PrpZ, a Salmonella enterica serovar Typhi serine/threonine protein phosphatase 2C with dual substrate specificity

Sio Mei Lai and Hervé Le Moual

Department of Microbiology and Immunology, McGill University, 3775 University Street, Montréal, Québec, Canada H3A 2B4

Correspondence
Hervé Le Moual
herve.le-moual{at}mcgill.ca


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Genes encoding eukaryotic-type protein kinases and phosphatases are present in many bacterial genomes. An ORF encoding a polypeptide with homology to protein phosphatases 2C (PP2Cs) was identified in the genomes of Salmonella enterica serovar Typhi strains CT18 and Ty2. This protein, termed PrpZ, is the first PP2C to be identified in enterobacteria. Analysis of the amino acid sequence revealed two distinct domains: the N-terminal segment containing motifs of the catalytic domain of PP2Cs and the C-terminal segment with unknown function. PrpZ was expressed in Escherichia coli as a histidine-tagged fusion protein (PrpZHis) and the purified protein was analysed for its ability to dephosphorylate various substrates. Using p-nitrophenyl phosphate as a substrate, optimal PrpZHis activity was observed at pH 9·5, with a strong preference for Mn2+ over Mg2+. Activity of PrpZHis was inhibited by EDTA, sodium fluoride, sodium phosphate and sodium pyrophosphate but unaffected by okadaic acid, indicating that PrpZ is a PP2C. Using synthetic phosphopeptides as substrates, PrpZHis could hydrolyse phosphorylated serine, threonine or tyrosine residues, with the highest catalytic efficiency (kcat/Km) for the threonine phosphopeptide. With phosphorylated myelin basic protein (MBP) as the substrate, Mn2+ was only twofold more efficient than Mg2+ in stimulating PrpZHis activity at pH 8·0. The ability of PrpZHis to remove the phosphoryl group from phosphotyrosine residues was confirmed by measuring the release of inorganic phosphate from phospho-Tyr MBP. Together, these data indicate that PrpZ has all the features of a PP2C with dual substrate specificity in vitro.


Abbreviations: BME, {beta}-mercaptoethanol; MBP, myelin basic protein; PNP, p-nitrophenol; PNPP, p-nitrophenyl phosphate; PP2C, protein phosphatase 2C; PPM, Mg2+- or Mn2+-dependent protein phosphatase; PPP, phosphoprotein phosphatase


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Reversible protein phosphorylation through the combined action of kinases and phosphatases is the most common mechanism for regulating protein function in both prokaryotes and eukaryotes (Hunter, 1995). In bacteria, phosphorylation usually occurs on histidine and aspartate residues through two-component regulatory systems (Stock et al., 1989). Phosphorylation on serine, threonine and tyrosine residues, which has long been considered to be specific to eukaryotes, has also been found to occur in bacteria (Wang & Koshland, 1978). Although some bacterial Ser/Thr or Tyr protein kinases or phosphatases, such as the isocitrate dehydrogenase kinase/phosphatase and the Ptk protein tyrosine kinase of Acinetobacter johnsonii, have no apparent eukaryotic counterpart (Doublet et al., 1999; LaPorte et al., 1989), others share sequence similarity with their eukaryotic counterparts. The first bacterial gene encoding a eukaryotic-type protein kinase (pkn1) was cloned from the Gram-negative soil bacterium Myxococcus xanthus (Muñoz-Dorado et al., 1991). Recently, genomics has revealed that most bacterial genomes contain at least a few genes encoding eukaryotic-type protein kinases and phosphatases (Kennelly, 2002, 2003; Shi et al., 1998; Shi, 2004).

Ser/Thr protein phosphatases are divided into two distinct families, the phosphoprotein phosphatases (PPPs) and the Mg2+- or Mn2+-dependent protein phosphatases (PPMs), based on the presence of signature motifs in their amino acid sequences (Barford et al., 1998; Cohen, 1989). Although they lack sequence similarity, PPPs and PPMs share similar protein architecture and contain two metal ions (Mg2+ and/or Mn2+) in their active site. PPMs can be distinguished from PPPs on the basis of their resistance to the inhibitor okadaic acid (Barford et al., 1998). Whereas the PPP family is divided into three distinct subfamilies, protein phosphatases 1, 2A and 2B, the PPM family consists only of protein phosphatases 2C (PP2Cs). The catalytic domain of PP2Cs consists of 250–300 amino acid residues and contains 11 conserved motifs (Bork et al., 1996). The three-dimensional structure of the human PP2C{alpha} revealed that these motifs contain highly conserved aspartate residues that are involved in the binding of the two metal ions. A metal-bound water molecule has been proposed to act as the nucleophile that attacks the substrate phosphoryl group (Das et al., 1996).

PP2Cs are distributed unevenly among bacterial species. The genomes of Streptomyces coelicolor A3(2) and Streptomyces avermitilis contain 49 and 48 PP2C genes, respectively (Shi & Zhang, 2004). The Bacillus subtilis and Synechocystis sp. strain PCC 6803 genomes contain five and eight PP2C genes, respectively (Kennelly, 2002; Shi, 2004). In contrast, no PP2C gene has been identified in the genomes of Escherichia coli or Salmonella enterica serovar Typhimurium strain LT2. Although some bacterial PP2Cs have been functionally characterized, the function of most of these proteins remains elusive. The SpoIIE PP2C of B. subtilis regulates sporulation by promoting the dephosphorylation of the SpoIIAA anti-anti-sigma factor (Duncan et al., 1995). In B. subtilis, the general stress response is controlled by two PP2Cs, RsbP and RsbU, that regulate the phosphorylation status of the RsbV anti-anti-sigma factor (Vijay et al., 2000). In Myxococcus xanthus, the Pph1 PP2C controls vegetative growth and development by interacting with the Ser/Thr kinase Pkn5 (Treuner-Lange et al., 2001). In Synechocystis sp. strain PCC 6803, the PphA PP2C mediates dephosphorylation of the PII signal transduction protein controlling nitrogen and carbon assimilation (Irmler & Forchhammer, 2001; Ruppert et al., 2002). The Stp1 PP2C of Streptococcus agalactiae has been shown to dephosphorylate an inorganic pyrophosphatase (Rajagopal et al., 2003). In Mycobacterium tuberculosis, the PstP PP2C (also known as MstP) plays a role in regulating cell division by dephosphorylating the Ser/Thr kinases PknA and PknB (Boitel et al., 2003; Chopra et al., 2003). Thus, bacterial PP2Cs control many diverse signalling pathways.

