Phosphorylation of Mycobacterium tuberculosis Ser/Thr Phosphatase by PknA and PknB

Background The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs. Principal Findings PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn2+-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr137, Thr141, Thr174 and Thr290 being the target residues of PknB while Thr137 and Thr174 are phosphorylated by PknA. The Mn2+-ion binding residues Asp38 and Asp229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstPc D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent. Conclusions and Significance This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.


Introduction
Mycobacterium tuberculosis has an array of proteins to ensure its existence during the course of infection. In order to thrive and maintain its homeostasis, the pathogen continuously influences its surroundings mainly through surface-located sensor proteins. Extracellular signals are communicated through the sensors to the cytosol leading to the appropriate cell responses. Apparently, a large number of pathogens employ reversible phosphorylation of proteins by kinases and phosphatases as a way of transmitting the signals from extracellular milieu which helps in their survival and pathogenicity [1][2][3][4]. Kinases carry out the phosphorylation by transferring the phosphate moiety on target proteins and phosphatases convert them back to the unphosphorylated state, either by dephosphorylating the substrate or by regulating the activity of kinases.
Apart from the well recognized two component systems targeting His/Asp residues in bacteria, Ser, Thr and Tyr residues are also the major targets for phosphorylation. M. tuberculosis is known to have 11 Ser/Thr protein kinases (STPKs PknA-L, except C), one tyrosine kinase (PtkA), one Ser/Thr phosphatase (PstP) and two tyrosine phosphatases (PtpA and PtpB) [5,6]. Till date a large number of mycobacterial proteins are shown to be regulated through phosphorylation by STPKs [7][8][9][10][11]. Some of these substrates are also known to be dephosphorylated by PstP [9,[11][12][13][14][15][16][17]. PstP is a PP2C phosphatase (PPM family) that strictly requires Mn 2+ -ion for its activity [13]. It is a membrane localized enzyme with intracellular catalytic domain of 237 amino acids joined by a juxtamembrane region to the extracellular domain of 191 residues with a single transmembrane helix [18]. Using multiwavelength anomalous diffraction studies, Pullen et al. determined the structure of the catalytic phosphatase domain of PstP [18]. PstP contains three metal-binding centers in its structure in contrast to two metal centers found in most of the PP2C phosphatases. Using atomic absorption spectroscopy and X-ray analysis, it has been shown that all the bound metal-ions are Mn 2+ . Similarities between Human Ser/Thr phosphatase PP2Ca and the mycobacterial enzyme have been explained on the basis of structural folds, metal binding and conserved residues [18]. Mutational analyses of PP2Ca have depicted the significance of certain conserved amino acid residues [19]. The corresponding residues in PstP are involved in binding to metal-ions and catalysis in addition to managing the binding and release of phosphate moiety. These residues in PP2Ca are critical for its activity [19] and thus, they are hypothesized to be important for PstP also.
The interesting feature of M. tuberculosis Ser/Thr signaling molecules is that both the essential STPKs, PknB (Rv0014c) and PknA (Rv0015c) and the only Ser/Thr phosphatase PstP (Rv0018c) are located in the same genomic cluster which is conserved in several mycobacterial species [6,9,20]. Transcriptional analysis in earlier studies revealed that PknA, PknB and PstP show similar expression profiles [20] and thus, implicate that strong regulation is required for their own functions as both the classes of enzymes functionally counteract each other. In this study, we show that the activity of PstP is modulated by phosphorylation. This is the first report on the regulation of any bacterial Ser/Thr phosphatase by post-translation modification. PstP was found to be phosphorylated differentially by PknA and PknB, both in vitro and in the surrogate host Escherichia coli. Additionally, we found that zinc ions (Zn 2+ ) and inorganic phosphate (Pi) can inhibit the activity of PstP which in turn affects the phosphorylation status of both the kinases and phosphatase.

Materials and Methods
Bacterial strains and growth conditions E. coli DH5a strain (Novagen) was used for cloning and BL21 (DE3) (Stratagene) was used for the expression of recombinant proteins. E. coli cells were grown and maintained with constant shaking (220 rpm) at 37uC in LB medium supplemented with 100 mg/ml ampicillin.

