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
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ABSTRACT |
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INTRODUCTION |
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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 250300 amino acid residues and contains 11 conserved motifs (Bork et al., 1996
). The three-dimensional structure of the human PP2C
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.
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METHODS |
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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 5200 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) -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 cm1 M1 (Li, 1984
). One unit of activity was defined as the release of 1 nmol PNP min1 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 7·07·5), Tris/HCl (pH 88·5) and CAPS/NaOH (pH 911). 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 MichaelisMenten 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 molybdatemalachite greenphosphate 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 [-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.
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RESULTS |
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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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DISCUSSION |
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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+ (110 mM) is at least one order of magnitude higher than that of Mn2+ (10100 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
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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 25 August 2004;
revised 15 December 2004;
accepted 26 December 2004.
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