Although no PP2C has been identified in enterobacteria, eukaryotic-type protein phosphatases belonging to other families have been characterized. The PrpA and PrpB proteins of E. coli and S. enterica are PPPs that regulate transcription of the htrA gene through the CpxR/CpxA two-component regulatory system (Missiakas & Raina, 1997; Shi et al., 2001). The S. enterica SptP and Yersinia YopH proteins, which are delivered into host cells by a type III secretion system, possess a C-terminal tyrosine phosphatase domain that modulates the host-cell response to infection (Bliska et al., 1991; Fu & Galan, 1999). The Wzb protein is a low-molecular-mass protein tyrosine phosphatase that dephosphorylates the Wzc protein tyrosine kinase involved in the synthesis and export of exopolysaccharides in E. coli (Vincent et al., 1999).

Recently, the genomes of S. enterica serovar Typhimurium strain LT2, serovar Typhi strain CT18 and serovar Typhi strain Ty2 have been sequenced (Deng et al., 2003; McClelland et al., 2001; Parkhill et al., 2001). Comparative genomics showed that about 600 genes are unique to serovar Typhi (Parkhill et al., 2001). In this study, we identified an ORF (sty4824 or t4521) that is present in both serovar Typhi CT18 and Ty2 but absent from serovar Typhimurium LT2. This ORF encodes a putative protein with sequence similarity to PP2Cs. We designated this gene prpZ and its product PrpZ. We characterized the enzymic properties of the recombinant PrpZHis protein and showed that it has all the hallmarks of PP2Cs. In addition, we provide evidence that PrpZHis has broader substrate specificity than most PP2Cs, since it shows unusual reactivity towards phosphotyrosine residues.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
E. coli strain XL-1 Blue (Stratagene) was used for all DNA manipulations. E. coli strain BL21(DE3) (Novagen) was used for protein expression. S. enterica serovar Typhi strain CT18 was kindly provided by Dr France Daigle, Université de Montréal, Canada. Bacterial cultures were grown at 37 °C with aeration in Luria–Bertani broth medium containing 100 µg ampicillin ml–1, when appropriate.

Cloning of the prpZ gene.
Chromosomal DNA was isolated from an overnight culture of S. enterica serovar Typhi strain CT18 using Genomic-tips (Qiagen). The prpZ gene, which corresponds to ORF sty4824 of the S. enterica serovar Typhi CT18 genome sequence, was amplified by PCR using an upstream primer carrying an NdeI restriction site (5'-CCTCCACCATATGTTTACAGAACGACTTGCTCGCTG-3') and a downstream primer carrying an XhoI site (5'-CCGGCTCGAGATCTTCATTTTTGTTTTCCTCATCCTTG-3'). The PCR was performed using the Pwo DNA polymerase (Roche Diagnostics), which possesses proofreading activity. The amplified 1200-bp DNA fragment was digested with restriction enzymes NdeI and XhoI and cloned into pET-20b(+) (Novagen) that had been digested with the same enzymes. The insert was sequenced and the translated sequence was identical to ORF sty4824 of S. enterica serovar Typhi strain CT18 (Parkhill et al., 2001). The resulting plasmid (pET-PrpZHis) encodes the PrpZHis protein harbouring a C-terminal tag of six histidine residues.

Expression and purification of the recombinant PrpZHis protein.
Cultures of E. coli BL21(DE3) cells transformed with the pET-PrpZHis plasmid were induced with 1 mM IPTG at an OD600 of 0·8. After 1 h of induction, cells were harvested by centrifugation at 5000 g for 20 min and resuspended in BugBuster protein extraction reagent (Novagen) supplemented with Benzoase (Novagen) according to the manufacturer's instructions. The suspension was incubated on a shaking platform for 10 min at room temperature and centrifuged at 16 000 g for 20 min. The supernatant, containing PrpZHis, was applied to a HiTrap chelating column (Amersham Biosciences) charged with Ni2+ and equilibrated in binding buffer (20 mM Tris/HCl, pH 8·0, 500 mM NaCl and 5 mM imidazole). After extensive washing, the protein was eluted with a linear gradient of 5–200 mM imidazole in binding buffer. Fractions containing PrpZHis were pooled, dialysed against final buffer (20 mM Tris/HCl, pH 8·0, 10 % v/v glycerol) and stored at –70 °C. Under these conditions, approximately 1 mg pure protein was obtained per litre of bacterial culture. Protein concentrations were measured with the Bio-Rad protein assay kit, using BSA as the standard. The PrpZHis protein was visualized on SDS-PAGE gels stained with Coomassie blue. For Western blotting, the recombinant protein was detected using an anti-His·Tag monoclonal antibody (Novagen) and the His·Tag AP LumiBlot system (Novagen) according to the manufacturer's instructions. Mass spectrometry (MS) analyses were performed on a Voyager DE matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) instrument (Applied Biosystems) at the Sheldon Biotechnology Centre (McGill University).

