Báo cáo khoa học: EmbR, a regulatory protein with ATPase activity, is a substrate of multiple serine⁄threonine kinases and phosphatase in Mycobacterium tuberculosis doc

EmbR, a regulatory protein with ATPase activity, is a substrate of multiple serine⁄threonine kinases and phosphatase in Mycobacterium tuberculosis Kirti Sharma1, Meetu Gupta1, Ananth Krupa2,*, Narayanaswamy Srinivasan2 and Yogendra Singh1 Institute of Genomics and Integrative Biology, Delhi, India Molecular Biophysics Unit, Indian Institute of Science, Bangalore Keywords ATPase; kinase; phosphatase; Mycobacterium; tuberculosis Correspondence Y Singh, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India Fax: +91 11 27667471 Tel: +91 11 27666156 E-mail: ysingh@igib.res.in *Present address Cancer Research UK, Clare Hall Laboratories, UK (Received 23 March 2006, revised 19 April 2006, accepted 24 April 2006) doi:10.1111/j.1742-4658.2006.05289.x Phosphorylation of the mycobacterial transcriptional activator, EmbR, is essential for transcriptional regulation of the embCAB operon encoding cell wall arabinosyltransferases This signaling pathway eventually affects the resistance to ethambutol (a frontline antimycobacterial drug) and the cell wall Lipoarabinomannan ⁄ Lipomannan ratio (an important determinant for averting the host immune response) In this study, further biochemical characterization revealed that EmbR, as a transcriptional regulator, interacts with RNA polymerase and possesses a phosphorylation-dependent ATPase activity that might play a role in forming an open complex between EmbR and RNA polymerase EmbR was recently shown to be phosphorylated by the cognate mycobacterial serine ⁄ threonine (Ser ⁄ Thr) kinase, PknH Using bioinformatic analysis and in vitro assays, we identified additional novel regulators of the signaling pathway leading to EmbR phosphorylation, namely the Ser ⁄ Thr protein kinases PknA and PknB A previously unresolved question raised by this signaling scheme is the fate of phosphorylated kinases and EmbR at the end of the signaling cycle Here we show that Mstp, a mycobacterial Ser ⁄ Thr phosphatase, antagonizes Ser ⁄ Thr protein kinase–EmbR signaling by dephosphorylating Ser ⁄ Thr protein kinases, as well as EmbR, in vitro Additionally, dephosphorylation of EmbR reduced its ATPase activity, interaction with Ser ⁄ Thr protein kinases and DNA-binding activity, emphasizing the antagonistic role of Mstp in the EmbR–Ser ⁄ Thr protein kinase signaling system Serine ⁄ threonine (Ser ⁄ Thr) protein kinases (STPKs) have emerged as crucial players for environmental sensing and physiological signaling in prokaryotes These kinases have been implicated in diverse control mechanisms, including stress responses, developmental changes and host–pathogen interactions, in several microorganisms The genome of Mycobacterium tuberculosis, the causative agent for tuberculosis, has shown the presence of 11 genes that code for putative STPKs and one gene (Mstp) that codes for Ser ⁄ Thr phospha- tase [1] These STPKs have been proposed to mediate signaling between mycobacteria and host cells to establish an environment that is favorable for the replication and survival of mycobacteria [2] Recent reviews have highlighted the importance of such signaling mediated by mycobacterial STPKs and identified them as potential drug targets [1,3,4] To date, eight of these STPKs or kinase domains have been expressed, purified and shown to be active in vitro [5–12] The mycobacterial STPKs regulate diverse processes by phosphorylating Abbreviations GST, glutathione S-transferase; RNAP, RNA polymerase; LAM ⁄ LM, Lipoarabinomannan ⁄ Lipomannan; SARP, Streptomyces coelicolor antibiotic regulatory gene; Ser ⁄ Thr, serine ⁄ threonine; STPKs, serine ⁄ threonine protein kinases FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS 2711 Regulation of EmbR activity by STPKs and Mstp K Sharma et al 120 100 80 EmbR EmbR∆N EmbR Heat inactivated 60 40 20 Results Interaction of EmbR with RNA polymerase and its phosphorylation dependent ATPase activity EmbR belongs to the Streptomyces coelicolor antibiotic regulatory gene (SARP) family of proteins, which are known to regulate genes involved in the biosynthesis of secondary metabolites through DNA binding to specific gene sequences Our previous results have demonstrated the positive regulatory effect of EmbR 2712 on transcription of the embCAB operon after its phosphorylation by PknH in vivo [18] Until very recently, little was known about the mechanism by which SARPs exerted their effect on gene expression Bioinformatic analysis revealed that the SARP family shares sequence homology with the OmpR ⁄ PhoB family, a large family of transcription factors that bind DNA through their winged