower efficiency even with concentrations of Mn2þ and Ca2þ as low as 0.1 mM (Fig. 4C). This increase is calcium-dependent because addition of EGTA is sufficient to return the TMKP1 activity to its initial level (Fig. 4B). Finally to confirm the observed regulatory effects of AtCaM1/ Ca2þ on TMKP1 activity, we performed a new series of phosphatase assays using the truncated form His_DCTMKP1. As shown in Fig. 5, the activity of His_DCTMKP1 remains unchanged in the presence of AtCaM1/Ca2þ. There is neither a negative (in the absence of Mn2þ) nor a positive effect (in the presence of Mn2þ) of AtCaM1/Ca2þ on the catalytic activity of the truncated TMKP1 protein, suggesting that the regulation of TMKP1 by AtCaM1/Ca2þ requires the CaM binding domain. All together, these data confirm that the catalytic activity of TMKP1 can be specifically activated by AtCaM1/Ca2þ in the presence of Mn2þ. 4. Discussion Although considered as major regulators of plant MAPKs, MKPs are still poorly characterized and their multiple functional properties remain largely undiscovered. In the present manuscript, we first showed that the in vitro activity of TMKP1, a wheat MPK phosphatase, is significantly enhanced by the presence of Mn2þ (up to 5 fold increase) and to a lesser extent by Mg2þ (up to 2.5 increase) but not by other cations (Suppl. Fig. 1). To our knowledge, this was not reported for other plant MKPs such as AtMKP1, NtMKP1 and OsMKP1 [22,24,25]. Other plant phosphatases including type 1 (PP1) and type 2C (PP2C) protein phosphatases were reported to be activated by these bivalent cations but most of
Fig. 4. Dual effects of the AtCaM1/Ca2þ complex on TMKP1 activity. (A) Inhibitory effect of the AtCaM1/Ca2þ complex on His_TMKP1 activity. TMKP1 activity was measured using a TMKP1/AtCaM1 molar ratio of 1:4, either in the absence or the presence of increasing concentrations of Ca2þ (0e2 mM) and EGTA (5 mM) as indicated. (B) Stimulatory effect of AtCaM1/Ca2þ complex on His_TMKP1 activity, in presence of 2 mM Mn2þ (C) Stimulatory effect of AtCaM1/Ca2þ complex on His_TMKP1 activity using increasing concentrations of Mn2þ. Assays were carried out according to the same buffer conditions as indicated in A. All data are mean values ± S.E of initial rate (103 mmol of OMF/min) from three independent assays. (***) indicates value significantly different from the control. Statistical significance was assessed by applying the student t-test at p < 0.01.
Fig. 5. The activity of His_DCTMKP1 is not affected by AtCaM1/Ca2þ. Phosphatase activities were measured according to the same conditions indicated before with a His_DCTMKP1/AtCaM1 molar ratio of 1:4. Assays were performed in the presence or the absence of AtCaM1/Ca2þ and increasing concentrations of Mn2þ as indicated. All data are mean values ± S.E of initial rate (103 mmol of OMF/min) from three independent assays.
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them are Mg2þ-dependent phosphatases [33,34]. Nevertheless, in Physcomitrella patens the catalytic activity of PCaMPP, a PP2C-like protein phosphatase with a CaM binding domain was shown to be Mn2þ-dependent [35]. The activation of the PCaMPP phosphatase was nearly saturated at Mn2þ concentration above 1 mM, whereas concentrations of Mg2þ as high as 5 mM have no effect on this activity. Therefore, as far as we know, we demonstrate here for the first time that Mn2þ can stimulate a plant MKP. An additional observation made from His_DCTMKP1 (where the C-terminal part including the CaM binding domain was deleted), points towards an increased phosphatase activity of this truncated form that is a 4 fold higher compared to the wild type protein. This deletion might have resulted in a novel protein conformation that is more suitable for phosphatase activity in vitro. Interestingly, this form can be still enhanced by Mg2þ and Mn2þ (using even lower concentrations compared to wild type protein). It is noteworthy that Mn2þ requirement for TMKP1 activation can be achieved even with mM range of concentrations. In fact, His_TMKP1 activity can be stimulated by Mn2þ concentrations ranging from 50 to 100 mM but only in the presence of AtCaM1/Ca2þ (Fig. 4). Therefore it is not excluded that TMKP1 can be activated in vivo by lower concentrations of Mn2þ (mM range) and the contribution of this cation in modulating plant phosphatases deserves further investigations. Another interesting feature of MKPs deals with the presence of CaM binding domains in their sequence. CaMs are ubiquitous Ca2þbinding proteins in eukaryotes that mediate the primary intracellular Ca2þ signaling pathways. Elevation in cytosolic Ca2þ concentration leads to the formation of active Ca2þ/CaM complexes which in turn modulate cellular functions by interacting with a variety of targets including transcription factors, protein kinases, phosphatases and ion transporters [16,36]. In plants, the functional relationship between phosphatases as well as protein kinases and Ca2þ/CaM complexes, remains poorly understood. In this study, we demonstrated that TMKP1 interacts in vitro