Gene manipulation
The genes coding for PknA c (rv0015c, representing the cytosolic region of 1-337aa) and PstP (rv0018c, PstP: 1-514aa) were PCR amplified using M. tuberculosis H37Rv genomic DNA. Resulting PCR products were digested with corresponding restriction enzymes and ligated into the vectors pProEx-HTc (Invitrogen) and/or pGEX-5X-3 (GE Healthcare Bio-Sciences) previously digested with the same enzymes. Htc-PknB c and Htc-PstP c were obtained as described earlier [9]. pGEX-PknB c was sub-cloned from Htc-PknB c using standard protocols under the same restriction sites. For cloning in dual-expression vector pETDuet-1 (Novagen), genes coding for PstP c or PstP c D38G were inserted in MCS1 having N-terminal His 6 -tag while kinases PknA and PknB (full length) were cloned in MCS2 with N-terminal MBP-tag (Maltose-binding protein tag upstream of the kinase). MBP-alone (without kinase) was taken as control vector having PstP c or PstP c D38G in MCS1. The protocols used for cloning in pETDuet-1 have been discussed earlier [21].
Mutagenesis of specific residues was carried out using the QuikChange XL site-directed mutagenesis kit (Stratagene) as per manufacturer's instructions. Mutants of PstP and PstP c were created as R20G, D38G and D229G using Htc-PstP and Htc-PstP c as templates. Htc-PstP c was utilized for the generation of Htc-PstP c t5a and Htc-PstP c t141e . Htc-pknB c was employed as template for generation of double mutant Htc-pknB c t171/173d . The details of all the primers and clones are provided in tables 1 and 2, respectively. The integrity of all clones was confirmed by DNA sequencing (TCGA, New Delhi).

Protein expression and purification
Proteins were expressed and purified from E. coli as described previously [9]. The purified proteins were assessed by SDS-PAGE and concentrations were estimated by Bradford assay (Bio-Rad).

In vitro kinase assays and phosphoamino acid analysis
In vitro phosphorylation of PstP c or its mutants (0.5-3 mg) by PknA c (0.5-1 mg) or PknB c (1-3 mg) was carried out in kinase buffer (20 mM PIPES [pH 7.2], 5 mM MnCl 2 , 5 mM MgCl 2 ) containing 2 mCi [c-32 P]ATP (BRIT, Hyderabad, India) followed by incubation at 25uC for 20 min. Reactions were terminated by 5X SDS sample buffer followed by boiling at 100uC for 5 min. Proteins were separated by 12% SDS-PAGE and analyzed by PhosphorImager (FLA 2000, Fuji). Zn 2+ and Pi were added to the kinase assay reactions as per requirement of the assay. For the visualization of phosphorylation signal on cleaved proteins, removal of recombinant tags was achieved by addition of TEV protease (for His 6 -tagged PstP/PstP c and their mutants) in TEV buffer (Tris-Cl [pH 8.5], 5 mM EDTA, 300 mM NaCl and 1 mM DTT) after the kinase reaction followed by an additional incubation for 2 hr at 20uC. For phosphoamino acid analysis, PstP c D38G was phosphorylated by PknB c and PknA c and cleaved with TEV protease as mentioned above, separated by SDS-PAGE and electroblotted onto Immobilon PVDF membrane (Millipore). Phosphoamino acid analysis by two-dimensional thin layer electrophoresis was performed as described earlier [9,22].
In vitro dephosphorylation and p-nitrophenol phosphate (pNPP) hydrolysis assays (2 mg each) were autophosphorylated by in vitro kinase assays and exposed to dephosphorylation by PstP c and PstP c D38G (1 mg). Reactions were stopped by adding 5X SDS sample buffer and boiled for 5 min at 100uC. The samples were separated by 12% SDS-PAGE and phosphorylated bands were observed and analysed by PhosphorImager. pNPP hydrolysis assay was performed as a measure of phosphatase activity. PstP c was added to a reaction mixture containing phosphatase assay buffer (50 mM Tris pH 8.0, 5 mM DTT, 4 mM MnCl 2 ) and 10 mM pNPP in a 96-well plate and incubated at 37uC for indicated time points and absorbance was read at 405 nm (Microplate reader, Bio-Rad). To assay the relative activity of PstP c and its phosphatase-deficient variants, increasing concentrations of enzymes were added to the reaction mix and processed as above. Alkaline phosphatase (Roche) and PknB c were taken as positive and negative controls, respectively, for the pNPP hydrolysis assays. Variations of PstP c activity by addition of Zn 2+ and Pi was assessed by adding ZnCl 2 or sodium phosphate [pH 7.2] to the reaction mixture as above, to achieve the indicated final concentrations. pETDuet-1 purified PstP c and PstP c D38G , coexpressed with or without kinases, were employed for pNPP-assays to measure the effect of phosphorylation on their activities.