helix-turn-helix motifs [23] In agreement, the recently reported structure of EmbR revealed that the structural elements relevant for function in OmpR are conserved in EmbR, including the transactivation loop, which mediates interactions with RNA polymerase (RNAP), the DNA recognition-helix and the ‘wing’ [24] Based on the presence of a transactivation loop, termed as the ‘a loop’ in the members of the OmpR ⁄ PhoB family [23], the possible interaction of EmbR with RNAP holoenzyme (holoRNAP) was experimentally investigated by ELISA, whereupon EmbR was found to interact with RNAP in a concentration-dependent manner (Fig 1) This observation corroborates the function of EmbR as a transcriptional activator of embCAB genes in view of the fact that OmpR family members are known to interact productively with RNAP for transcriptional activation of their target genes [23] EmbRDN, a deletion mutant lacking DBD and thus the ‘a loop’, failed to interact with holo-RNAP, thereby suggesting that the EmbR–RNAP interaction was specific (Fig 1) Relative ELISA signal distinct substrates, including proteins implicated in regulating cell division and morphology [13–15], an ABC transporter [10,16], mediators of glutamate ⁄ glutamine metabolism [17] and a transcriptional regulator, EmbR [9,18] In addition, the Ser ⁄ Thr phosphatase, Mstp, dephosphorylates two Ser ⁄ Thr kinases (PknA and PknB) and has been implicated in regulating the cell division of M tuberculosis [19,20] One of the major gaps in our knowledge concerns identification of the key substrates of protein kinases and phosphatases and how their phosphorylation ⁄ dephosphorylation contributes to the changes in cell physiology evoked in response to particular signals PknH, a mycobacterial Ser ⁄ Thr kinase unique to the members of M tuberculosis complex [7], has been shown to phosphorylate the cognate regulatory protein, EmbR [9] Recently, we reported that phosphorylated EmbR serves as a transcriptional activator for arbinosyltransferases encoded by embCAB genes [18] embCAB is a gene cluster involved in arabinan synthesis and represents ethambutol targets in M tuberculosis [21] Our results also revealed that EmbR phosphorylation affects two important physiological phenomena, namely the Lipoarabinomannan ⁄ Lipomannan (LAM ⁄ LM) ratio, which is an important determinant of mycobacterial virulence and resistance to ethambutol (a frontline antituberculosis drug) [18] Concomitantly, AvenueGay and co-workers have shown that deletion of pknH results in a hypervirulent phenotype and also suggested a role of PknH in mediating NO toxicity [22] Thus, part of the signal transduction by PknH ⁄ EmbR has been elucidated This study shows that EmbR is a substrate for multiple STPKs, as well as a substrate for Mstp In addition, we show, for the first time, that a phosphorylation-dependent ATPase activity is associated with EmbR Dephosphorylation of EmbR by Mstp reduces its ATPase activity, interaction with STPKs and DNA-binding activity towards promoter regions of embCAB genes, revealing the antagonistic role of the phosphatase in the EmbR–STPK signaling system 0.5 EmbR (mg/ml) Fig EmbR–RNA polyerase (RNAP) interaction, as investigated by ELISA RNAP holoenzyme containing the principal sigma factor, sigA, was purified from Mycobacterium smegmatis Holo-RNAP (100 ngỈwell)1) coated in wells was incubated with EmbR ⁄ EmbRDN at graded concentrations Unbound EmbR was removed and holoRNAP bound EmbR was quantified using anti-EmbR Ig FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS K Sharma et al Regulation of EmbR activity by STPKs and Mstp A comparison of EmbR with its closest homologue, AfsR, a transcriptional activator of Streptomyces [25], revealed the absence of any defined ATPase domain in EmbR Besides, no such domain was identified in the recently published 3D structure of EmbR [24] However, after a closer examination of its amino acid sequence, certain altered nucleotide-binding consensus sequences were identified in EmbR (Fig 2A) Therefore, the ability of EmbR to bind and hydrolyze nucleotide triphosphates (NTPs) was investigated by three methods (Fig 2) Interestingly, EmbR showed distinct ATPase and GTPase activities, with ATP preferred over GTP as a substrate (Fig 2B) No phosphate was released when ADP was used as a substrate, indicating A ATP that EmbR is not a phosphatase These results showed that despite the absence of consensus nucleotide-binding motifs, EmbR exhibits ATPase and GTPase activities STPK-mediated phosphorylation of a transcriptional activator whose function depends on ATP hydrolysis is emerging as a central theme in prokaryotic signal transduction systems [25,26] Therefore, the effect of PknH-mediated phosphorylation on the in vitro ATPase activity