Phosphatase assays using PNPP as a substrate.
Tablets of PNPP (Sigma) were dissolved immediately before use. Unless specified, reactions were performed in 200 µl assay buffer containing 50 mM CAPS/NaOH (pH 9·5), 0·02 % (v/v) {beta}-mercaptoethanol (BME), 10 mM PNPP and 5 mM MnCl2 or MgCl2. Reactions were initiated by adding purified PrpZHis and incubated for 30 min at 37 °C. The release of p-nitrophenol (PNP) was followed by measuring the A405 using a PowerWave microplate spectrophotometer (Bio-Tek Instruments). The molecular absorbance coefficient of PNP under these assay conditions was 17 800 cm–1 M–1 (Li, 1984). One unit of activity was defined as the release of 1 nmol PNP min–1 at 37 °C. Specific activity was defined as the number of enzyme units (µg protein)–1. Under these conditions, release of PNP was linear for about 60 min. The optimal pH for PNPP hydrolysis was determined with 50 mM concentrations of the following buffers: MES/NaOH (pH 6·5), HEPES/NaOH (pH 7·0–7·5), Tris/HCl (pH 8–8·5) and CAPS/NaOH (pH 9–11). The optimal concentrations of Mn2+ or Mg2+ for enzyme activity were determined in 50 mM CAPS/NaOH (pH 9·5) by varying the concentration of MnCl2 or MgCl2 in the assay buffer. The effect of various inhibitors on PrpZHis activity was assayed in the presence of 5 mM MnCl2 in 50 mM Tris/HCl (pH 8·5) to prevent precipitation of some inhibitors at higher pH. Kinetic parameters of PrpZHis with PNPP as the substrate were determined in 50 mM CAPS/NaOH (pH 9·5) in the presence of 5 mM MnCl2 using substrate concentrations ranging from 0·1 to 25 mM. Data were fitted to the Michaelis–Menten equation by non-linear regression analysis.

Phosphatase assays using phosphopeptides.
Phosphatase activity of PrpZHis was measured by using various synthetic phosphopeptides as substrates. Phosphorylated peptides were purchased from Promega [RRA(pT)VA] and Upstate Biotechnology [RRA(pS)VA and RRLIEDAE(pY)AARG]. The release of inorganic phosphate (Pi) was monitored by measuring the absorbance of the molybdate–malachite green–phosphate complex using the Serine/Threonine Phosphatase assay system (Promega). Reactions were performed in 50 µl assay buffer containing 50 mM CAPS/NaOH (pH 9·5), 0·02 % (v/v) BME, 1 mM phosphopeptide and 5 mM MnCl2. Reactions were initiated by adding 40 ng purified PrpZHis, incubated for 30 min at 37 °C and stopped by adding 50 µl of a molybdate dye/additive mixture. After 15 min incubation at room temperature to allow colour development, the A600 was measured. The amount of Pi generated was determined by using a standard curve. Optimum pH for enzyme activity against the phosphothreonine peptide was determined as described above. For the determination of kinetic parameters, initial velocities were measured at substrate concentrations ranging from 10 to 800 µM.

Phosphatase assays using phosphorylated myelin basic protein (MBP).
MBP was radiolabelled at Ser/Thr residues using the cAMP-dependent protein kinase (PKA) (New England Biolabs) or at tyrosine residues using the Abl protein tyrosine kinase (New England Biolabs). Reactions were performed in the presence of [{gamma}-32P]ATP as specified by the manufacturer. Phosphorylated MBPs were purified by TCA precipitation and extensive dialysis against buffer containing 25 mM Tris/HCl (pH 7·5), 0·1 mM EDTA, 2 mM DTT and 0·01 % (v/v) Brij 35. Assays were performed by incubating purified PrpZHis with 1·8 µM [32P]MBP in 50 mM Tris/HCl (pH 8·0), 0·02 % (v/v) BME and 5 mM MnCl2. At various time points, reactions (15 µl) were stopped by the addition of 4x Laemmli SDS sample buffer (250 mM Tris/HCl, pH 6·8, 8 % SDS, 40 % glycerol, 0·02 % bromophenol blue, 4 % BME). Reaction products were applied to 15 % SDS-PAGE gels. Gels were dried under vacuum and exposed to a phosphor screen (Kodak). Radiolabelled products were visualized using an FX scanner (Bio-Rad) and quantified by image analysis using the Quantity One software (Bio-Rad). Optimal pH for PrpZHis activity against [32P]MBP was determined as described above. For the determination of kinetic parameters, initial velocities were measured at substrate concentrations ranging from 0·5 to 50 µM. Reactions were stopped by the addition of 20 % TCA. Following centrifugation, the release of radiolabelled Pi was determined by scintillation counting.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
PrpZ is a member of the PPM family
The prpZ gene (ORF sty4824) was identified from the S. enterica serovar Typhi CT18 genome sequence (Parkhill et al., 2001). It encodes the PrpZ protein, which consists of 387 amino acid residues with an estimated molecular mass of 42·7 kDa and an isoelectric point of 5·6. Analysis of the PrpZ amino acid sequence revealed the presence of two domains. The N-terminal domain, which consists of approximately 260 amino acid residues, shows homology to PP2Cs (Fig. 1a). The C-terminal domain, consisting of 127 amino acid residues, shares no homology with any known protein (Fig. 1a). A BLASTP sequence similarity search using the PrpZ N-terminal domain (residues 1–260) identified more than 100 bacterial homologues from both Gram-negative and Gram-positive species. The amino acid sequence of the PrpZ phosphatase domain is 23–28 % identical to the various bacterial homologues and 18 % identical to the human PP2C{alpha}. Multiple sequence alignments reveal that PrpZ contains the motifs that are conserved among PP2Cs (data not shown) (Bork et al., 1996). Importantly, these conserved motifs contain the invariant residues involved in the binding of both the two metal ions (Asp 42, Asp 66, Asp 202 and Asp 240) and the phosphate group of substrates (Arg 37) (Das et al., 1996). Thus, sequence similarity and conservation of the essential catalytic residues between serovar Typhi PrpZ and human PP2C{alpha} suggest that PrpZ is a PP2C of the PPM family.