with a plant calmodulin (AtCaM1) in a calcium dependent manner as previously reported for other plant MKPs such as AtMKP1, NtMKP1 and OsMKP1 [22e24]. It was assumed that the binding to CaM may activate the phosphatase activity of these MKPs. Thus, the Arabidopsis DsPTP1 activity for p-nitrophenyl phosphate is stimulated by CaM [37]. Similarly, AtMKP1 is catalytically activated by CaM/Ca2þ [22]. However, this CaM/Ca2þ mediated activation of plant MKPs cannot be considered as a general rule as in tobacco the binding of CaM has no effect on NtMKP1 phosphatase activity [25]. In our work, using experimental conditions similar to those reported for other plant MKPs, we showed that the activation of TMKP1 activity by the AtCaM1/Ca2þ complex is observed only in the presence of Mn2þ. In the absence of Mn2þ, the activity was unexpectedly inhibited by the AtCaM1/Ca2þ complex in a doseedependent manner. Therefore, to date, TMKP1 is the first plant MKP which can be inhibited by calmodulin. However, it has been previously reported that the Arabidopsis Ser/Thr protein phosphatase 7 (PP7) which is also a CaM binding phosphatase is inhibited by calmodulin [38]. Moreover, when Mn2þ is replaced by Mg2þ, we didn't observe any activation of the TMKP1 activity by AtCaM1/Ca2þ, beyond the level registered under control conditions (Suppl. Fig. 4), suggesting that this positive effect is rather due to Mn2þ. This Mn2þ-mediated positive effect of CaM/Ca2þ is specific and seems to require a direct interaction of CaM with CaM binding domain in the C-terminal part of TMKP1. This hypothesis was reinforced by further phosphatase assays using the truncated form His_DCTMKP1 mixed to AtCaM1/ Ca2þ. In this case neither an inhibition (in the absence of Mn2þ) nor an activation (in the presence of Mn2þ) of the activity could be observed. Such data serves as a proof of concept for dual regulatory
effects (conditioned by Mn2þ) of CaM/Ca2þ on TMKP1 that depend likely on specific interaction via the CaM binding domain. How can Mn2þ mediate the activation of the TMKP1 activity by CaM/Ca2þ remains an open question. It is plausible that Mn2þ might modify the TMKP1 conformation to promote CaM binding in a way that leads ultimately to the activation of the phosphatase activity. In this regard, since Mg2þ cannot fully replace Mn2þ we can speculate that the affinity of this cation to TMKP1 is too weak to induce the required conformational change leading to CaM binding and to TMKP1 activation. All together, these results strongly suggest that Mn2þ acts as a cofactor in the regulation of a plant MKP by a CaM/ Ca2þ complex. Such finding leads us to revise our understanding of the regulatory function of CaM/Ca2þ on plant MKPs and novel studies dealing with the effect of this complex (CaM/Ca2þ and Mn2þ) on the activity of plant MKPs should be indeed carried out to investigate the mechanisms involved. 5. Conclusions In conclusion, the data obtained in this study regarding TMKP1 provide evidence for a novel regulatory mechanism where AtCaM1/ Ca2þ promotes the catalytic activity of the wheat MKP in the presence of Mn2þ. While TMKP1 activity can be modulated by calcium and CaM in a dose dependent manner, the activation by Mn2þ itself does not appear as a doseeresponse effect but rather occurs as an “oneoff” control. Further investigations should help to understand the contribution of such a regulatory mechanism in the regulation of the MAPK signaling pathways controlling plant responses to abiotic or biotic stresses. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the PHC Utique program between Paul Sabatier (Toulouse, the University of Sfax and the Universite France), and by core funding provided by the Ministry of Higher Education and Scientific Research (Tunisia) and the CNRS (France). This work was performed in CBS and supported by the French Laboratory of Excellence project “TULIP” (ANR-10-LABX-41; ANR11-IDEX-0002-02). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2014.10.021. References [1] M. Camps, A. Nichols, S. Arkinstall, Dual specificity phosphatases: a gene family for control of MAP kinase function, FASEB J. 14 (2000) 6e16. [2] R.J. Dickinson, S.M. Keyse, Diverse physiological functions for dual-specificity MAP kinase phosphatases, J. Cell. Sci. 119 (2006) 4607e4615. [3] S.M. Keyse, Dual-specificity MAP kinase phosphatases (MKPs) and cancer, Cancer Metastasis Rev. 27 (2008) 253e261. [4] Y. Zhang, J.N. Blattman, N.J. Kennedy, J. Duong, T. Nguyen, Y. Wang, J. Roger, G. Davis, D. Philip, R.A. Flavell, C. Dong, Regulation of innate and adaptive immune responses by MAP kinase phosphatase 5, Nature 430 (2004) 793e797. [5] G.R. Christie, D.J. Williams, F. Macisaac, R.J. Dickinson, I. Rosewell, S.M. Keyse, The dual-specificity protein phosphatase DUSP9/MKP-4 is essential for placental function but is not required for normal embryonic development, Mol. Cell. Biol. 25 (2005) 8323e8333. [6] R. Ulm, E. Revenkova, G.P. di Sansebastiano, N. Bechtold, J. Paszkowski, Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis, Genes. Dev. 15 (2001) 699e709.