of EmbR was analyzed While purified EmbR showed an ATPase activity of 0.040 nmol of phosphatmin)1Ỉlg)1 EmbR protein, the phosphorylated form of EmbR displayed an ATPase activity of 0.257 nmol of phosphatmin)1Ỉlg)1 EmbR (i.e about ATP + EmbR 10 20 30 (min) Percent of original ATP hydrolyzed Pi ATP Nucleotide binding motif Consensus EmbR 237 40 20 GXXXXGKT 226 GAYRRVKT Consensus EmbR 60 10 20 Time (min) DXXG DDLG a B b C ATP + EmbR GTP + EmbR nmoles of Pi per mg protein nmol of Pi per ug EmbR 30 ATP + heat inactivated EmbR GTP + heat inactivated EmbR EmbR Phosphorylated EmbR 0 20 40 60 80 100 Time (min) 20 40 60 80 100 Time (min) Fig (A) ATPase activity of EmbR (a) EmbR was incubated with [32P]ATP[cP] for various time periods, and the release of 32Phosphate (32Pi) was monitored by TLC Also shown are altered nucleotide-binding motifs in EmbR (b) EmbR was incubated with [32P]ATP[cP] for various time intervals (0–30 min) The filter binding assay was performed as described in the Experimental procedures The ATP hydrolyzed at each time point is shown as a percentage of the original [32P]ATP[cP] before incubation at 37 °C (B) Time courses of ATP and GTP hydrolysis by EmbR The release of Pi was measured, using the malachite green method, at various time points The Pi release was assayed when ATP or GTP was used as a substrate of EmbR Each time point is the average of the values obtained from three independent experiments (C) Effect of phosphorylation on ATPase activity ATPase activities of EmbR and phosphorylated EmbR were compared The phosphorylated EmbR sample was prepared by in vitro phosphorylation as described in the Experimental procedures FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS 2713 Regulation of EmbR activity by STPKs and Mstp K Sharma et al For further characterization of EmbR and the associated phosphorelay in M tuberculosis, the ability of mycobacterial cell lysate to phosphorylate purifed EmbR was analyzed Resin-bound EmbR was incubated with whole cell lysate of M tuberculosis in the presence of [32P]ATP[cP] and it was observed that the cell lysate of M tuberculosis harbors the ability to phosphorylate EmbR (Fig 3A) Boiling of cell lysate, or treatment with a kinase inhibitor, staurosporine, resulted in the complete loss of its ability to phosphorylate EmbR (Fig 3A) These observations indicated that the EmbR phosphorylating activity in the whole cell lysate of M tuberculosis is caused by the presence of STPK(s) Recently, it has been shown that EmbR is phosphorylated in vitro by PknH [9] Therefore, it can be anticipated that mycobacterial cell lysate devoid of PknH should not phosphorylate EmbR Interestingly, whole cell lysate of M tuberculosis, pre-incubated with anti-PknH Ig and thus neutralized for PknH, was also able to phosphorylate EmbR (Fig 3A) At the same time, the anti-PknH Ig was able to prevent the phosphorylation of EmbR by purified PknH All these observations suggested the presence of additional STPK(s) that can phosphorylate EmbR An important clue towards other STPK(s) capable of phosphorylating EmbR came from cross-genomic comparisons of bacterial protein kinases to identify homologues of kinases with known substrates This study revealed PknB of M tuberculosis as the closest homolog of AfsK, an STPK-phosphorylating AfsR Considering the homology of AfsR with EmbR, as well as the significant sequence similarity observed between catalytic domains of AfsK and PknB (38% identity), it was interesting to study EmbR as a possible substrate of PknB The in vitro assays revealed that autophosphorylated PknB phosphorylates EmbR, whereas heatinactivated PknB does not (Fig 3B) In fact, EmbR has previously been suggested as one of the targets for a signal transduction pathway mediated by PknA and PknB If so, this pathway could link cell division and peptidoglycan synthesis with arabinogalactan synthesis, another process essential for growth [27] PknA, an STPK present in the same operon as PknB, was also 2714 sp ori ne kn H kn H uro +A Pk nH nH Pk Ly s ate +A nti -P nti -P st a mM at e +1 ate Ly s Ly s Bo ile d te sa Ly Co ntr ol Phosphorylation of EmbR by multiple Ser ⁄ Thr kinases in M tuberculosis A EmbR B 4 PknA EmbR Autoradiogram SDS-PAGE PknB EmbR Autoradiogram SDS-PAGE (a) Bound Radioactivity (cpm) sixfold higher) (Fig 2C) The ATPase activity of EmbR probably provides energy to catalyze the isomerization of the closed complex between EmbR and RNAP to a transcriptionally competent open complex, as is proposed for AfsR [25] 80000 PknA PknB 60000 40000 20000 0.5 10 20 30 Kinase 30 30 (min) + Heat inactivated 20mM EDTA Kinase (b) Fig EmbR, a substrate for multiple serine ⁄ threonine protein kinases (STPKs) (A) Phosphorylation of EmbR with Mycobacterium tuberculosis