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Fig. 1. Expression and purification of recombinant PrpZHis PP2C. (a) Schematic representation of the PrpZ protein phosphatase. The N-terminal domain consists of the PP2C catalytic domain. The C-terminal domain is of unknown function. (b)–(c) Coomassie-blue-stained SDS-PAGE (b) and Western-blot analysis (c) of crude extracts and purified PrpZHis. The PrpZHis protein was detected using a monoclonal antibody directed against the C-terminal His tag. PrpZHis migrated with an apparent molecular mass of 51 kDa. Lanes: 1, crude extract prepared from uninduced E. coli cells; 2, crude extract prepared from induced E. coli cells; 3, purified PrpZHis (3 µg purified protein). Positions of molecular mass markers are indicated on the left.

 
Cloning, expression and purification of PrpZHis
To determine whether the prpZ gene encoded a functional protein phosphatase, the ORF corresponding to the PrpZ protein was PCR-amplified and cloned into expression vector pET20b(+) to generate plasmid pET-PrpZHis. The recombinant PrpZHis protein consists of the complete coding sequence of PrpZ fused to a C-terminal tag of six histidine residues. E. coli BL21(DE3) cells transformed with plasmid pET-PrpZHis were analysed for production of PrpZHis by SDS-PAGE. Upon IPTG induction, a protein that migrated with an apparent molecular mass of 51 kDa was detected in the soluble fraction of cell lysates (Fig. 1b, lane 2). To confirm that the overproduced protein corresponded to PrpZHis, a Western-blot analysis was performed using a monoclonal antibody directed against the C-terminal His tag. As shown in Fig. 1(c), the PrpZHis protein was detected by the antibody and migrated with an apparent molecular mass of 51 kDa. The calculated molecular mass of PrpZHis (43·8 kDa) is less than the apparent molecular mass deduced from SDS-PAGE (51 kDa). Following purification to apparent homogeneity by Ni2+-NTA chromatography (Fig. 1b, lane 3), the PrpZHis protein was subjected to MS analysis. The mass determined by MS (43 827 Da) was identical to the theoretical mass of PrpZHis (43·8 kDa), indicating that PrpZHis runs aberrantly on SDS-PAGE.

Phosphatase activity of PrpZHis using PNPP or a phosphothreonine peptide as substrates
PP2Cs have been shown to require divalent cations such as Mg2+ or Mn2+ for activity (Cohen, 1989). First, we determined the optimal pH for PrpZHis activity in the presence of 5 mM MnCl2 or MgCl2 using PNPP as a substrate. In the presence of MnCl2, the pH profile showed a maximum at pH 9·5 (Fig. 2a). In comparison, when MgCl2 was substituted for MnCl2, PrpZHis activity at pH 9·5 was reduced 40-fold (Fig. 2a). In the presence of 5 mM MgCl2, PrpZHis was essentially inactive at neutral pH and the optimal pH was 10·5 (Fig. 2a). To determine which divalent cations are effective in activating PrpZHis phosphatase activity, assays were performed in the presence of increasing concentrations of MgCl2, MnCl2, CaCl2 and ZnCl2. The release of PNP was greatly induced by the addition of Mn2+, with 2 mM MnCl2 stimulating half-maximal activity of PrpZHis (Fig. 2b). In contrast, Mg2+ barely stimulated PrpZHis activity (Fig. 2b). No activity was detected with Ca2+ or Zn2+ (data not shown). Thus, with PNPP as the substrate, PrpZHis shows a strong preference for Mn2+ over Mg2+ and its activity is optimal at alkaline pH.



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Fig. 2. Hydrolysis of PNPP by purified PrpZHis. (a) Influence of pH on PrpZHis activity. The phosphatase activity of PrpZHis was measured under standard conditions using PNPP (10 mM) as a substrate. PrpZHis (46 nM) was incubated in the presence of 5 mM MnCl2 ({bullet}) or 5 mM MgCl2 ({blacksquare}) as described in Methods. (b) Effect of Mn2+ and Mg2+ on PrpZHis activity. The phosphatase activity of PrpZHis was measured under optimal pH conditions using PNPP as a substrate. PrpZHis (0·4 µg) was incubated in 50 mM CAPS/NaOH (pH 9·5) in the presence of increasing concentrations of MnCl2 ({bullet}) or in the same buffer at pH 10·5 in the presence of increasing concentrations of MgCl2 ({blacksquare}). Specific activity is defined as nmol PNP released min–1 (µg protein)–1.

 
To examine the phosphatase activity of PrpZHis further, we used a synthetic peptide substrate containing a phosphothreonine residue [RRA(pT)VA]. Enzymic activity was determined by measuring the release of Pi as described in Methods. In the presence of 5 mM MnCl2, maximum PrpZHis activity towards the phosphothreonine peptide was observed at pH 9·0 (Fig. 3a). Strikingly, we found that PrpZHis activity was stimulated to similar levels when 5 mM MgCl2 was substituted for MnCl2 (Fig. 3a). In the presence of 5 mM MgCl2, the optimal pH for PrpZHis activity was 9·0–9·5. Maximal PrpZHis activity was achieved at 5 mM MnCl2 or MgCl2 (data not shown). These data indicate that Mn2+ and Mg2+ have a similar effect on the dephosphorylation of the phosphothreonine peptide. Thus, the metal-ion requirement of PrpZHis depends on the substrate used.