Metabolic labeling in E. coli
The procedure described by Kumar et al. was followed for metabolic labeling [23]. E. coli (BL21-DE3) transformants harbouring either pETDuet-PstP c /PstP c D38G -mbp or pETDuet-PstP c /PstP c D38G -mbpPknA or pETDuet-PstP c /PstP c D38G -mbpPknB were grown in 5 ml LB medium containing 100 mg/ml ampicillin to an O.D 600 of ,0.6. The cells were induced with 1 mM IPTG and further grown for 4 hr at 16uC. Cultures were harvested, washed with 5 ml of M9 medium [pH 7.0] without phosphate salts (for 1 L: NH 4 Cl-1 g, NaCl-0.5 g, 20% Glucose-10 ml, MgSO 4 .7H 2 O-1 ml, Thiamine-HCl-1 ml, CaCl 2 -1 ml). The cells were resuspended in 2 ml of M9 media supplemented with 1 mCi of [ 32 P]orthophosphoric acid (BRIT, Hyderabad, India), 100 mg/ml ampicillin and 1 mM IPTG and further grown at 16uC for 4 hr. Under specific conditions, Sodium phosphate [pH 7.2] (2 mM) or ZnCl 2 (4 mM) were added to M9 media and subsequent processing steps of metabolic labeling. The cells were harvested and lysed by sonication in the lysis buffer containing phosphate-buffered saline, 5% glycerol and protease inhibitor cocktail. The cell lysate was clarified and the lysates containing His 6 -fusion protein were incubated with lysis buffer equilibrated Ni 2+ -NTA affinity beads for 2 hr at 4uC. The beads were then thoroughly washed with lysis buffer containing 20 mM imidazole and resuspended in 5X SDS sample buffer followed by boiling for 15 min. The samples were resolved on SDS-PAGE followed by autoradiography.

D38G
PknB c and PknA c were employed for in vitro kinase assay using 50 mM cold ATP and PstP c D38G . The samples were run on 12% SDS-PAGE, stained with Coomassie Brilliant Blue and de-stained. Bands corresponding to PstP c D38G were excised from the gel and washed with MilliQ water. The samples were processed for identification of phosphorylation sites by using Thermo-Finnagen LTQ electrospray instrument (Proteomics Core Facility, Children's Hospital, Boston). The detailed protocol of sample processing for identification of phosphorylation sites has been provided in File S1.

Generation of polyclonal antibodies for PstP c in rabbit and immunoblotting
Polyclonal antibodies against PstP c were generated in rabbit. To confirm the presence of PstP c /PstP c D38G in Ni 2+ -NTA pulled-out proteins after metabolic labeling by western blot analysis, the samples were resolved by SDS-PAGE along with positive (purified PstP c ) and negative controls (GST-PknB c ) and transferred onto nitrocellulose membrane (Bio-Rad). Standard procedure for immunoblotting was followed [9,11]. The blots were developed using SuperSignal R West Pico Chemiluminescent Substrate kit (Pierce Protein Research Products) according to manufacturer's instructions.