cell lysate Resin-bound EmbR was incubated in the presence of [32P]ATP[cP] and under the indicated experimental conditions, as described in the Experimental procedures After elution, EmbR was run on SDS ⁄ PAGE and its phosphorylation was visualized by autoradiography (B) Phosphorylation of EmbR by PknA and PknB (a) In vitro kinase assays were performed to examine the ability of PknA (upper half) and PknB (lower half) to phosphorylate EmbR in the presence of [32P]ATP[cP] The labeled proteins were separated by SDS ⁄ PAGE and visualized by autoradiography or Coomassie Blue staining Lane 1, EmbR; lane 2, PknA (upper half) or PknB (lower half); lane 3, PknA or PknB incubated with EmbR; lane 4, heat-inactivated PknA or PknB incubated with EmbR (b) For resin-bound assays, EmbR bound to Ni-nitrilotriacetic acid resin was incubated with purified PknA ⁄ PknB in the presence of [32P]ATP[cP] for the indicated time periods and conditions Shown is the bound radioactivity in counts per minute FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS K Sharma et al tested for its ability to phosphorylate EmbR and it came as an expected finding that PknA also phosphorylated EmbR (Fig 3B) Incubation of EmbR alone in the presence of [32P]ATP[cP], as a negative control, did not yield any phosphorylated product The phosphorylation of EmbR by these kinases was found to be specific, as other mycobacterial kinases, such as PknG and PknI, could not phosphorylate EmbR under similar conditions (data not shown) Thus, it was confirmed that EmbR acts as a substrate of three mycobacterial STPKs, viz PknH, PknA and PknB Regulation of EmbR activity by STPKs and Mstp A PknH Mstp SDS-PAGE SDS-PAGE Mstp EmbR B 100 Remaining radioactivity (%) EmbR is phosphorylated by three STPKs, which themselves are believed to autophosphorylate in response to environmental perturbations This kinase-mediated signaling should be ‘switched off’ when it is not required Returning to the inactive ⁄ resting state would require either the synthesis of new proteins or the dephosphorylation of the existing phosphorylated species The only reported Ser ⁄ Thr phosphatase of M tuberculosis, Mstp, is known to dephosphorylate PknA and PknB, thereby acting as a regulator of these kinases [19,20] Therefore, it was tempting to examine whether Mstp could dephosphorylate PknH in addition to PknA and PknB, all of which are involved in EmbR phosphorylation We also examined the ability of Mstp to dephosphorylate EmbR directly to ‘switch off’ signaling at the effector level As described in the Experimental procedures, two methods were employed to examine dephosphorylation of PknH by Mstp, namely resin-bound and in-solution dephosphorylation assays The prephosphorylated substrates for dephosphorylation assays were prepared using resin-based phosphorylation reactions in the presence of [32P]ATP[cP] In-solution phosphatase assays revealed that incubation with Mstp led to a decrease in the intensity of bands corresponding to prephosphorylated substrates, namely, PknH and EmbR, thus confirming that PknH and EmbR are substrates of Mstp in vitro A reaction set with heatinactivated Mstp served as a negative control (Fig 4A) Incubation with heat-inactivated Mstp had no effect EmbR phosphorylation status when compared with the control phosphorylated EmbR with no addition of Mstp (data not shown) For resin-bound dephosphorylation assays, autophosphorylated PknH and phosphorylated EmbR bound to resin were incubated with purified Mstp (or heat-inactivated Mstp) in phosphatase buffer Incubation with Mstp resulted in 73% and 79% dephospho- Autoradiogram Autoradiogram Phosphorylated forms of PknH and EmbR are substrates of Mstp in vitro PknH EmbR 80 60 40 20 0 10 + Mstp 30 30 − Mstp 30 (min) Heat inactivated Mstp Fig Dephosphorylation of EmbR and PknH by Mstp (A) In vitro dephosphorylation was analyzed by incubating prephosphorylated PknH (upper half) or EmbR (lower half) with Mstp (lane 2) or heatinactivated Mstp (lane 1) The reaction products were resolved by SDS ⁄ PAGE and the loss of labeling was visualized by autoradiography Right, Coomassie Blue staining; left, corresponding autoradiogram It is unclear why there are protein double bands of EmbR and Mstp on SDS ⁄ PAGE (B) For resin-bound assays, PknH or EmbR bound to beads was prephosphorylated in the presence of [32P]ATP[cP], as described in the Experimental procedures Mstpmediated dephosphorylation of resin-bound prephosphorylated PknH or EmbR was assessed by measuring the reduction in the substrate-bound radioactivity after incubation for the indicated time periods and under the experimental conditions described Shown is the residual PknH- and EmbR-associated radioactivity Each value is the average of two individual