Using bioinformatic analysis and in vitro assays, we identi-fied additional novel regulators of the signaling pathway leading to EmbR phosphorylation, namely the Ser⁄ Thr protein kinases
Trang 1substrate of multiple serine⁄threonine kinases and
phosphatase in Mycobacterium tuberculosis
Kirti Sharma1, Meetu Gupta1, Ananth Krupa2,*, Narayanaswamy Srinivasan2and Yogendra Singh1
1 Institute of Genomics and Integrative Biology, Delhi, India
2 Molecular Biophysics Unit, Indian Institute of Science, Bangalore
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
tuber-culosis, the causative agent for tubertuber-culosis, 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 estab-lish an environment that is favorable for the replica-tion 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
mycobacteri-al STPKs regulate diverse processes by phosphorylating
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, inter-acts 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 identi-fied 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 kin-ases and DNA-binding activity, emphasizing the antagonistic role of Mstp
in the EmbR–Ser⁄ Thr protein kinase signaling system
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.
Trang 2distinct substrates, including proteins implicated in
regulating cell division and morphology [13–15], an
ABC transporter [10,16], mediators of glutamate⁄
gluta-mine 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
arbinosyltransf-erases encoded by embCAB genes [18] embCAB is a
gene cluster involved in arabinan synthesis and
repre-sents 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
anti-tuberculosis drug) [18] Concomitantly,
Avenue-Gay 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
phosphory-lation-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
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
on transcription of the embCAB operon after its phos-phorylation by PknH in vivo [18] Until very recently, little was known about the mechanism by which SARPs exerted their effect on gene expression Bio-informatic 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 func-tion 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 (holo-RNAP) 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 dele-tion 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)
0 20 40 60 80 100 120
EmbR (mg/ml)
EmbR
EmbR Heat inactivated EmbR∆N
Fig 1 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 ngÆwell)1) coated in wells was incubated with EmbR ⁄ EmbRDN
at graded concentrations Unbound EmbR was removed and holo-RNAP bound EmbR was quantified using anti-EmbR Ig.
Trang 3A 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]
How-ever, after a closer examination of its amino acid
sequence, certain altered nucleotide-binding consensus
sequences were identified in EmbR (Fig 2A)
There-fore, the ability of EmbR to bind and hydrolyze
nuc-leotide 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
that EmbR is not a phosphatase These results showed that despite the absence of consensus nucleotide-bind-ing motifs, EmbR exhibits ATPase and GTPase activ-ities
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 ATP-ase activity of EmbR was analyzed While purified EmbR showed an ATPase activity of 0.040 nmol of phosphateÆmin)1Ælg)1 EmbR protein, the phosphoryl-ated form of EmbR displayed an ATPase activity of 0.257 nmol of phosphateÆmin)1Ælg)1 EmbR (i.e about
B
0
1
2
3
4
5
ATP + EmbR GTP + EmbR ATP + heat inactivated EmbR GTP + heat inactivated EmbR
Time (min)
C
0 1 2 3 4 5 6
Time (min)
EmbR Phosphorylated EmbR
0 20 40 60
Nucleotide binding motif
Consensus GXXXXGKT EmbR 226GAYRRVKT
Consensus DXXG EmbR 237DDLG
ATP
A ATP + EmbR
1 10 20 30 (min)
Pi
ATP
b a
Time (min)
Fig 2 (A) ATPase activity of EmbR (a) EmbR was incubated with [ 32 P]ATP[cP] for various time periods, and the release of 32 Phosphate ( 32 Pi) was monitored by TLC Also shown are altered nucleotide-binding motifs in EmbR (b) EmbR was incubated with [ 32 P]ATP[cP] for var-ious 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 hydro-lysis 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 experi-ments (C) Effect of phosphorylation on ATPase activity ATPase activities of EmbR and phosphorylated EmbR were compared The phos-phorylated EmbR sample was prepared by in vitro phosphorylation as described in the Experimental procedures.