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[24] S. Katou, K. Kuroda, S. Seo, Y. Yanagawa, T. Tsuge, M. Yamasaki, A. Miyao, H. Hirochika, Y. Ohashi, A calmoudulin-binding mitogen-activated protein kinase phosphatase is induced by wounding and regulates the activities of stress-related mitogen-activated protein kinases in rice, Plant Cell Physiol. 48 (2007) 332e344. [25] S. Katou, E. Karita, H. Yamakawa, S. Seo, I. Mitsuhara, K. Kuchitsu, Y. Ohashi, Catalytic activation of the plant MAPK phosphatase NtMKP1 by its physiological substrate salicylic acid-induced protein kinase but not by calmodulins, J. Biol. Chem. 280 (2005) 39569e39581. [26] I. Zaïdi, C. Ebel, M. Touzri, E. Herzog, J.L. Evrard, A.C. Schmit, K. Masmoudi, M. Hanin, TMKP1 is a novel wheat stress responsive MAP kinase phosphatase localized in the nucleus, Plant Mol. Biol. 73 (2010) 325e338. [27] I. Zaïdi, A. Gonzalez, M. Touzri, M.C. Alvarez, J. Ramos, K. Masmoudi, J. Arino, M. Hanin, The wheat MAP kinase phosphatase 1 confers higher lithium tolerance in yeast, FEMS Yeast Res. 12 (2012) 774e784. [28] N. Janski, E. Herzog, A.C. Schmit, Identification of a novel small Arabidopsis protein interacting with gamma-tubulin complex protein 3, Cell. Biol. Int. 32 (2008) 546e548. [29] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,, Ann. Biochem. 72 (1976) 248e254. [30] H. Cheng, Q. Gao, M. Jiang, Y. Ma, X. Ni, L. Guo, W. Jin, G. Cao, C. Ji, K. Ying, W. Xu, S. Gu, Y. Ma, Y. Xie, Y. Mao, Molecular cloning and characterization of a novel human protein phosphatase, LMW-DSP3, Int. J. Biochem. Cell. Biol. 35 (2003) 226e234. [31] T.A. Reiter, N.J. Reiter, F. Rusnak, Mn2þ is a native metal ion activator for bacteriophage lambda protein phosphatase, Biochemistry 41 (2002) 15404e15409. [32] D.M. Bers, C.W. Patton, R. Nuccitelli, A practical guide to the preparation of Ca2þ buffers, Methods Cell. Biol. 99 (2010) 1e26. [33] R.D. Smith, J.C. Walker, Plant protein phosphatases, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47 (1996) 101e125. [34] A. Schweighofer, H. Hirt, I. Meskiene, Plant PP2C phosphatases: emerging functions in stress signaling, Trends Plant Sci. 9 (2004) 236e243. [35] D. Takezawa, Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein, J. Biol. Chem. 278 (2003) 38076e38083. [36] T.A. DeFalco, K.W. Bender, W.A. Snedden, Breaking the code: Ca2þ sensors in plant signaling, Biochem. J. 425 (2009) 27e40. [37] J.H. Yoo, M.S. Cheong, C.Y. Park, B.C. Moon, M.C. Kim, Y.H. Kang, H.C. Park, M.S. Choi, J.H. Lee, W.Y. Jung, H.W. Yoon, W.S. Chung, C.O. Lim, S.Y. Lee, M.J. Cho, Regulation of the dual specificity protein phosphatase, DsPTP1,through interactions with calmodulin, J. Biol. Chem. 279 (2004) 848e858. [38] M.A. Kutuzov, N. Bennett, A.V. Andreeva, Interaction of plant protein Ser/Thr phosphates PP7 with calmodulin, Biochem. Biophys. Res. Commun. 289 (2001) 634e640.
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Research paper
The activity of the wheat MAP kinase phosphatase 1 is regulated by manganese and by calmodulin nie Robe c, d, Benoit Ranty c, d, Christian Mazars c, d, Mouna Ghorbel a, c, Ikram Zaidi a, Euge Jean-Philippe Galaud c, d, **, Moez Hanin a, b, * a
Laboratory of Plant Protection and Improvement, Center of Biotechnology of Sfax, BP1177, 3018 Sfax, Tunisia University of Sfax, Institute of Biotechnology, BP “1175”, 3038 Sfax, Tunisia Universit e de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences V eg etales, BP 42617, F-31326 Castanet-Tolosan, France d CNRS, UMR 5546, BP 42617, F-31326 Castanet-Tolosan, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 October 2014 Accepted 15 October 2014 Available online 30 October 2014
MAPK phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes and play key roles in the regulation of different cellular processes. However in plants, little is known about the regulation of these Dual Specific Phosphatases (DSPs) by Ca2þ and calmodulin (CaM). Here, we showed that the wheat MKP (TMKP1) harboring a calmodulin (CaM) binding domain, binds to CaM in a Ca2þ-dependent manner. In addition, TMKP1 exhibited a phosphatase activity in vitro that is specifically enhanced by Mn2þ and to a lesser extent by Mg2þ, but without any synergistic effect between the two bivalent cations. Most interestingly, CaM/Ca2þ complex inhibits the catalytic activity of TMKP1 in a CaM-dose dependent manner. However, in the presence of Mn2þ this activity is enhanced by CaM/Ca2þ complex. These dual regulatory effects seem to be mediated via interaction of CaM/Ca2þ to the CaM binding domain in the Cterminal part of TMKP1. Such effects were not reported so far, and raise a possible role for CaM and Mn2þ in the regulation of plant MKPs during cellular response to external signals. te française de biochimie et biologie Mole culaire (SFBBM). All rights © 2014 Elsevier B.V. and Socie reserved.
Keywords: Calmodulin Ca2þ Enzyme regulation MAP kinase phosphatase Mn2þ
1. Introduction In all eukaryotes, mitogen-activated protein kinases (MAPKs) belong to a conserved family of proteins that regulate a large number of physiological processes, including cell proliferation, differentiation, development, and stress responses. MAPKs are activated by phosphorylation cascades relayed on three interlinked components, a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a terminal MAP kinase (MAPK). MAPKs are activated by dual phosphorylation of Thr and Tyr residues within their T-X-Y consensus sequence by the dual-specificity MAPKKs.
Abbreviations: DSP, dual specificity phosphatase; MKP, MAP kinase phosphatase; CaM, calmodulin; OMFP, 3-O-methylfluorescein phosphate. * Corresponding author. Laboratory of Plant Protection and Improvement, Center of Biotechnology of Sfax, BP1177, 3018 Sfax, Tunisia. Tel./fax: þ216 74 875 818. de Toulouse, UPS, UMR 5546, Laboratoire de ** Corresponding author. Universite ge tales, BP 42617, F-31326, Castanet-Tolosan, France. Recherche en Sciences Ve Tel.: þ33 562 34323828; fax: þ33 562 34323802. E-mail addresses: [email protected] (J.-P. Galaud), [email protected] (M. Hanin).