Identification of the residues critical for the activity of PstP
On the basis of structural data available for PstP and alignment with the residues important for Human PP2Ca activity [18], PstP c mutants were generated using site-directed mutagenesis. These residues include the Mn 2+ -ion binding sites-Asp 38 and Asp 229 and phosphate (Pi) binding residue-Arg 20 ( Figure 1A). In the resulting mutants, these sites were converted to Glycine (PstP c D38G , PstP c D229G and PstP c R20G ). The activity of these mutants was compared using chromogenic substrate pNPP. To confirm the authenticity of the assay, increasing concentrations of alkaline phosphatase were utilized as a positive control while PknB c was used as negative control ( Figure S1). The pNPP assay with increasing amounts of PstP c -mutants showed that the mutation of Asp 38 and Asp 229 to Gly resulted in .90% loss of the dephosphorylation activity of PstP c , while the PstP c R20G mutant lost about 60% of its activity ( Figure 1B and 1C). Thus, Arg 20 , Asp 38 and Asp 229 were identified as the residues required for optimum activity of PstP. To confirm that the loss in activity was specifically due to mutagenesis of Asp 38 , Asp 229 and Arg 20 , irrelevant residues (Thr 5 and Thr 141 ) in PstP c were mutagenized to generate PstP c T5A and PstP c T141E . The relative activities of these mutants were compared with the native enzyme through pNPP-assay ( Figure S2). There were no significant changes observed in the mutants in comparison to PstP c , thus reinforcing the importance of Arg 20 , Asp 38 and Asp 229 residues.

Phosphatase activity of PstP c and its mutants
The dephosphorylation potential of PstP c and its mutants was also assessed by their ability to dephosphorylate PknB c in a timedependent dephosphorylation ( Figure 2A) and pNPP hydrolysis assays ( Figure S3). PstP c R20G dephosphorylated the autophosphorylated PknB c to some extent, whereas substantial loss of phosphatase activity was observed with PstP c D38G and PstP c D229G ( Figure 2A). The activity of PstP c D229G was relatively higher than that of PstP c D38G as opposed to the observation in pNPP-assays ( Figures 1C and S3). Similar observations have been reported earlier where the activity of an enzyme, specifically Ser/Thr phosphatases, is shown to be dependent on the nature of substrate [24][25][26]. pNPP is an artificial substrate while PknB is a natural substrate of PstP, which may be recognized and subsequently dephosphorylated more optimally. Additionally, in this case, the activity of the phosphatase also depends on the activity of PknB, as discussed in later sections. The assays were also performed using autophosphorylated PknA c which showed similar results (data not shown). Surprisingly, in this assay, additional phosphorylated bands corresponding to the size of PstP c D38G were observed when incubated with kinase for longer time. No such bands were observed with PstP c , PstP c R20G and PstP c D229G at the given concentrations. To further assess this observation, PknA c or PknB c were incubated with increasing concentrations of PstP c . Interestingly, PstP c was phosphorylated by PknA c at higher kinase to phosphatase ratio ( Figure 2B). An increase in the concentration of PstP c resulted in complete dephosphorylation of both the proteins. This serendipitous observation intrigued us to explore whether PstP is a target of Ser/Thr protein kinases. Due to strong dephosphorylation activity of PstP, it was difficult to achieve the phosphotransfer on native phosphatase. Therefore, further studies were carried out with the mutants of PstP that were deficient in phosphatase activity.
Phosphorylation of PstP c D38G , PstP c D229G and PstP c

R20G
After identification of the residues critical for PstP c activity and measuring the activity of corresponding mutants, the phosphorylation status of PstP c mutants was studied. PknA and PknB were employed for the phosphorylation assays. PstP c D38G and PstP c D229G were efficiently phosphorylated by both PknA c and PknB c ( Figure 3A), whereas faint signal on PstP c R20G was observed owing to its partial phosphatase activity. Phosphorylation of PstP c (at 3 mg concentration) was not observed by in vitro kinase assay as it completely dephosphorylated PknA c and PknB c , making them inactive (heat-inactive PstP c was found to be phosphorylated-data not shown). To confirm that the observed phosphorylation is on PstP c -mutants and not on the N-terminally attached His 6 -tag, TEV-protease cleavage of the tag was performed after the kinase assays. Phosphorylation was confirmed to be specifically localized on the cleaved substrate protein (data not shown). Additionally, the R20G, D38G and D229G mutants were also created in full length PstP construct and pNPP-hydrolysis assays and phosphorylation reactions were also confirmed using full length PstP and its mutants (data not shown).

