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. 2011 Mar 9;6(3):e17871.
doi: 10.1371/journal.pone.0017871.

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

Andaleeb Sajid  1 , Gunjan Arora, Meetu Gupta, Sandeep Upadhyay, Vinay K Nandicoori, Yogendra Singh
Affiliations

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

Andaleeb Sajid et al. PLoS One. .

Abstract

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 Zn(2+)-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr(137), Thr(141), Thr(174) and Thr(290) being the target residues of PknB while Thr(137) and Thr(174) are phosphorylated by PknA. The Mn(2+)-ion binding residues Asp(38) and Asp(229) are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c) (D38G) are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+)/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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Critical residues of PstP.
(A) Schematic representation of PstP with critical residues (Arg20, Asp38 and Asp229) being highlighted with upward arrows. (B) Activity profiles of PstPc and its mutants: Activity assays were performed by pNPP-hydrolysis mediated by PstPc, PstPc R20G, PstPc D38G and PstPc D229G. Increasing concentrations of proteins were taken with constant substrate concentration (10 mM pNPP) and incubated at 37°C for 30 mins. As shown in the graph, the mutants had lost phosphatase activity to different extents. Activity is calculated as a measure of µmoles of pNPP hydrolyzed per min. at a given enzyme concentration. (C) The relative activity of all the phosphatase variants (5 µg each, 30 min.) showed that PstPc D38G and PstPc D229G had lost >90% of activity while PstPc R20G lost ∼60% of the activity as compared to PstPc. The error bars indicate the SD of three individual experiments.
Figure 2
Figure 2. Dephosphorylation by PstPc and its mutants.
(A) Autoradiogram showing autophosphorylated PknBc, exposed to dephosphorylation by PstPc, PstPc R20G, PstPc D38G and PstPc D229G. Time-dependent dephosphorylation was performed with 1 µg of phosphatase after carrying out autophosphorylation of PknBc (2 µg) in an in vitro kinase assay. Noticeably, PstPc D38G was observed to be phosphorylated with increasing time points (3rd panel from the top). (B) Autoradiogram showing phosphorylation of PstPc by PknAc (1 µg). Increasing concentrations of PstPc were used to measure the extent of dephosphorylation. Unexpectedly, the phosphatase itself got phosphorylated at higher kinase to phosphatase ratio, though kinase was completely dephosphorylated. No phosphorylation was observed at higher PstPc concentrations.
Figure 3
Figure 3. Phosphorylation of PstPc and its mutants by PknA and PknB.
(A) Phosphorylation of PstPc and its mutants (3 µg each) by 2 µg PknBc (upper panel) and 0.5 µg PknAc (middle panel). PstPc D38G and PstPc D229G were efficiently phosphorylated by both the kinases due to loss of phosphatase activity. Phosphorylation on PstPc R20G mutant was low due to its partial phosphatase activity. The corresponding SDS-PAGE is shown (lowest panel) as a loading control. (B) Phosphoamino acid analysis by 2D-TLE illustrates that both PknAc (upper panel) and PknBc (lower panel) phosphorylates PstPc D38G on Thr residues. (C) Sites of phosphorylation of PknBc (blue) and PknAc (green) in PstPc D38G were identified by mass spectrometric analysis. PknBc phosphorylates PstPc D38G majorly on four Thr residues-Thr137, Thr141, Thr174 and Thr290 while two Thr residues were phosphorylated by PknAc-Thr137 and Thr174.
Figure 4
Figure 4. Co-expression analysis of STPKs and PstPc/PstPc D38G.
(A) Metabolic labeling of PstPc: PstPc co-expressed with MBP-PknA (lane 2) or MBP-PknB (lane 3) gets phosphorylated in E. coli under native conditions while PstPc co-expressed with MBP alone (lane 1) was not phosphorylated. (B) Metabolic labeling of PstPc D38G: PstPc D38G co-expressed with MBP-PknA (lane 2) or MBP-PknB (lane 3) gets phosphorylated in E. coli while PstPc D38G co-expressed with MBP alone (lane 1) was not phosphorylated. As expected, the intensity of phosphorylation on PstPc D38G was comparatively higher than that of PstPc. (C) Relative activity profile of pETDuet1 purified PstPc and (D) PstPc D38G: pNPP assays were performed with PstPc and PstPc D38G (1 µg each) purified from pETDuet1 co-expressing MBP or MBP-PknA/PknB. The dephosphorylation potential of phosphorylated PstPc and PstPc D38G (co-expressed with either kinase) is higher than that of unphosphorylated protein. For PstPc D38G, activity was evaluated over long time points due to its low dephosphorylation activity. Activity is calculated as a measure of µmoles of pNPP hydrolyzed per µg of protein at a given time. The error bars indicate the SD of three individual experiments. (E) Relative dephosphorylation of PknAc by pETDuet-1 purified PstPc D38G: Autophosphorylated PknAc was incubated for 30 mins with unphosphorylated and phosphorylated PstPc D38G and the extent of dephosphorylation was assessed by in vitro dephosphorylation assays. The image obtained after autoradiography was analyzed by ImageGauge software (Fuji) and relative intensity of phosphorylation was measured: (1) PknAc alone, (2) PknAc+MBP-PstPc D38G, (3) PknAc+PstPc D38G phosphorylated by PknA and (4) PknAc+PstPc D38G phosphorylated by PknB. As shown, the PknA-phosphorylated PstPc D38G dephosphorylated the kinase to a greater extent in comparison to the unphosphorylated PstPc D38G. The error bars represent the SD of the three individual experiments. The corresponding autoradiogram is shown in Figure S4.
Figure 5
Figure 5. Factors affecting PstP activity.
(A) Auto-dephosphorylation of PstPc: Autoradiogram showing phosphorylation by PknBc. PstPc and PstPc D38G (3 µg each) were used for in vitro phosphorylation assay by PknBc and PknBc T171/173D (2 µg each). Since PknBc T171/173D cannot be dephosphorylated by PstPc, lack of signal signifies auto-dephosphorylation of phosphatase. PstPc D38G was used as positive control to show that PknBc T171/173D is active. Regulation of PstPc activity: pNPP assay showing the effect on activity of PstPc (1 µg) by (B) Zn2+ and (C) Pi. pNPP assay was carried out for 30 mins and activity was calculated as a measure of µmoles of pNPP hydrolyzed per min per µg of protein. The error bars show SD of three independent experiments. (D) Phosphorylation of PstPc: Autoradiogram showing the phosphorylation of PstPc (1 µg) by GST-PknAc (left panel) and GST-PknBc (right panel) in presence of 0.2 mM Zn2+ and 0.5 mM Pi. Since His6-tagged STPKs were not resolved properly from PstPc on SDS-PAGE (Figure S5), the assay was also performed with GST-tagged kinases having higher molecular weights. (E) Metabolic labeling of PstPc by PknA and PknB in E. coli in presence of Zn2+ and Pi: Phosphorylation level of PstPc was observed to be increased when Zn2+ (4 mM) and Pi (2 mM) were added during the culture conditions and subsequent processing steps. The autoradiograms obtained after SDS-PAGE were analyzed by ImageGauge software and intensity of the band corresponding to PstPc phosphorylation without any added factor was taken as 100%. Relative phosphorylation is depicted in the bar graph.
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