However, this phosphorylation needs to be tightly regulated [1,2] and protein phosphatases are known to act as important negative regulators of MAPK signaling pathways as they control the magnitude and duration of MAPK activities. They can be ranged into three major groups: tyrosine phosphatases (PTPS), serineethreonine phosphatases (PSTPs) and DSP (Ser/Thr and Tyr) phosphatases that include MAPK phosphatases (MKPs). Given that full inactivation of MAPKs requires dual dephosphorylation, MKPs are an important group of phosphatases dedicated to the regulation of MAPK signaling [1e3]. It has been previously reported that defects in such MKPs have a broad range of detrimental effects in multicellular eukaryotes including mammals [4,5] and plants [6e8]. In plants, whereas MAPKs constitute a large family, the number of MKP encoding genes per genome is very limited [9,10]. The Arabidopsis genome encodes only five MKPs namely DUALSPECIFICITY PROTEIN TYROSINE PHOSPHATASE 1 (DsPTP1), MAP KINASE PHOSPHATASE 2 (MKP2), INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5), PROPYZAMIDE HYPERSENSITIVE 1 (PHS1) and MAP KINASE PHOSPHATASE 1 (MKP1) [10]. Experimental evidence confirmed that all these phosphatases are active since they interact with and dephosphorylate MAPKs [11e15].
http://dx.doi.org/10.1016/j.biochi.2014.10.021 te française de biochimie et biologie Mole culaire (SFBBM). All rights reserved. 0300-9084/© 2014 Elsevier B.V. and Socie
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M. Ghorbel et al. / Biochimie 108 (2015) 13e19
Calmodulins (CaMs) are highly conserved calcium binding proteins in eukaryotic cells in both amino acid composition and number (148aa) [16]. Considered as an ubiquitous intracellular Ca2þ sensor involved in the transduction of a variety of extracellular signals in eukaryotes [17], CaM contains four canonical calciumbinding motifs called EF-hands encompassing the well-known helix-loop-helix structure. Each motif can bind a single Ca2þ ion that relays Ca2þ signals into downstream effectors influencing a broad range of cellular processes including Ca2þ homeostasis [18], stress responses such as cold, wind, wounding and pathogenic attacks, and gene regulation [19e21]. Interestingly, AtMKP1 from Arabidopsis, as well as tobacco (NtMKP1) and rice (OsMKP1) orthologs were identified as CaM-binding proteins [22e24]. In addition, it has been reported that the in vitro phosphatase activity of AtMKP1 using the artificial substrate OMFP (3-O-methylfluorescein phosphate) increases in the presence of CaM in the reaction medium [22]. In contrast, the catalytic activity of NtMKP1 did not vary by adding CaM, while it is increased in the presence of its substrates such as WIPK and SIPK, two orthologs of AtMPK3 and AtMPK6 respectively [25]. Collectively, these results indicate that the molecular mechanisms regulating the activity of MKPs may be complex and can vary between plant species. We recently identified the first MAPK phosphatase (TMKP1) in durum wheat. TMKP1 harbors the four characteristic domains of MKPs i.e. a dual specificity protein phosphatase (DSP) catalytic domain, a gelsolin-like domain, a putative CaM-binding domain (CaMBD), and a serine-rich region, indicating that TMKP1 could be the closest ortholog of the Arabidopsis AtMKP1 [26]. TMKP1 accumulates in nuclear/nucleolar compartments and controls the subcellular localization of its MAPK partners TMPK3 and TMPK6. In vitro studies have also demonstrated that TMKP1 is an active phosphatase that physically interacts with TMPK3 and TMPK6. Furthermore, heterologous functional analysis showed that the overexpression of TMKP1 in Saccharomyces cerevisiae, leads to salt stress tolerance (especially LiCl) that is dependent on the phosphatase activity of the protein [27]. In this study, we provide experimental evidence that TMKP1 activity is enhanced by the presence of Mn2þ and to a lesser extent by Mg2þ and that TMKP1 binds to CaM. Most importantly, the CaM/ Ca2þ complex was shown to stimulate the phosphatase activity of TMKP1 in the presence of Mn2þ. These data were never described so far and suggest a possible contribution of calmodulin and Mn2þ in the modulation of wheat MKP phosphatase activity in plant cells. 2. Methods 2.1. Overexpression and purification of recombinant proteins in Escherichia coli To produce the recombinant proteins GST_TMKP1, GST_TMKP1C214G (dead phosphatase mutant form), and GST_AtCaM1, the ORFs of TMKP1 (accession no. EU502843), TMKP1C214G, and AtCaM1 (At5g37780) were amplified by PCR with the Pfu Taq polymerase, digested by the appropriate restriction enzymes, and cloned in-frame with a GST tag either into the pGEX-6P1 or pGEX2TK expression vectors (Amersham Biosciences). For the production of the recombinant proteins His_TMKP1 and His_TMKP1C214G, the corresponding ORFs were cloned in frame with the polyhistidine tag in the pET28a expression vector (Novagen, Madison, WI, USA). A truncated form His_DCTMKP1 that harbors only the first 380 amino acids (where the C-terminal part including the predicted CaM binding domain was deleted) was derived from the recombinant His_TMKP1 by a digestion with HindIII restriction enzyme and following a self religation of the corresponding plasmid pHis_TMKP1. The resulting constructs pGST_TMKP1,
pGST_TMKP1C214G, pGST_AtCaM1, pHis_TMKP1, pHis_TMKP1C214G and pHis_DCTMKP1 were introduced into the Rosetta E. coli strain (DE3) (Novagen). The production of these different recombinant proteins was induced by 0.8 mM isopropyl b-D-thiogalactopyranoside (IPTG) for 4 h at 37 C. Recombinant proteins were extracted and purified by affinity chromatography either on Glutathione Sepharose 4B beads (Amersham Biosciences) as previously described [28] or on nickel columns (GE Healthcare) according to described procedures. Protein quantification was performed using the Bradford method [29] and the correct size of recombinant proteins was checked by SDS-PAGE electrophoresis. 2.2. Phosphatase activity The phosphatase assays were performed as previously described [22,25e27] using 1 mg of purified recombinant GST_TMKP1, GST_TMKP1C214G, His_TMKP1C214G His_TMKP1, or His_DCTMKP1 with 3-O-methylfluorescein phosphate (OMFP, Sigma) as a substrate. Phosphatase activities were assayed by measuring the amount of OMF released by absorbance at l ¼ 477 nm in the presence or absence of calmodulin as well as bivalent cations such as Mn2þ, Mg2þ, Ca2þ, Fe2þ, Zn2þ and Cu2þ. 2.3. GST pull down assay Prior to binding, Gluthatione Sepharose 4B beads were washed with the appropriate buffer (TriseHCl 20 mM; pH 7.4, EDTA 1 mM, DTT 0.5 mM, NaCl 150 mM, 0.5% Triton, PMSF 1 mM), and equilibrated with the same buffer. Then, the beads were incubated with 10 mg of GST_AtCaM1 or GST for 2 h at 4 C and washed to eliminate the non fixed proteins. The His_TMKP1 or His_DCTMKP1 proteins (15 mg) were then incubated with the immobilized proteins overnight at 4 C. After extensive washes, proteins were dissociated from the beads by boiling them in TriseHCl 50 mM, pH 6.8, DTT 1 mM, SDS 2%, glycerol 10%, bromophenol blue 0.1% and separated by SDSePAGE. The His_TMKP1 and His_DCTMKP1 proteins were finally detected by western blot using the anti-TMKP1 antibody as previously described [26]. As controls, fractions the His_TMKP1 and of His_DCTMKP1 used in the pull-down assays, were also analyzed by western blot. 3. Results 3.1. Phosphatase activity of TMKP1 is enhanced by Mn2þ Our previous phosphatase assays using OMFP as a substrate have shown that the catalytic activity of GST_TMKP1 is weak [26] as it was also reported for the activity of AtMKP1 [22] and AtMKP2 [8].