reactions and representative of three experiments rylation of PknH and EmbR, respectively, in 30 (Fig 4B) Our earlier studies, characterizing Mstp, revealed that PknA and PknB, the endogenous substrate kinases present in an operon with Mstp, were 75% and 79% dephosphorylated after incubation with Mstp for 60 min, respectively [20] In concordance, in this study, we observed that Mstp dephosphorylates PknH and EmbR at comparable levels FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS 2715 Regulation of EmbR activity by STPKs and Mstp K Sharma et al Dephosphorylation of phosphorylated EmbR decreases its DNA-binding activity Our earlier results showed that EmbR is physically and functionally engaged as a mediator of embCAB activation by PknH in vivo [18] The embA and embB genes can be expressed from their own individual promoters [28]; however, synthesis of a polycistronic mRNA encoding the three Emb proteins has also been reported in M tuberculosis [29] PknH-mediated phosphorylation of EmbR is crucial for its interaction with upstream regions of emb genes [18] In view of our observation that phosphorylated EmbR is a substrate of Mstp, the modulation of its DNA-binding activity upon dephosphorylation by this phosphatase was examined The dephosphorylated form of EmbR was prepared by in vitro dephosphorylation of prephosphorylated EmbR, and the binding of phosphorylated ⁄ dephosphorylated EmbR to the upstream region of embCAB genes was examined by the gel mobility shift assay Following its dephosphorylation, the strength of DNA binding by EmbR decreased many fold, with lg of dephosphorylated protein bringing about a similar mobility shift as seen for 0.3 lg of phosphorylated EmbR (Fig 5A) Thus, while phosphorylation enhances the DNAbinding activity of EmbR, the dephosphorylated form of EmbR was incapable of binding the promoter regions of emb genes at low concentrations, in agreement with the belief that EmbR itself in the phosphorylated form interacts with upstream regions of emb genes Furthermore, it suggests that Mstp acts as an antagonist of the STPK–EmbR signal relay Moreover, as one would anticipate, the dephosphorylation of phosphorylated EmbR also reduces the level of ATPase activity equivalent to that of unphosphorylated protein (data not shown) Mstp-mediated dephosphorylation of PknB ⁄ PknA ⁄ PknH inhibits their interaction with EmbR To further comprehend the role of Mstp, the effect of Mstp-mediated dephosphorylation of kinases on their specific interaction with endogenous substrate, EmbR, was examined using a glutathione S-transferase (GST) pull-down assay To analyze the PknA–EmbR interaction (Fig 5B), the soluble fraction containing His– EmbR was incubated with either prephosphorylated GST–PknA (lane 2) or dephosphorylated (using Mstp) GST–PknA (lane 3) As controls, EmbR was incubated with glutathione–Sepharose, either with GST (lane 4) or alone (lane 5), in NaCl ⁄ Pi buffer The binding assay 2716 was performed as described in the Experimental procedures When pre-incubated with phosphorylated PknA, EmbR was recovered in the soluble fraction eluted from glutathione–Sepharose (lane 2) EmbR was not recovered in control experiments when it was incubated either alone (lane 5) or in the presence of GST (lane 4) Therefore, the complex was formed only via the phosphorylated form of PknA The absence of recovery of EmbR upon pre-incubation with dephosphorylated PknA in this assay (lane 3) indicated, by comparison with lane 2, that the Mstp-mediated dephosphorylation abrogates the interaction of EmbR with PknA (Fig 5B) Similar assays were performed with PknB and PknH to show that Mstp inhibits the interaction of STPKs– EmbR by directly dephosphorylating these kinases (data not shown) In accordance with our observations, previous reports have shown that the PknH–EmbR interaction does not take place when a kinase mutant, incapable of autophosphorylation, was used [9] Discussion Phosphorylation-dependent signal transduction between PknH and its cognate DNA-binding transcription regulator, EmbR, triggers the regulation of mycobacterial embCAB genes and consequently influences ethambutol resistance and the LAM ⁄ LM ratio The present study shows that EmbR serves as a substrate of multiple STPKs If each of the kinases senses its own signal amongst a plethora of environmental cues, as is known for eukaryotic protein kinases, EmbR makes an integrator of the signals (Fig 6) The idea of one response regulator protein communicating with multiple sensor kinases is not unusual, as exemplified by the PhoR ⁄ PhoM ⁄ PhoB system in Escherichia coli [30] and the AfsK ⁄ PkaG ⁄ PknL ⁄ AfsR system in S coelicolor [25] Shared communication links help the organism to integrate diverse signals into a global