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Fig. 3. Dephosphorylation of synthetic phosphopeptides by purified PrpZHis. (a) Influence of pH on PrpZHis activity. The phosphatase activity of PrpZHis was measured under standard conditions using the peptide RRA(pT)VA (1mM) as a substrate. PrpZHis (18 nM) was incubated in the presence of 5 mM MnCl2 ({bullet}) or 5 mM MgCl2 ({blacksquare}). Buffers are described in Methods. (b) Michaelis–Menten plot of phosphopeptide dephosphorylation against substrate concentration. The activity of PrpZHis was measured in 50 mM CAPS/NaOH (pH 9·5) in the presence of 5 mM MnCl2. PrpZHis (18 nM) was incubated with RRA(pT)VA ({bullet}), RRA(pS)VA ({circ}) or RRLIEDAE(pY)AARG ({blacktriangleup}). Specific activity is defined as pmol Pi released min–1 (µg protein)–1.

 
To confirm that PrpZHis is a PP2C, we examined the effect of various inhibitors of protein phosphatases, acid phosphatases and alkaline phosphatases on PrpZHis activity using PNPP or the phosphothreonine peptide as substrates (Table 1). Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, did not inhibit PrpZHis activity. The same results were obtained for trifluoperazine, an inhibitor of protein phosphatases 2B, and ammonium molybdate, a general inhibitor of PPPs. Levamisole and sodium tartrate, which respectively inhibit alkaline phosphatases and acid phosphatases, had no effect on PrpZHis activity. As shown in Table 1, inhibition of PrpZHis was detected only with non-specific protein phosphatase inhibitors like sodium fluoride, sodium pyrophosphate and sodium phosphate when present at millimolar concentrations. As expected from a metal-dependent protein phosphatase, inhibition was also observed with EDTA. Altogether, these data confirm that PrpZHis has all the features of a PP2C.


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Table 1. Effect of various inhibitors on the catalytic activity of PrpZHis

Assays were performed in 50 mM Tris/HCl (pH 8·5) in the presence of 5 mM MnCl2 using 10 mM PNPP or 1 mM RRA(pT)VA as substrate.

 
Substrate specificity of PrpZHis
To examine the substrate specificity of PrpZHis, we used synthetic peptide substrates containing a phosphothreonine [RRA(pT)VA], a phosphoserine [RRA(pS)VA] or a phosphotyrosine [RRLIEDAE(pY)AARG] residue. Kinetic parameters of PrpZHis for these synthetic peptides were determined (Table 2). The PrpZHis phosphatase showed a fivefold reduction in catalytic efficiency (kcat/Km) for the serine phosphopeptide, compared with the threonine phosphopeptide. This decreased catalytic efficiency for the phosphoserine peptide was due to both an increase in Km and a decrease in kcat (Table 2). These data indicate that PrpZHis displays a preference for the phosphothreonine peptide. As shown in Fig. 3(b), PrpZHis was able to release Pi from the phosphotyrosine peptide, which is not a usual substrate for PP2Cs. Because the phosphotyrosine peptide substrate was not present at saturating levels (i.e. Km>1 mM), the kinetic parameters could not be determined with good accuracy. Compared with PNPP, we found that the phosphothreonine and phosphoserine peptides are better substrates for PrpZHis, with kcat/Km values approximately one order of magnitude higher (Table 2). Overall, these data show that PrpZHis displays a preference for phosphothreonine residues and is also able to dephosphorylate phosphotyrosine residues.


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Table 2. Kinetic parameters of PrpZHis with different substrates

For PNPP and phosphopeptides, assays were performed in 50 mM CAPS/NaOH (pH 9·5) in the presence of 5 mM MnCl2. For MBP, assays were performed in 50 mM Tris/HCl (pH 8·0) in the presence of 5 mM MnCl2.

 
PrpZHis dephosphorylates phospho-Ser/Thr and phospho-Tyr MBP
To examine the ability of PrpZHis to dephosphorylate protein substrates, we performed phosphatase assays using [32P]MBP phosphorylated at either Ser/Thr residues or Tyr residues. First, we determined the optimal pH of the reaction by incubating [32P]MBP phosphorylated at Ser/Thr residues with purified PrpZHis in the presence of 5 mM MnCl2 or MgCl2 and measuring the release of [32P]Pi. In the presence of MnCl2, optimal PrpZHis activity occurred at pH 8·0, since higher pHs induced precipitation of [32P]MBP (Fig. 4a). In the presence of MgCl2, optimal activity was observed at pH 9·5 (Fig. 4a). Interestingly, substantial amounts of [32P]MBP were dephosphorylated at neutral pH, regardless of the divalent cation present in the reaction (Fig. 4a). Similar pH optima were obtained for [32P]MBP phosphorylated at Tyr residues (data not shown). Substrate specificity of PrpZHis was further determined by measuring the dephosphorylation of [32P]MBP phosphorylated at Ser/Thr residues or Tyr residues over time (Fig. 4b). Assays were performed at pH 8 in the presence of 5 mM MnCl2. In the absence of PrpZHis, [32P]MBP was stable for at least 20 min under these experimental conditions (data not shown). Addition of purified PrpZHis stimulated the dephosphorylation of phospho-Ser/Thr MBP as well as phospho-Tyr MBP (Fig. 4b). The initial rate of dephosphorylation of [32P]MBP phosphorylated at Tyr residues was slightly slower than that of [32P]MBP phosphorylated at Ser/Thr residues (Fig. 4b). Similar results were obtained when reactions were performed in the presence of MgCl2 (data not shown). Dephosphorylation of [32P]MBP was strictly dependent on the presence of Mn2+ or Mg2+, since the presence of 10 mM EDTA inhibited the reaction (data not shown). Kinetic parameters were also determined for [32P]MBP phosphorylated at either Ser/Thr residues or Tyr residues. As shown in Table 2, the kcat/Km value for phospho-Tyr MBP was slightly lower than that for phospho-Ser/Thr MBP. These data confirmed the ability of PrpZHis in vitro to dephosphorylate protein substrates phosphorylated at Tyr residues. Taken together, these data show that PrpZHis is a PP2C with dual substrate specificity.