Table 1 Phosphatase activity of various forms of the recombinant TMKP1 protein fused either to GST or to His tags. Assays were performed as already described [27] in reaction buffers containing either the OMFP (500 mM) as a substrate alone or with 1 mg of GST, GST_TMKP1, His_TMKP1 (native or heat denatured), or dead phosphatase mutated form GST_TMKP1C214G. Data presented are mean values ± S.E of initial rate (103 mmol of OMF/ min) from three independent assays. Enzyme
OMF release (103 mmol/min)
e GST Native His_TMKP1 Native GST_TMKP1 Heat-denatured GST_TMKP1 GST_TMKP1C214G
0.02 0.02 1.10 1.10 0.02 0.03
± ± ± ± ± ±
0.02 0.01 0.01 0.01 0.02 0.02
M. Ghorbel et al. / Biochimie 108 (2015) 13e19
For this reason, we have first determined the initial reaction rate (Vo) by measuring enzyme's kinetics of the purified recombinant proteins GST_TMKP1 and His_TMKP1 during the first 10 min. As shown in Table 1, the Vo for both proteins is as expected, fairly the same and relatively low (103 mmoles of OMF released per min) indicating that with His or GST tags added to TMKP1, the activity remains constant. We also confirmed that the phosphatase activity depends on the tertiary structure of the protein since protein denaturation by heat treatment completely abolishes the phosphatase activity as it was the case with the dead phosphatase mutated form GST_TMKP1C214G [27]. Because it is known that phosphatase activities can be modulated by bivalent cations [30,31], different enzyme assays were performed with TMKP1 in the presence of 2 mM of Mn2þ, Mg2þ, Ca2þ, Fe2þ, Zn2þ or Cu2þ. We noticed that the catalytic activity is not significantly modified by bivalent cations such as Ca2þ, Fe2þ, Zn2þ, Cu2þ (Suppl. Fig. 1). In contrast, TMKP1 activity is significantly stimulated in the presence of 2 mM Mn2þ or Mg2þ. Thus, a doseeresponse assay was performed with these two cations separately and the results showed that the activity of TMKP1 is enhanced by increasing Mn2þ (Fig. 1A) or Mg2þ (Fig. 1B) concentrations. The maximal activity of TMKP1 (5-fold higher than in control
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conditions) is reached using 2 mM Mn2þ (Fig. 1A) which corresponds in our experimental conditions to ~1 mM of free Mn2þ as estimated using Maxchelator program (http://maxchelator. stanford.edu/webmaxc/webmaxcS.htm) and according to Bers et al. [32]. The catalytic activity of TMKP1 starts to increase only with 1.5 mM of Mn2þ (4 fold increase) Fig. 1A. Therefore, we performed additional series of phosphatase assays with intermediate Mn2þ concentrations ranging from 1 to 1.5 mM. As shown in Fig. 1A (inset), no significant increase in the TMKP1 activity was registered with up to 1.4 mM of Mn2þ, and an increase was only observed in the presence of 1.5 mM of Mn2þ. This result suggests that the phosphatase activity of TMKP1 does not increase in a gradual manner by increasing Mn concentrations but rather suddenly above a threshold level of this cation (1.5 mM). It is worth to note that Mn2þ stimulation is specific and not artifactual since no activity could be detected when similar assays are performed using the dead phosphatase mutated forms GST_TMKP1C214G or His_TMKP1C214G, in the presence of increasing Mn2þ concentrations (Suppl. Table 1). In the presence of Mg2þ (Fig. 1B), the activity of TMKP1 also increases in a dose-dependent manner, but to a lesser extent than with Mn2þ (with a maximum of 2.5 fold increase using 3 mM Mg2þ compared to the basal activity). Therefore, Mn2þ appears more efficient than Mg2þ on TMKP1 activity in vitro and was used in the further experiments. In order to explore any additive, synergistic or competitive effects between the two cations, TMKP1 activity was measured after adding a mixture of 2 mM Mn2þ and 3 mM Mg2þ. As shown in Table 2, the TMKP1 activity increases about 4 fold similar to what was obtained with 2 mM Mn2þ alone indicating that there is no synergistic effect between the two cations. This result suggests that Mn2þ and Mg2þ might share a similar mechanism of action on TMKP1 activity but with a different efficiency. 3.2. TMKP1 binds to the Arabidopsis calmodulin AtCaM1 in Ca2þ dependent manner Similarly to other plant MKPs, TMKP1 protein sequence analysis revealed the presence of two putative CaM binding motifs in the Cterminus of TMKP1. The first motif is highly conserved and located at the position 398-449, whereas the second one is less conserved and positioned from residues 618 to 669. In order to investigate whether TMKP1 binds CaM, in vitro CaM binding assays were performed using AtCaM1, a CaM isoform almost identical to known wheat CaM sequences sharing 97% identity (Suppl. Fig. 2). To perform a GST pull down assay, the recombinant proteins His_TMKP1, His_DCTMKP1 and GST_AtCaM1 were first purified (Fig. 2A). Then, the purified His_TMKP1 was mixed with the Sepharose bound GST_AtCaM1 and the interaction between the two proteins was investigated by immunoblotting the membrane using the TMKP1 antibody. As shown in Fig. 2B, the
Table 2 Stimulatory effects of Mn2þ and Mg2þ on the phosphatase activity of the recombinant protein GST_TMKP1. Assays were performed in a reaction buffer containing the OMFP (500 mM) as a substrate in the absence or presence of various cation compositions. Data presented are mean values ± S.E of initial rate (103 mmol of OMF/min) from three independent assays.