response In analogy with its closest homolog – AfsR of S coelicolor [25] – EmbR also possesses a phosphorylation-dependent ATPase activity despite the absence of any conserved nucleotide-binding motifs in its amino acid sequence The enhancement of DNA-binding activity of EmbR upon phosphorylation [18], and its ability to interact with RNAP, is similar to that of many other OmpR family members [23] The energy supplied by the intrinsic low ATPase activity of unphosphorylated EmbR is thought to be insufficient to overcome the activation energy barrier to open complex formation From the present study, together with the similarity of EmbR with AfsR and other FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS K Sharma et al Regulation of EmbR activity by STPKs and Mstp A P-Em bR 0.3 P-EmbR deP-EmbR ( µg) 0.3 embA -189/+135 deP-EmbR P-EmbR ( µg) 0.3 deP-EmbR ( µg) embC -205/+112 embB -194/+128 B LANE Dephosphorylated PknA GST EmbR Mo l Weig ht Marker Prephosphorylated PknA + Glutathione Sepharose + + + + - + - - - + - - + - - - - - - + - - - + + + + - + Anti His Blot EmbR Fig (A) Dephosphorylation of phosphorylated EmbR decreases its DNA-binding activity towards the promoter region of embCAB genes A total of 0.3 lg of phosphorylated EmbR (P-EmbR), or an increasing amount of dephosphorylated EmbR, was incubated with 32P-labeled upstream regions of embC, embA and embB genes at °C for 30 After incubation, complexes and free DNA were separated by nondenaturing polyacrylamide gels and subjected to autoradiography The positions of EmbR-bound (solid arrow) and free (open arrow) probes are shown The numbers represent nucleotides relative to the translation start codon of the specific emb gene (B) Mstp-mediated dephosphorylation of PknB ⁄ PknA ⁄ PknH inhibits their interaction with EmbR The interaction of PknA with EmbR was analyzed using a pull-down assay The presence of either a protein or glutathione–Sepharose is indicated by ‘+’ and the absence by ‘–’ The soluble fraction of Escherichia coli cells expressing recombinant His–EmbR was incubated with either prephosphorylated glutathione S-transferase (GST)–PknA (lane 2) or dephosphorylated (using Mstp) GST–PknA (lane 3), each bound to glutathione–Sepharose resin As controls, prephosphorylated GST–PknA was incubated with glutathione–sepharose in the absence of EmbR (lane 6) In addition, EmbR was incubated with glutathione–Sepharose, either with GST (lane 4) or alone (lane 5) GST complexes were pulled down with glutathione–Sepharose, separated by SDS ⁄ PAGE, and transferred onto a nitrocellulose membrane before detection of recombinant poly histidine-tagged EmbR fusion protein (lower part) Purified EmbR was run as a positive control (lane 7) and lane represents the molecular weight marker OmpR family members, we infer that the association of phosphorylation-modulated ATPase activity and DNA binding ensures that phosphorylation of EmbR is primarily coupled to the formation of site-specific open complex during transcriptional initiation Conversely, Mstp antagonizes this signaling by individually dephosphorylating all three kinases as well as EmbR The phosphorylation-dependent enhancement in DNA binding and ATPase activity of EmbR is reversed as a consequence of its dephosphorylation by Mstp Moreover, Mstp-mediated dephosphorylation of kinases abrogates their interaction with EmbR, thus emphasizing the antagonistic role played by Mstp in the EmbR–STPK signaling cascade In conclusion, by demonstrating multiple STPKmediated phosphorylation and Mstp-mediated dephosphorylation of EmbR, our findings add other upstream effectors to the EmbR-mediated signaling FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS 2717 Regulation of EmbR activity by STPKs and Mstp K Sharma et al SIGNAL X PknA SIGNAL A SIGNAL Y PknB SIGNAL Z PknH (Sensor Kinases) EXTR ACE LLUL AR Autophosphorylation LAM / LM Ratio EMB Resistance Mstp Dephosphorylation INTR ACE LLUL AR P EmbR Arabinan Metabolism Dephosphorylation P EmbR RNA Polymerase Increased transcription of embCAB genes Closed complex P ATP hydrolysis RNA Polymerase P Open complex Fig A hypothetical scheme for the regulation of arabinan metabolism by the Mstp serine ⁄ threonine protein kinases (STPKs) ⁄ EmbR ⁄ embCAB system in Mycobacterium tuberculosis By analogy with eukaryotic signal transduction, we speculate that multiple STPKs autophosphorylate on sensing certain external stimuli and transfer the signal to EmbR by means of phosphorylation EmbR phosphorylation triggers the transcriptional activation of embCAB genes and consequently influences ethambutol resistance and the LAM ⁄ LM ratio On the contrary, the Ser ⁄ Thr phosphatase, Mstp, antagonizes the STPK ⁄ EmbR signaling system The environmental stimuli that