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Fig. 4. Dephosphorylation of [32P]MBP by PrpZHis. (a) Influence of pH on PrpZHis activity against phospho-Ser/Thr MBP. PrpZHis (15·6 nM) was incubated with 1·8 µM [32P]MBP in the presence of 5 mM MnCl2 ({bullet}) or 5 mM MgCl2 ({blacksquare}). Buffers are described in Methods. After 10 min of incubation, reactions were stopped by the addition of 4x Laemmli SDS sample buffer. Reaction products were analysed on 15 % SDS-PAGE. Amounts of radiolabelled Pi released were quantified with a PhosphorImager. (b) Kinetics of dephosphorylation of MBP phosphorylated either at Ser/Thr residues ({bullet}) or at Tyr residues ({circ}). PrpZHis (15·6 nM) was incubated with 1·8 µM [32P]MBP in 50 mM Tris/HCl (pH 8·0) in the presence of 5 mM MnCl2. At indicated time points, reactions were stopped by the addition of 4x Laemmli SDS sample buffer and analysed as indicated in (a).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we characterized the enzymic properties of PrpZ, a novel eukaryotic-type Ser/Thr protein phosphatase identified by the sequencing of the S. enterica serovar Typhi CT18 and Ty2 genomes (Deng et al., 2003; Parkhill et al., 2001). We showed that the purified recombinant PrpZHis protein is a functional protein phosphatase. PrpZHis displays the hallmarks of PP2Cs, as indicated by its amino acid sequence similarity to other PP2Cs, its Mn2+- or Mg2+-dependent enzymic activity and its insensitivity to okadaic acid. In addition, we found that PrpZHis possesses the ability to dephosphorylate phosphotyrosine residues, in vitro, which is unusual for a PP2C. PrpZ is the first PP2C to be identified in enterobacteria.

Comparison of PrpZHis reactivity towards different substrates (PNPP, phosphopeptides and phosphorylated MBP) revealed important differences with respect to pH range and metal-ion requirement. Using PNPP as a substrate, the highest specific activity of PrpZHis was observed at pH 9·5 in the presence of MnCl2 (Fig. 2a). With the same substrate, alkaline pH optima were also observed for the B. subtilis PrpC and the Synechocystis PCC 6803 PphA PP2Cs (Obuchowski et al., 2000; Ruppert et al., 2002). Although PrpZHis was barely active towards PNPP and the phosphothreonine peptide at neutral pH (Figs 2a and 3a), we found that it readily dephosphorylates [32P]MBP at pH 7·5 (Fig. 4a). In good agreement, both the B. subtilis PrpC and the Synechocystis PCC 6803 PphA PP2Cs were shown to dephosphorylate protein substrates at a pH close to 7·5 (Obuchowski et al., 2000; Ruppert et al., 2002). Thus, our data show that PrpZHis is active at physiological pH (pH 7·5) towards a protein substrate like [32P]MBP.

Like other PP2Cs, PrpZHis activity is strictly dependent on Mn2+ or Mg2+ in vitro. Using PNPP as the substrate, PrpZHis showed a strong preference for Mn2+ (Fig. 2b). This preference for Mn2+ over Mg2+, with PNPP as a substrate, is similar to that of other PP2Cs like the Stp1 phosphatases of Streptococcus agalactiae and Pseudomonas aeruginosa (Mukhopadhyay et al., 1999; Rajagopal et al., 2003), the B. subtilis PrpC phosphatase (Obuchowski et al., 2000), the Myxococcus xanthus Pph1 phosphatase (Treuner-Lange et al., 2001), the Synechocystis PCC 6803 PphA phosphatase (Ruppert et al., 2002) and the PstP (MstP) phosphatase of Mycobacterium tuberculosis (Chopra et al., 2003). Using the phosphothreonine peptide as a substrate, we found that Mg2+ is as effective as Mn2+ in catalysing substrate dephosphorylation (Fig. 3a). Similar differences in metal requirement were also observed for other bacterial PP2Cs. For example, Mg2+ was only twofold less effective than Mn2+ in stimulating the Myxococcus xanthus Pph1 activity towards phosphopeptides, whereas hydrolysis of PNPP was strictly Mn2+-dependent (Treuner-Lange et al., 2001). Altogether, these data show that information obtained with the substrate PNPP may not reflect the enzymic properties obtained with other substrates like phosphopeptides or phosphorylated proteins.

An important question is to identify the metal ion used in vivo by PrpZ. The intracellular concentration of Mg2+ (1–10 mM) is at least one order of magnitude higher than that of Mn2+ (10–100 µM) (Finney & O'Halloran, 2003). Based on our finding that both Mg2+ and Mn2+ can stimulate the dephosphorylation of [32P]MBP at neutral pH (Fig. 4a), it appears most likely that Mg2+ is the main physiologically relevant metal ion for PrpZ activity in vivo. In enterobacteria, levels of intracellular Mn2+ have been reported to vary over two orders of magnitude, reaching the millimolar range under appropriate environmental conditions (Kehres & Maguire, 2003). Thus, it cannot be ruled out that increasing concentrations of intracellular Mn2+ may further stimulate PrpZ activity. Variations in the intracellular concentration of Mn2+ might be one of the mechanisms used in vivo to regulate the activity of PrpZ.