Activity assays of pETDuet1-purified PstP c and PstP c D38G
The activity profiles of PstP c and PstP c D38G co-expressed with and without PknA/PknB, were evaluated. According to the pNPP assays, the activity of phosphorylated PstP c (co-expressed with PknA or PknB) was higher than that of unphosphorylated phosphatase (co-expressed with MBP alone) ( Figure 4C). The phenomenon was also confirmed by measuring the activity of PstP c D38G . As already discussed, PstP c D38G had retained about 10% of the dephosphorylation activity as a result of which, it was phosphorylated efficiently by kinases. The relative activity of phosphorylated PstP c D38G with PknA/PknB and unphosphorylated protein was measured for 420 min. Interestingly, the activity of phosphorylated PstP c D38G was remarkably higher than that of unphosphorylated protein, thus the similar profile as that of PstP c was observed ( Figure 4D). Also, the activity of PknA phosphorylated phosphatase was even more than the protein phosphorylated by PknB. Noticeably, the increase in phosphatase activity after phosphorylation may also account for the observed increase in the activity of PstP c D229G in the time-dependent dephosphorylation assays (Figure 2A).
The dephosphorylation of in vitro autophosphorylated PknA c was assessed by PstP c D38G +MBP, PstP c D38G +MBP-PknA and PstP c D38G +MBP-PknB. As expected, due to higher activity of phosphorylated PstP c D38G , intensity of phosphorylation on PknA c was low as compared to the reaction containing unphosphorylated PstP c D38G +MBP (Figures 4E and S4). Also, since PknA-phosphorylated PstP c D38G was more active than PknB-phosphorylated PstP c D38G ( Figure 4D), the extent of dephosphorylation was more in lane 3 as compared to lane 4.

Auto-dephosphorylation of PstP c
Next, we tried to understand whether the inability of PstP c to be effectively phosphorylated was due to its dephosphorylation activity on the kinases resulting in their inactivation or it was due to auto-dephosphorylation. Consequently, phosphomimetic mutants of PknB c were generated for the Thr residues of activation loop in catalytic domain [12], forming PknB c T171/173D which cannot be dephosphorylated by PstP c on Thr 171 and Thr 173 . As reported by Boitel et al., PknB does not lose phosphorylation signals after mutagenesis of Thr 171 and Thr 173 . Through a series of careful analysis of single and double mutants of PknB, it has been shown that PknB can be additionally phosphorylated on Ser 166 and/or Ser 169 residues [12]. Thus, we utilized PknB c and PknB c T171/173D , that were autophosphorylated in an in vitro kinase assay using [c-32 P]ATP, before incubation with PstP c . Phosphorylation of PstP c was still not observed with constitutively active PknB c T171/173D , as confirmed by phosphotransfer observed on PstP c D38G ( Figure 5A). This suggests that PstP c can dephosphorylate itself. Additionally, PknB c T171/173D was completely dephosphorylated in presence of PstP c , suggesting that PstP could also dephosphorylate the surplus sites Ser 166 /Ser 169 .

Identification of the factors affecting the activity of PstP
The phosphorylation of PstP suggested that additional factors may be involved in the cellular milieu that can regulate and control the phosphatase activity, preceding its phosphorylation. In general, phosphatases are known to be affected by a number of factors like metal-cations, Pi, creatine phosphate (CP) and ATP/ADP ratio. PstP c activity assay was carried out in the presence of selected factors. Interestingly, activity of PstP c was reduced in the presence of Zn 2+ and Pi, as assessed by pNPP assay. Reduction of almost 50% activity was observed at 0.2 mM Zn 2+ ( Figure 5B) and 0.5 mM Pi ( Figure 5C). Maximum inhibition of PstP c was observed at 1 mM Zn 2+ and 4 mM Pi. Inhibition by Zn 2+ at .1 mM was not calculable due to protein precipitation in the reaction mixture.