Fig. 1. Stimulatory effects of Mn2þ and Mg2þ on the in vitro phosphatase activity of the recombinant GST_TMKP1. TMKP1 activity was assayed with 1 mg of recombinant GST_TMKP1 and 500 mM of OMFP as a substrate, in the presence of increasing concentrations of Mn2þ (A and inset), or Mg2þ (B). Values are means of initial rates (103 mmol of OMF/min) ± S.E from three independent experiments.
Cation concentration
OMF release (103 mmol/min)
0 5 mM Mn2þ 5 mM Mg2þ 2 mM Mn2þ/3 mM Mg2þ
1.10 4.70 2.60 4.16
± ± ± ±
0.01 0.10 0.30 0.20
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Fig. 2. AtCaM1 binds to TMKP1 in vitro. (A) SDS-PAGE analyses of the purified recombinant proteins His_TMKP1 (left panel), His_DCTMKP1 (middle panel) and GST_AtCaM1 (right panel). Protein extracts from non-induced (lane1) and IPTGinduced (lane2) E. coli cells expressing pHis_TMKP1 are also presented. Positions of the purified proteins are indicated by arrows. The size of protein standards is given in kDa on the left side. (B) Pulled down proteins were analyzed by immunoblotting with anti-TMKP1 antibody (left panel). Eluates from GST-bound sepharose beads incubated with His_TMKP1 and GST (lane 1); eluates from ungrafted beads incubated with purified His_TMKP1 (lane 2); eluates from GST_AtCaM1-bound sepharose beads alone (lane 3); eluates from GST_AtCaM1 bound sepharose beads incubated with His_TMKP1 without (lane 4) or with 2 mM Ca2þ (lane 5), and in the presence of 2 mM Ca2þ and 5 mM EGTA (lane 6); eluates from GST_AtCaM1-bound sepharose beads incubated with His_DCTMKP1 in the presence of 2 mM Ca2þ (lane 7). Inputs of His_TMKP1 and His_DCTMKP1 were also run as controls (right panel). Positions of molecular weight markers are indicated on the left side of each panel. Experiments were repeated three times with identical results.
His_TMKP1 was pulled down by the GST_AtCaM1 (but not with GST alone). This interaction appears to be Ca2þ dependent since the signal corresponding to TMKP1 is higher when the GST_AtCaM1 was supplemented with 2 mM Ca2þ (Fig. 2B lane 5) while it disappears when the chelating agent EGTA was added to the complex AtCaM1/Ca2þ (Fig. 2B lane 6). Moreover, a truncated form His_DCTMKP1 which has lost the C-terminal part including the CaM binding domain, couldn't be pulled down as expected by GST_AtCaM1/Ca2þ (Fig. 2B lane 7), confirming that TMKP1 interaction to CaM is specific and requires the conserved CaM-binding motifs. Therefore TMKP1 interacts in vitro with AtCaM1 in a Ca2þdependent manner as already reported for other plant MKPs such as AtMKP1, OsMKP1 and NtMKP1 [22e24].
Fig. 3. Comparative analysis of the phosphatase activities of His_TMKP1 and His_DCTMKP1. Phosphatase assays were performed as indicated in Fig. 1, with 1 mg of His_TMKP1 or His_DCTMKP1 in the presence of increasing concentrations of Mn2þ (A) or Mg2þ (B). Values are means of initial rates (103 mmol of OMF/min) ± S.E from three independent experiments.
We then investigated whether the deletion of the C-terminal part containing the CaM binding domain, affects the activity of TMKP1. Interestingly, His_DCTMKP has an activity 4 fold higher than the wild type one in normal assay conditions (without Mn2þ or Mg2þ) (Fig. 3). This finding indicates that the C-terminal part of the enzyme exerts a still unknown and constitutive inhibitory effect on enzyme activity. When mixed with increasing concentrations of Mn2þ or Mg2þ (0.5 mMe5 mM), the activity was also stimulated but to different extents in comparison to the full length protein. Indeed, the activity of His_DCTMKP1 reached its maximum with only 0.5 mM of Mn2þ (versus 2 mM with His_TMKP1) (Fig. 3A). While in the presence of Mg2þ, the activity of the truncated protein is stimulated by adding 1 mM (instead of 1.5 mM for His_TMKP1) and reaches the maximum (2.5-fold) with 2 mM (Fig. 3B).