activate PknH ⁄ PknB ⁄ PknA and Mstp have yet to be identified network Mediated by the action of STPKs and Mstp, we demonstrate the modulation of ATPase activity and DNA-binding activity of EmbR as a possible physical mechanism to modulate its regulatory effect on emb genes On the basis of the results obtained so far, we present a hypothetical model for the regulation of arabinan metabolism by the Mstp ⁄ STPKs ⁄ EmbR ⁄ embCAB system in M tuberculosis (Fig 6) Collectively, these observations provide another example for the mutual regulation of protein Ser ⁄ Thr kinases and protein Ser ⁄ Thr phosphatases In vivo studies and further functional characterization to comprehend the role of these merging pathways in mycobacterial pathogenicity are in progress and are expected to provide intriguing insights into the significance of corresponding signaling events in M tuberculosis 2718 Experimental procedures Bacterial culture and growth conditions Mycobacterial strains were grown in Middlebrook 7H9 broth supplemented with 0.5% glycerol and 10% albumin ⁄ dextrose ⁄ catalase at 37 °C, with shaking at 220 r.p.m., for 3–4 weeks The E coli strains were grown in Luria–Bertani (LB) broth or on LB agar plates at 37 °C with shaking at 220 r.p.m Plasmid construction, mutagenesis and protein purification GST-tagged PknH and PknHK45M mutant protein were used from previous studies [7] EmbR and EmbDN were expressed as His-tagged fusion proteins and purified under FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS K Sharma et al denaturing conditions using Ni-nitrilotriacetic acid resin, as per the manufacturer’s instructions and as described previously [18] PknA, PknB and Mstp were also purified under denaturing conditions as described in previous studies [20] ELISA The M smegmatis RNAP holoenzyme, containing the principal sigma factor, sigA, was kindly provided by Prof Anil K Tyagi Purified Holo-RNAP was coated on a 96-well ELISA plate (100 ngỈwell)1) His–EmbR or His–EmbRDN fusion proteins were incubated, at three concentrations, with the coated protein overnight at °C in buffer comprising 10 mm Tris HCl, 150 mm NaCl, pH 7.5, 0.5% Tween 20 (TBS-T), after which the plates were washed and developed as described previously [31] In vitro kinase assay The in vitro kinase reactions routinely contained 500 ng of the enzyme in the kinase buffer (25 mm Tris, pH 7.4, 10 mm MgCl2, mm dithiothreitol) with lg of EmbR and lCi of [32P]ATP[cP] and incubated for 30 at 37 °C The reactions were stopped by the addition of SDS sample buffer, and proteins were separated by 1D gel electrophoresis, electroblotted onto nitrocellulose membranes and visualized by autoradiography For resin-bound kinase assays, purified EmbR was phosphorylated by PknH in the kinase buffer, as described previously [20] The counts associated with resin-bound EmbR are a measure of its phosphorylation by the kinase To prepare phosphorylated substrates for dephosphorylation reactions, the phosphorylated EmbR was eluted from Ni-nitrilotriacetic acid beads using elution buffer (200 mm imidazole in 50 mm Na phosphate, pH 7.0, 100 mm NaCl and 10% glycerol) Similarly, GST–PknH was autophosphorylated and eluted from glutathione–Sepharose 4B, as described previously for PknA and PknB [20] After elution, phosphorylated EmbR and PknH were dialyzed against buffer (40 mm Tris, pH 7.6, and 10% glycerol) and stored at )20 °C until further use To monitor phosphorylation of EmbR by mycobacterial lysate, lg of resin-bound EmbR was incubated with 10 lg of whole cell lysate of M tuberculosis in the presence of 15 lCi of [32P]ATP[cP] in 25 mm Tris, pH 7.4, 10 mm MgCl2, mm dithiothreitol (TMD) buffer and 50 mm sodium fluoride (Ser ⁄ Thr phosphatase inhibitor) for 30 at room temperature The effects of boiling the whole cell lysate on its ability to phosphorylate EmbR was examined by boiling the whole cell lysate for 10 min, before incubation with resinbound EmbR The effect of kinase inhibitor was investigated by pre-incubating the whole cell lysate with mm staurosporine The effect of anti-PknH Ig was examined by pre-incubating the whole cell lysate with anti-PknH Ig (1 : 500 dilution) for 20 before incubation with resin-bound EmbR Regulation of EmbR activity by STPKs and Mstp In vitro phosphatase assay For resin-bound assays, dephosphorylation of phosphorylated PknH and EmbR by Mstp was examined by measuring the release of 32Phosphate (32Pi) Glutathione– Sepharose 4B beads bound to phosphorylated GST–PknH, or Ni-nitrilotriacetic acid beads bound to His-EmbR, (2.5 lg each) were incubated with Mstp (1 lg) for different time periods After incubation, the beads were washed twice with wash buffer to remove liberated 32Pi and the proteins were eluted at 65 °C using elution buffer (1% SDS and 50 mm EDTA) for 10 min, as reported previously [20] Radioactivity was measured using a scintillation counter A decrease in the counts of phosphorylated PknH ⁄ EmbR in the presence of Mstp is a measure of the dephosphorylation activity of Mstp The in vitro dephosphorylation of PknH ⁄ EmbR by Mstp was also analyzed by using phosphorylated PknH ⁄ EmbR that was eluted from affinity resin Phosphorylated PknH ⁄ EmbR (2 lg) was incubated with Mstp (1 lg) in 50 mm TrisHCl, pH 8.0, mm MnCl2 and 0.5 mm dithiothreitol, for 30 at 30 °C, the mixtures were resolved by SDS ⁄ PAGE and the loss of labeling was visualized by autoradiography ATPase activity measurements The malachite green ATPase assay The reaction buffer contained 10 lL of 10· TMD buffer, 10 lL of mgỈmL)1 BSA, lL of 100 mm ATP ⁄ GTP and 71 lL of H2O Five microlitres of purified dephosphorylated or phosphorylated EmbR (1 mgỈmL)1) was added to the reaction buffer and incubated at 37 °C At various time points (0, 5, 10, 20, 40 and 80 min), 10 lL of the reaction mixture was added to 80 lL of freshly prepared malachite green-ammonium molybdate reagent [three volumes of 0.045% malachite-green hydrochloride, one volume of 4.2% ammonium molybdate tetrahydrate in m HCl and 0.02 volume of 1% Triton X-100] After at room temperature, 10 lL of 34% citric acid was added to stop the colour development and the absorbance at 660 nm was measured The amounts of enzymatically released inorganic phosphate in triplicate samples were measured photometrically by referring to a standard curve, which was prepared with dilutions of a standard solution The ATPase activity of purified EmbR was also assayed by polyethyleneimine-TLC, as described previously [32] ATPase activity was also determined, as described previously, by a filter binding assay [32] Gel mobility shift assay The protein–DNA binding assay was performed as described previously [18] 32P-labeled probe DNA was prepared by end labeling using polynucleotide kinase, and FEBS Journal 273 (2006) 2711–2721 ª 2006 The Authors Journal compilation ª 2006 FEBS 2719 Regulation of EmbR activity by STPKs and Mstp K Sharma et al labeled PCR products, representing different promoter regions, were incubated with various amounts of phosphorylated and dephosphorylated EmbR at °C for 30 After incubation, complexes and free DNA were resolved by 5% nondenaturing polyacrylamide gels Gels were dried and subjected to autoradiography PknA ⁄ PknB –EmbR interaction assay (GST pulldown assays) The resin-bound phosphorylated GST–PknA, or the dephosphorylated (using Mstp) form of PknA (10 lg each), was incubated with a soluble fraction (5 lg of protein) of E coli cells expressing EmbR, at 25 °C for h in mL of NaCl ⁄ Pi buffer The protein–resin complex was washed six times with mL of NaCl ⁄ Pi each wash The proteins thus retained on the beads were eluted with elution buffer (50 mm Tris ⁄ HCl, pH 8.0, mm MgCl2, mm dithiothreitol, 15 mm glutathione) Eluted fractions were precipitated with trichloroacetic acid, resuspended in 30 lL of SDS-loading buffer and boiled for The proteins were then resolved on a 10% SDS–polyacrylamide gel, electroblotted onto a poly(vinylidene difluoride) membrane and probed with anti-His Ig conjugated to horseradish peroxidase (HRP) to detect the poly histidine-tagged EmbR fusion protein As a control, lg of EmbR was incubated either with 10 lg of GST bound to resin or to resin alone in NaCl ⁄ Pi buffer Similar assays were performed to study the interaction of EmbR with PknB and PknH Acknowledgements Financial support was provided by CSIR (SMM 0003) RNAP was a kind gift from Prof Anil K Tyagi, University of Delhi, Delhi Studentships of KS, MG and AK were supported by CSIR, India NS is an International Senior Fellow of the Wellcome Trust, UK References Av-Gay Y & Everett M (2000) The eukaryotic-like Ser ⁄ Thr protein kinases of Mycobacterium tuberculosis Trends Microbiol 5, 238–244 Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, Huygen K, Klebl B, Thompson C, Bacher G & Pieters J (2004) Protein kinase G from pathogenic mycobacteria promotes survival within macrophages Science 304, 1800–1804 Koul A, Herget T, Klebl B & Ullrich A (2004) Interplay between mycobacteria and host signalling pathways Nat Rev Microbiol 2, 189–202 2720 Sharma K, Chopra P & Singh Y (2004) Recent advances towards identification of new drug targets for 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localization Microbiology 147, 2307–2314 12 Gopalaswamy R, Narayanan PR & Narayanan S (2004) Cloning, overexpression, and. .. heat-inactivated Mstp) in phosphatase buffer Incubation with Mstp resulted in 73% and 79% dephospho- Autoradiogram Autoradiogram Phosphorylated forms of PknH and EmbR are substrates of Mstp in