Phosphorylation of PstP c in the presence of Zn 2+ and Pi
The inhibition of PstP c in the presence of Zn 2+ and Pi provided a condition that could favour the phosphorylation of PstP c by STPKs. PstP c was indeed phosphorylated by PknA c and PknB c in presence of 0.2 mM Zn 2+ or 0.5 mM Pi ( Figure 5D), under in vitro conditions. Since the phosphorylated bands of His 6 -tagged PknA c / PknB c and PstP c were not able to resolve on SDS-PAGE ( Figure  S5), the assay was performed with GST-tagged kinases and similar results were obtained. To further assess the effects of Zn 2+ and Pi, metabolic labeling of PstP c by co-expressed kinases PknA and PknB was performed in E. coli in the presence of Zn 2+ (4 mM) and Pi (2 mM) ( Figure 5E). Phosphorylation of PstP c was indeed enhanced in the presence of Zn 2+ by ,40%-50%. The enhancement in phosphorylation in the presence of Pi was not as prominent (,10%-20%), possibly due to competition of phosphate ions with [ 32 P]orthophosphoric acid. Nevertheless, as . The error bars represent the SD of the three individual experiments. The corresponding autoradiogram is shown in Figure S4. doi:10.1371/journal.pone.0017871.g004 a proof of principle, Zn 2+ and Pi were identified as the novel regulators which can inhibit the activity of PstP c and facilitate its phosphorylation.