3.3. Effects of AtCaM1 on TMKP1 activity It is well established that CaM is able to interact with target proteins and to modulate their activities [16]. Consequently, we investigated the effect of AtCaM1 binding on the phosphatase activity of TMKP1, using in vitro assays in the absence of Mn2þ as reported in the previous studies [22,25e27]. Moreover and in order to avoid any possible in vitro dimerization between the two
M. Ghorbel et al. / Biochimie 108 (2015) 13e19
recombinant proteins through their GST tags, we used in our phosphatase assays GST_AtCaM1 and His_TMKP1 forms. AtCaM1 alone did not modify TMKP1 activity but surprisingly the addition of calcium results in a significant reduction of the phosphatase activity of TMKP1 using a TMKP1/AtCaM1 molar ratio of 1:4 (Fig. 4A). This activity started to decrease with Ca2þ concentrations as low as 100 mM and inhibition reaches its maximum level using 2 mM of Ca2þ corresponding to a ~70% decrease of the Vo. This inhibition was also shown to be AtCaM1 dose-dependent using
17
molar ratios ranging from 1:2 to 1:7 (Suppl. Fig. 3). Moreover, the addition of EGTA a well known Ca2þ chelator, restores the activity of TMKP1 in presence of AtCaM1/Ca2þ whereas EGTA alone does not alter the TMKP1 activity (Fig. 4). This result indicates that Ca2þ is necessary for the calmodulin inhibition of TMKP1. As Mn2þ was shown to enhance the TMKP1 phosphatase activity (Fig. 1A), we evaluated also the effects of AtCaM1/Ca2þ on the TMKP1 activity in the presence of Mn2þ. Remarkably, in a buffer containing 2 mM of Mn2þ, addition of AtCaM1 and calcium rather increases TMKP1 activity by about 2-fold comparatively to the activity measured only in the presence of Mn2þ (Fig. 4B). This stimulatory effect occurs albeit with lower efficiency even with concentrations of Mn2þ and Ca2þ as low as 0.1 mM (Fig. 4C). This increase is calcium-dependent because addition of EGTA is sufficient to return the TMKP1 activity to its initial level (Fig. 4B). Finally to confirm the observed regulatory effects of AtCaM1/ Ca2þ on TMKP1 activity, we performed a new series of phosphatase assays using the truncated form His_DCTMKP1. As shown in Fig. 5, the activity of His_DCTMKP1 remains unchanged in the presence of AtCaM1/Ca2þ. There is neither a negative (in the absence of Mn2þ) nor a positive effect (in the presence of Mn2þ) of AtCaM1/Ca2þ on the catalytic activity of the truncated TMKP1 protein, suggesting that the regulation of TMKP1 by AtCaM1/Ca2þ requires the CaM binding domain. All together, these data confirm that the catalytic activity of TMKP1 can be specifically activated by AtCaM1/Ca2þ in the presence of Mn2þ. 4. Discussion Although considered as major regulators of plant MAPKs, MKPs are still poorly characterized and their multiple functional properties remain largely undiscovered. In the present manuscript, we first showed that the in vitro activity of TMKP1, a wheat MPK phosphatase, is significantly enhanced by the presence of Mn2þ (up to 5 fold increase) and to a lesser extent by Mg2þ (up to 2.5 increase) but not by other cations (Suppl. Fig. 1). To our knowledge, this was not reported for other plant MKPs such as AtMKP1, NtMKP1 and OsMKP1 [22,24,25]. Other plant phosphatases including type 1 (PP1) and type 2C (PP2C) protein phosphatases were reported to be activated by these bivalent cations but most of
Fig. 4. Dual effects of the AtCaM1/Ca2þ complex on TMKP1 activity. (A) Inhibitory effect of the AtCaM1/Ca2þ complex on His_TMKP1 activity. TMKP1 activity was measured using a TMKP1/AtCaM1 molar ratio of 1:4, either in the absence or the presence of increasing concentrations of Ca2þ (0e2 mM) and EGTA (5 mM) as indicated. (B) Stimulatory effect of AtCaM1/Ca2þ complex on His_TMKP1 activity, in presence of 2 mM Mn2þ (C) Stimulatory effect of AtCaM1/Ca2þ complex on His_TMKP1 activity using increasing concentrations of Mn2þ. Assays were carried out according to the same buffer conditions as indicated in A. All data are mean values ± S.E of initial rate (103 mmol of OMF/min) from three independent assays. (***) indicates value significantly different from the control. Statistical significance was assessed by applying the student t-test at p < 0.01.
Fig. 5. The activity of His_DCTMKP1 is not affected by AtCaM1/Ca2þ. Phosphatase activities were measured according to the same conditions indicated before with a His_DCTMKP1/AtCaM1 molar ratio of 1:4. Assays were performed in the presence or the absence of AtCaM1/Ca2þ and increasing concentrations of Mn2þ as indicated. All data are mean values ± S.E of initial rate (103 mmol of OMF/min) from three independent assays.