Using synthetic phosphopeptides, we showed that PrpZHis displays a preference for phosphothreonine over phosphoserine (Table 2). A similar preference for phosphothreonine residues has also been reported for the Synechocystis PCC 6803 PphA and the Myxococcus xanthus Pph1 PP2Cs (Ruppert et al., 2002; Treuner-Lange et al., 2001). Unexpectedly, we found that PrpZHis also dephosphorylates the phosphotyrosine peptide (Table 2). The ability of PrpZHis to dephosphorylate phosphotyrosine was confirmed with MBP phosphorylated at Tyr residues (Fig. 4b). These data indicate that PrpZHis has dual specificity in vitro. In contrast, the Mycobacterium tuberculosis PstP (MstP) PP2C showed little or no activity with phosphotyrosine protein substrates (Boitel et al., 2003; Chopra et al., 2003). To date, the PphA protein of Synechocystis PCC 6803 is the only PP2C that has been reported to dephosphorylate phosphotyrosine residues in vitro (Ruppert et al., 2002). This ability to dephosphorylate phosphotyrosine residues has also been observed for other Ser/Thr phosphatases of the PPP family. For example, PrpE of B. subtilis and the protein Ser/Thr phosphatase encoded by bacteriophage {lambda} showed activity towards phosphotyrosine (Barik, 1993; Iwanicki et al., 2002). Thus, until the physiological substrate(s) of PrpZ is identified and characterized, we cannot rule out the possibility that PrpZ might hydrolyse phosphotyrosine residues in vivo.

The physiological function of PrpZ is unknown. Like the S. enterica SptP and Yersinia YopH tyrosine phosphatases, PrpZ might be a type III effector protein injected into host cells through one of the two S. enterica type III secretion systems. This possibility is unlikely, because PrpZ does not contain the N-terminal domain of type III effector proteins that is essential for both targeting to the secretion apparatus and chaperone recognition (Smith et al., 2001). Alternatively, PrpZ might be a cytosolic protein that modulates the phosphorylation level of a still unknown protein substrate. This substrate might be a protein whose function is regulated through reversible phosphorylation by PrpZ and an opposing protein kinase. Many of the genes encoding bacterial PP2Cs are genetically linked with Ser/Thr protein kinase genes. Accordingly, two ORFs encoding proteins with homology to Ser/Thr protein kinases (sty4822 and sty4823) were found in the same genomic region as the prpZ gene. Thus, it is most likely that PrpZ and these two protein kinases are part of the same signalling pathway. Recently, a microarray study showed that transcription of the prpZ gene decreased by more than threefold upon cell exposure to hydrogen peroxide, suggesting that PrpZ might be involved in a signalling pathway controlling oxidative stress (Porwollik et al., 2003). Since prpZ and the neighbouring protein kinase genes are present in S. enterica serovar Typhi but absent from the closely related S. enterica serovar Typhimurium, understanding the roles of PrpZ and the opposing protein kinases is likely to provide important insights into differences in lifestyle and/or pathogenesis between serovar Typhi and serovar Typhimurium.


   ACKNOWLEDGEMENTS
 
This work was supported by Discovery Grant 217482 from the Natural Sciences and Engineering Research Council (NSERC). H. L. M. was the recipient of a fellowship from Fonds de la Recherche en Santé du Québec (FRSQ). S. M. L. was the recipient of a studentship from FRSQ. We thank Dr France Daigle for providing strain CT18. We appreciate the contribution of Dr Martin Olivier and Dr Maritza Jaramillo in the early stages of this project.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Barford, D., Das, A. K. & Egloff, M. P. (1998). The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27, 133–164.[CrossRef][Medline]

Barik, S. (1993). Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda. Proc Natl Acad Sci U S A 90, 10633–10637.[Abstract/Free Full Text]

Bliska, J. B., Guan, K. L., Dixon, J. E. & Falkow, S. (1991). Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci U S A 88, 1187–1191.[Abstract/Free Full Text]

Boitel, B., Ortiz-Lombardia, M., Duran, R., Pompeo, F., Cole, S. T., Cervenansky, C. & Alzari, P. M. (2003). PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis. Mol Microbiol 49, 1493–1508.[CrossRef][Medline]

Bork, P., Brown, N. P., Hegyi, H. & Schultz, J. (1996). The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues. Protein Sci 5, 1421–1425.[Abstract/Free Full Text]

Chopra, P., Singh, B., Singh, R. & 9 other authors (2003). Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB. Biochem Biophys Res Commun 311, 112–120.[CrossRef][Medline]

Cohen, P. (1989). The structure and regulation of protein phosphatases. Annu Rev Biochem 58, 453–508.[CrossRef][Medline]

Das, A. K., Helps, N. R., Cohen, P. T. & Barford, D. (1996). Crystal structure of the protein serine/threonine phosphatase 2C at 2·0 Å resolution. EMBO J 15, 6798–6809.[Abstract]

Deng, W., Liou, S. R., Plunkett, G., III, Mayhew, G. F., Rose, D. J., Burland, V., Kodoyianni, V., Schwartz, D. C. & Blattner, F. R. (2003). Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18. J Bacteriol 185, 2330–2337.[Abstract/Free Full Text]

Doublet, P., Vincent, C., Grangeasse, C., Cozzone, A. J. & Duclos, B. (1999). On the binding of ATP to the autophosphorylating protein, Ptk, of the bacterium Acinetobacter johnsonii. FEBS Lett 445, 137–143.[CrossRef][Medline]