Discussion
The coordinated regulation of Ser/Thr protein kinases and phosphatases is essential for maintaining the appropriate equilibrium of protein phosphorylation. Membrane associated kinases and phosphatases are known or hypothesized to be regulated by external stimulus. It is of great relevance to decipher the regulatory mechanisms especially in the systems like M. tuberculosis where one Ser/Thr phosphatase PstP is accountable for the effects caused by 11 STPKs. In general, the processes involved in regulating the phosphatases include some external signals, variation in pH [27], cellular concentrations of ATP, ADP, Pi (or their ratios) [28,29], cytosolic cations like Mn 2+ , Zn 2+ , Mg 2+ , Ca 2+ [13,27,[29][30][31] and post-translation modifications (phosphorylation, methylation) [28,30,[32][33][34][35][36][37][38][39]. Present study demonstrates an example of PknA and PknB mediated regulation of PstP through inter-dependent phosphorylation-dephosphorylation reactions. Regulation of phosphatases by phosphorylation is a critical step for cell signaling pathways. It is also associated with feedback phenomena in case where phosphatases are phosphorylated by the kinases that are in turn dephosphorylated by the same phosphatase. Certain examples illustrate the phosphorylation of PP2C phosphatases such as rat Mg 2+ -dependent protein phosphatase a (MPPa) by casein kinase II [39], Soybean kinase associated protein phosphatase (Soybean KAPP) [37], Oryza sativa KAPP [40], but these have not been detailed in terms of feedback regulation. PstP has conserved domain architecture of PP2C-phosphatases (PPM family). PPM family phosphatases play an imperative role in a number of systems described earlier [41][42][43][44][45][46][47][48]. Except a few PP2C-phosphatases like Human PP2Ca [49] and Arabidopsis KAPP [50], not much is known about other members of this family. For PstP, we have previously shown that PknA and PknB are the targets for dephosphorylation by PstP and detailed the basic biochemical requirements of this enzyme along with its membrane localization [13]. In a later study, Pullen et al. resolved the crystal structure of PstP catalytic domain and described the most important features of this molecule having characteristic PP2C-fold along with three-metal binding centers that associate with Mn 2+ [18]. The discovery of third-metal centre was a unique feature of PstP as other PP2C phosphatases were found to have two metal-binding centres. In the recent studies, the PP2Cphosphatases of Streptococcus agalactiae and Thermosynechococcus elongatus have been shown to have a similar third-metal binding centre [51,52]. The third metal ion center in PstP is proposed to be involved in structural perturbations leading to altered phosphoprotein recognition profiles.
In this study, three conserved residues were selected for generation of site-directed mutants in PstP c , on the basis of similarity with Human phosphatase PP2Ca [18]. Arg 20 (PP2Ca Arg 33 ) is responsible for hydrolysis of phosphate moiety from pSer/pThr residues in target proteins. Asp 38 (PP2Ca Asp 60 ) and Asp 229 (PP2Ca Asp 282 ) constitute a part of Mn 2+ -metal centers and coordinate with the two critical Mn 2+ . Mutations of Asp 38 and Asp 229 affected the activity of PstP rendering it active to minimal level, though R20G mutant retained about 40% activity. Thus, the residues that are involved in Mn 2+ -ion binding and hydrolysis of phosphate are deciphered to be critical for its activity. Accordingly, the extent of phosphorylation of each mutant was dependent on the remaining dephosphorylation activity, so that PstP c D38G and PstP c D229G were efficiently phosphorylated by PknA and PknB.
Association with metals is crucial for PP2C phosphatases and any perturbation with inherently associated metals may lead to altered functional profile. The minimum requirement for PstP c activity is the presence of Mn 2+ [13]. For PP2C-class of phosphatases, divalent ions other than Mn 2+ /Mg 2+ can inhibit their activity by competitively replacing the Mn 2+ in the core enzyme structure [27] and Zn 2+ are the most potent regulators, having comparable ionic radii with that of Mn 2+ . PstP c was partially inactive in the presence of 0.2 mM ZnCl 2 and displayed lower activity on increasing the Zn 2+ -ion concentration upto 2 mM, as observed by pNPP assays. In vitro kinase assays with PknA c and PknB c in presence of Zn 2+ resulted in phosphorylation of PstP c . Also, there was increase in phosphorylation of PstP c during metabolic labeling by PknA and PknB in the presence of Zn 2+ added in the E. coli culture. These results indicate that in mycobacterial cell, if cytosolic Zn 2+ concentration increases, it may inhibit PstP perhaps leading to its phosphorylation. In an elaborative elemental analysis, Wagner et al. have reported that during infection, intravacuolar Zn 2+ -ion concentration increases from 0.037 mM to 0.46 mM in macrophages infected with M. tuberculosis [53]. Although there is no report of concomitant increase in mycobacterial Zn 2+ -ion concentration, it can only be speculated that if these changes in vacuolar ionic concentrations alter the mycobacterial ionic profile, a condition may develop where the enzymes that respond to Zn 2+ (like PstP) can be activated or deactivated.