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M. Ghorbel et al. / Biochimie 108 (2015) 13e19
them are Mg2þ-dependent phosphatases [33,34]. Nevertheless, in Physcomitrella patens the catalytic activity of PCaMPP, a PP2C-like protein phosphatase with a CaM binding domain was shown to be Mn2þ-dependent [35]. The activation of the PCaMPP phosphatase was nearly saturated at Mn2þ concentration above 1 mM, whereas concentrations of Mg2þ as high as 5 mM have no effect on this activity. Therefore, as far as we know, we demonstrate here for the first time that Mn2þ can stimulate a plant MKP. An additional observation made from His_DCTMKP1 (where the C-terminal part including the CaM binding domain was deleted), points towards an increased phosphatase activity of this truncated form that is a 4 fold higher compared to the wild type protein. This deletion might have resulted in a novel protein conformation that is more suitable for phosphatase activity in vitro. Interestingly, this form can be still enhanced by Mg2þ and Mn2þ (using even lower concentrations compared to wild type protein). It is noteworthy that Mn2þ requirement for TMKP1 activation can be achieved even with mM range of concentrations. In fact, His_TMKP1 activity can be stimulated by Mn2þ concentrations ranging from 50 to 100 mM but only in the presence of AtCaM1/Ca2þ (Fig. 4). Therefore it is not excluded that TMKP1 can be activated in vivo by lower concentrations of Mn2þ (mM range) and the contribution of this cation in modulating plant phosphatases deserves further investigations. Another interesting feature of MKPs deals with the presence of CaM binding domains in their sequence. CaMs are ubiquitous Ca2þbinding proteins in eukaryotes that mediate the primary intracellular Ca2þ signaling pathways. Elevation in cytosolic Ca2þ concentration leads to the formation of active Ca2þ/CaM complexes which in turn modulate cellular functions by interacting with a variety of targets including transcription factors, protein kinases, phosphatases and ion transporters [16,36]. In plants, the functional relationship between phosphatases as well as protein kinases and Ca2þ/CaM complexes, remains poorly understood. In this study, we demonstrated that TMKP1 interacts in vitro with a plant calmodulin (AtCaM1) in a calcium dependent manner as previously reported for other plant MKPs such as AtMKP1, NtMKP1 and OsMKP1 [22e24]. It was assumed that the binding to CaM may activate the phosphatase activity of these MKPs. Thus, the Arabidopsis DsPTP1 activity for p-nitrophenyl phosphate is stimulated by CaM [37]. Similarly, AtMKP1 is catalytically activated by CaM/Ca2þ [22]. However, this CaM/Ca2þ mediated activation of plant MKPs cannot be considered as a general rule as in tobacco the binding of CaM has no effect on NtMKP1 phosphatase activity [25]. In our work, using experimental conditions similar to those reported for other plant MKPs, we showed that the activation of TMKP1 activity by the AtCaM1/Ca2þ complex is observed only in the presence of Mn2þ. In the absence of Mn2þ, the activity was unexpectedly inhibited by the AtCaM1/Ca2þ complex in a doseedependent manner. Therefore, to date, TMKP1 is the first plant MKP which can be inhibited by calmodulin. However, it has been previously reported that the Arabidopsis Ser/Thr protein phosphatase 7 (PP7) which is also a CaM binding phosphatase is inhibited by calmodulin [38]. Moreover, when Mn2þ is replaced by Mg2þ, we didn't observe any activation of the TMKP1 activity by AtCaM1/Ca2þ, beyond the level registered under control conditions (Suppl. Fig. 4), suggesting that this positive effect is rather due to Mn2þ. This Mn2þ-mediated positive effect of CaM/Ca2þ is specific and seems to require a direct interaction of CaM with CaM binding domain in the C-terminal part of TMKP1. This hypothesis was reinforced by further phosphatase assays using the truncated form His_DCTMKP1 mixed to AtCaM1/ Ca2þ. In this case neither an inhibition (in the absence of Mn2þ) nor an activation (in the presence of Mn2þ) of the activity could be observed. Such data serves as a proof of concept for dual regulatory
effects (conditioned by Mn2þ) of CaM/Ca2þ on TMKP1 that depend likely on specific interaction via the CaM binding domain. How can Mn2þ mediate the activation of the TMKP1 activity by CaM/Ca2þ remains an open question. It is plausible that Mn2þ might modify the TMKP1 conformation to promote CaM binding in a way that leads ultimately to the activation of the phosphatase activity. In this regard, since Mg2þ cannot fully replace Mn2þ we can speculate that the affinity of this cation to TMKP1 is too weak to induce the required conformational change leading to CaM binding and to TMKP1 activation. All together, these results strongly suggest that Mn2þ acts as a cofactor in the regulation of a plant MKP by a CaM/ Ca2þ complex. Such finding leads us to revise our understanding of the regulatory function of CaM/Ca2þ on plant MKPs and novel studies dealing with the effect of this complex (CaM/Ca2þ and Mn2þ) on the activity of plant MKPs should be indeed carried out to investigate the mechanisms involved. 5. Conclusions In conclusion, the data obtained in this study regarding TMKP1 provide evidence for a novel regulatory mechanism where AtCaM1/ Ca2þ promotes the catalytic activity of the wheat MKP in the presence of Mn2þ. While TMKP1 activity can be modulated by calcium and CaM in a dose dependent manner, the activation by Mn2þ itself does not appear as a doseeresponse effect but rather occurs as an “oneoff” control. Further investigations should help to understand the contribution of such a regulatory mechanism in the regulation of the MAPK signaling pathways controlling plant responses to abiotic or biotic stresses. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the PHC Utique program between Paul Sabatier (Toulouse, the University of Sfax and the Universite France), and by core funding provided by the Ministry of Higher Education and Scientific Research (Tunisia) and the CNRS (France). This work was performed in CBS and supported by the French Laboratory of Excellence project “TULIP” (ANR-10-LABX-41; ANR11-IDEX-0002-02). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2014.10.021. References [1] M. Camps, A. Nichols, S. Arkinstall, Dual specificity phosphatases: a gene family for control of MAP kinase function, FASEB J. 14 (2000) 6e16. [2] R.J. Dickinson, S.M. Keyse, Diverse physiological functions for dual-specificity MAP kinase phosphatases, J. Cell. Sci. 119 (2006) 4607e4615. [3] S.M. Keyse, Dual-specificity MAP kinase phosphatases (MKPs) and cancer, Cancer Metastasis Rev. 27 (2008) 253e261. [4] Y. Zhang, J.N. Blattman, N.J. Kennedy, J. Duong, T. Nguyen, Y. Wang, J. Roger, G. Davis, D. Philip, R.A. Flavell, C. Dong, Regulation of innate and adaptive immune responses by MAP kinase phosphatase 5, Nature 430 (2004) 793e797. [5] G.R. Christie, D.J. Williams, F. Macisaac, R.J. Dickinson, I. Rosewell, S.M. Keyse, The dual-specificity protein phosphatase DUSP9/MKP-4 is essential for placental function but is not required for normal embryonic development, Mol. Cell. Biol. 25 (2005) 8323e8333. [6] R. Ulm, E. Revenkova, G.P. di Sansebastiano, N. Bechtold, J. Paszkowski, Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis, Genes. Dev. 15 (2001) 699e709.
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