Duncan, L., Alper, S., Arigoni, F., Losick, R. & Stragier, P. (1995). Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division. Science 270, 641–644.[Abstract]

Finney, L. A. & O'Halloran, T. V. (2003). Transition metal speciation in the cell: insights from the chemistry of metal ion receptors. Science 300, 931–936.[Abstract/Free Full Text]

Fu, Y. & Galan, J. E. (1999). A Salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion. Nature 401, 293–297.[CrossRef][Medline]

Hunter, T. (1995). Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225–236.[CrossRef][Medline]

Irmler, A. & Forchhammer, K. (2001). A PP2C-type phosphatase dephosphorylates the PII signaling protein in the cyanobacterium Synechocystis PCC 6803. Proc Natl Acad Sci U S A 98, 12978–12983.[Abstract/Free Full Text]

Iwanicki, A., Herman-Antosiewicz, A., Pierechod, M., Seror, S. J. & Obuchowski, M. (2002). PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity. Biochem J 366, 929–936.[Medline]

Kehres, D. G. & Maguire, M. E. (2003). Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria. FEMS Microbiol Rev 27, 263–290.[CrossRef][Medline]

Kennelly, P. J. (2002). Protein kinases and protein phosphatases in prokaryotes: a genomic perspective. FEMS Microbiol Lett 206, 1–8.[CrossRef][Medline]

Kennelly, P. J. (2003). Archaeal protein kinases and protein phosphatases: insights from genomics and biochemistry. Biochem J 370, 373–389.[CrossRef][Medline]

LaPorte, D. C., Stueland, C. S. & Ikeda, T. P. (1989). Isocitrate dehydrogenase kinase/phosphatase. Biochimie 71, 1051–1057.[CrossRef][Medline]

Li, H. C. (1984). Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+. J Biol Chem 259, 8801–8807.[Abstract/Free Full Text]

McClelland, M., Sanderson, K. E., Spieth, J. & 23 other authors (2001). Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413, 852–856.[CrossRef][Medline]

Missiakas, D. & Raina, S. (1997). Signal transduction pathways in response to protein misfolding in the extracytoplasmic compartments of E. coli: role of two new phosphoprotein phosphatases PrpA and PrpB. EMBO J 16, 1670–1685.[Abstract/Free Full Text]

Mukhopadhyay, S., Kapatral, V., Xu, W. & Chakrabarty, A. M. (1999). Characterization of a Hank's type serine/threonine kinase and serine/threonine phosphoprotein phosphatase in Pseudomonas aeruginosa. J Bacteriol 181, 6615–6622.[Abstract/Free Full Text]

Muñoz-Dorado, J., Inouye, S. & Inouye, M. (1991). A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium. Cell 67, 995–1006.[CrossRef][Medline]

Obuchowski, M., Madec, E., Delattre, D., Boel, G., Iwanicki, A., Foulger, D. & Seror, S. J. (2000). Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family. J Bacteriol 182, 5634–5638.[Abstract/Free Full Text]

Parkhill, J., Dougan, G., James, K. D. & 38 other authors (2001). Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413, 848–852.[CrossRef][Medline]

Porwollik, S., Frye, J., Florea, L. D., Blackmer, F. & McClelland, M. (2003). A non-redundant microarray of genes for two related bacteria. Nucleic Acids Res 31, 1869–1876.[Abstract/Free Full Text]

Rajagopal, L., Clancy, A. & Rubens, C. E. (2003). A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth, cell segregation, and virulence. J Biol Chem 278, 14429–14441.[Abstract/Free Full Text]

Ruppert, U., Irmler, A., Kloft, N. & Forchhammer, K. (2002). The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein PII. Mol Microbiol 44, 855–864.[CrossRef][Medline]

Shi, L. (2004). Manganese-dependent protein O-phosphatases in prokaryotes and their biological functions. Front Biosci 9, 1382–1397.[Medline]

Shi, L. & Zhang, W. (2004). Comparative analysis of eukaryotic-type protein phosphatases in two streptomycete genomes. Microbiology 150, 2247–2256.[CrossRef][Medline]

Shi, L., Potts, M. & Kennelly, P. J. (1998). The serine, threonine, and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait. FEMS Microbiol Rev 22, 229–253.[CrossRef][Medline]

Shi, L., Kehres, D. G. & Maguire, M. E. (2001). The PPP-family protein phosphatases PrpA and PrpB of Salmonella enterica serovar Typhimurium possess distinct biochemical properties. J Bacteriol 183, 7053–7057.[Abstract/Free Full Text]

Smith, C. L., Khandelwal, P., Keliikuli, K., Zuiderweg, E. R. & Saper, M. A. (2001). Structure of the type III secretion and substrate-binding domain of Yersinia YopH phosphatase. Mol Microbiol 42, 967–979.[CrossRef][Medline]

Stock, J. B., Ninfa, A. J. & Stock, A. M. (1989). Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53, 450–490.[Medline]

Treuner-Lange, A., Ward, M. J. & Zusman, D. R. (2001). Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development. Mol Microbiol 40, 126–140.[CrossRef][Medline]

Vijay, K., Brody, M. S., Fredlund, E. & Price, C. W. (2000). A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the {sigma}B transcription factor of Bacillus subtilis. Mol Microbiol 35, 180–188.[CrossRef][Medline]

Vincent, C., Doublet, P., Grangeasse, C., Vaganay, E., Cozzone, A. J. & Duclos, B. (1999). Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol 181, 3472–3477.[Abstract/Free Full Text]

Wang, J. Y. & Koshland, D. E., Jr (1978). Evidence for protein kinase activities in the prokaryote Salmonella typhimurium. J Biol Chem 253, 7605–7608.[Abstract]

Received 25 August 2004; revised 15 December 2004; accepted 26 December 2004.



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