End-product inhibition of enzymes is a well established phenomenon to prevent the accumulation of a particular metabolite. In case of reversible reactions, end-product accumu-lation can change the direction of the reaction. Similarly, Pi is known to inhibit a number of phosphatases [27,42,49] and in present study, PstP c mediated pNPP hydrolysis is inhibited by Pi. To confirm that this effect is not limited to pNPP, in vitro kinase assays and metabolic labeling in E. coli showed PstP c to be phosphorylated by PknA and PknB in presence of Pi because of its inhibition. Pi content is indicative of nutrient availability and energy status of the cell. In general, high Pi is associated with energy-starved conditions, when all the ATP is depleted and metabolite homeostasis is in unbalanced state. Such conditions usually arise during late-log and stationary phases in culture conditions.
Metabolic labeling by [ 32 P]orthophosphoric acid in the presence of co-expressed STPK (PknA or PknB) in E. coli lead to the specific phosphorylation of PstP c and PstP c D38G . Co-expression in pETDuet-1 has previously been utilized extensively to assess the interaction of mycobacterial STPKs with their cognate substrates in the surrogate host E. coli [21,23]. Such dual-expression systems are increasingly becoming useful for analysis of protein-protein interactions specifically for challenging systems like mycobacteria [54]. Activity assays of the pETDuet-1 purified PstP c and PstP c D38G revealed the higher activity of PknA-phosphorylated phosphatase as compared to the PknB-phosphorylated protein.
Prominent variations in the activity of phosphorylated and unphosphorylated PstP c D38G were observed with phosphorylated protein being proficient to hydrolyze pNPP to a greater extent (,15-fold higher) in contrast to the unphosphorylated protein.
The difference in the activities of phosphorylated and unphosphorylated PstP c was not as prominent as that of PstP c D38G (,2-3 fold higher). These differences may be attributed to the fact that PstP c may get auto-dephosphorylated to a greater extent than PstP c D38G during expression and purification procedures. Higher activity of phosphorylated phosphatase is suggestive of reverse regulation of signaling cascade emanating from the kinases. In the constitutively active state, STPKs perform their regular functions and phosphorylate the target substrates following the stimulus. This may ultimately lead to the phosphorylation of PstP. The resulting increase in the activity of phosphatase may itself act as a control mechanism for kinases, eventually impeding the continued effect of that particular stimulus. The overall process has to be dynamic due to auto-dephosphorylation of PstP, eventually ceasing the effect of signaling cascade. In the conditions of high Zn 2+ or high Pi content of the cell, PstP may not be active and will allow the kinase to work at its maximal activity. The proposed phosphorylation of PstP in such conditions may act as a mechanism to overcome the inhibition of PstP, hence balancing the cellular signaling pathways. Figure S1 pNPP-assay. To confirm the authenticity of pNPP assay, increasing amounts of alkaline phosphatase (0-100 ng) was used a positive control and PknB c (0-5 mg) was used as a negative control. The assay was performed for 30 mins at 37uC and the activity is calculated as mmoles of pNPP hydrolyzed per min at a given amount of enzyme used. As clearly evident, alkaline phosphatase showed very high activity while no such activity was detected in PknB c . (TIF) Figure S2 Effect of mutations on the activity of PstP c . To show that the loss in activity of PstP c was specifically due to mutations of Arg 20 , Asp 38 and Asp 229 , PstP c was mutagenized on irrelevant residues Thr 5 and Thr 141 to Ala and Glu, respectively and pNPP hydrolysis was performed for 30 mins at 37uC. Activity of PstP c was taken as 100% and relative activity was calculated. As evident from the bar graph, there were no significant changes in the activity of the mutants PstP c T5A and PstP c T141E as compared to PstP c . (TIF) Figure S3 Time-dependent pNPP-assay. pNPP-hydrolysis was performed in a time-dependent manner for 30 mins using PstP c , PstP c R20G , PstP c D38G and PstP c D229G variants (2 mg each) at 37uC. Alkaline phosphatase (2 ng) was used a positive control and PknB c (5 mg) was used as a negative control. Activity was calculated as nmoles of pNPP hydrolyzed per mg of enzyme used at a given time and depicted in logarithmic scale. Nevertheless, the results are essentially similar as that of time-dependent dephosphorylation of PknB c (Figure 2A). (TIF) Figure S4 In vitro dephosphorylation activity of pET-Duet-1 purified PstP c D38G . Autophosphorylated PknA c was incubated with unphosphorylated and phosphorylated PstP c D38G .

Supporting Information
As shown in the autoradiogram, the PknA-phosphorylated PstP c D38G dephosphorylated the kinase to a greater extent in comparison to the unphosphorylated PstP c D38G . The image was also analyzed by ImageGauge software and corresponding values are depicted by bar-graph ( Figure 4E). (TIF) Figure S5 Phosphorylation of PstP c . Autoradiogram showing the phosphorylation of PstP c (1 mg) by His 6 -tagged STPKs PknA c (upper panel) and PknB c (lower panel) in presence of 0.2 mM Zn 2+ and 0.5 mM Pi. Due to overlapping molecular weights of PknA c and PknB c with PstP c , the bands were not separated properly. Still, the phosphotransfer on PstP c was evident in presence of Zn 2+ and Pi by both the kinases. The reaction was also performed with GST-tagged STPKs to clearly depict the reaction ( Figure 5D). (TIF) File S1 Detailed protocol of sample processing for identification of phosphorylation sites. (DOC)