Trang 4sixfold higher) (Fig 2C) The ATPase activity of
EmbR probably provides energy to catalyze the
isome-rization of the closed complex between EmbR and
RNAP to a transcriptionally competent open complex,
as is proposed for AfsR [25]
Phosphorylation of EmbR by multiple Ser⁄ Thr
kinases in M tuberculosis
For further characterization of EmbR and the
asso-ciated phosphorelay in M tuberculosis, the ability of
mycobacterial cell lysate to phosphorylate purifed
EmbR was analyzed Resin-bound EmbR was
incu-bated 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,
staurosp-orine, 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
myco-bacterial 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
phosphory-lation of EmbR by purified PknH All these
observa-tions 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
com-parisons of bacterial protein kinases to identify
homo-logues of kinases with known substrates This study
revealed PknB of M tuberculosis as the closest
homo-log of AfsK, an STPK-phosphorylating AfsR
Consid-ering the homology of AfsR with EmbR, as well as the
significant sequence similarity observed between
cata-lytic 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
autophosphor-ylated PknB phosphorylates EmbR, whereas
heat-inactivated 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
Lysate + 1m
M stau
ro sporin
e
EmbR
A
(a)
PknA
EmbR
1 2 3 4 1 2 3 4
PknB
EmbR
Autoradiogram
SDS-PAGE
(b)
0 20000 40000 60000
PknB
Kinase inactivatedHeat
Kinase
+ 20m M EDTA
B
L ysate B oiled Lysate Lysate+ A
nti -P H
P kn H
P kn
H + A nti -P H
Co ntro l
Fig 3 EmbR, a substrate for multiple serine ⁄ threonine protein kin-ases (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 con-ditions, as described in the Experimental procedures After elution, EmbR was run on SDS ⁄ PAGE and its phosphorylation was visual-ized 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 Coo-massie 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.
Trang 5tested for its ability to phosphorylate EmbR and it
came as an expected finding that PknA also
phosphor-ylated 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
myco-bacterial STPKs, viz PknH, PknA and PknB
Phosphorylated forms of PknH and EmbR are
substrates of Mstp in vitro
EmbR is phosphorylated by three STPKs, which
them-selves 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
de-phosphorylation of the existing phosphorylated species
The only reported Ser⁄ Thr phosphatase of M
tubercu-losis, 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’
sign-aling 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
sub-strates 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
heat-inactivated 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,
auto-phosphorylated PknH and phosphorylated EmbR
bound to resin were incubated with purified Mstp (or
heat-inactivated Mstp) in phosphatase buffer
Incuba-tion with Mstp resulted in 73% and 79%
dephospho-rylation of PknH and EmbR, respectively, in 30 min (Fig 4B) Our earlier studies, characterizing Mstp, revealed that PknA and PknB, the endogenous sub-strate 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
PknH
Mstp
Mstp EmbR
Autoradiogram
SDS-PAGE
A
B
0 20 40 60 80
EmbR
Heat inactivated Mstp
Fig 4 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 heat-inactivated Mstp (lane 1) The reaction products were resolved by SDS ⁄ PAGE and the loss of labeling was visualized by autoradiogra-phy Right, Coomassie Blue staining; left, corresponding autoradio-gram 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 [ 32 P]ATP[cP], as described in the Experimental procedures Mstp-mediated 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.
Trang 6Dephosphorylation 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
pro-moters [28]; however, synthesis of a polycistronic
mRNA encoding the three Emb proteins has also been
reported in M tuberculosis [29] PknH-mediated
phos-phorylation 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
prephospho-rylated EmbR, and the binding of phosphoprephospho-rylated⁄
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
2 lg of dephosphorylated protein bringing about a
similar mobility shift as seen for 0.3 lg of
phosphoryl-ated EmbR (Fig 5A)
Thus, while phosphorylation enhances the
DNA-binding activity of EmbR, the dephosphorylated form
of EmbR was incapable of binding the promoter
regions of emb genes at low concentrations, in
agree-ment with the belief that EmbR itself in the
phosphor-ylated 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
unphosphory-lated 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
interac-tion (Fig 5B), the soluble fracinterac-tion 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⁄ Pibuffer The binding assay
was performed as described in the Experimental proce-dures 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 incuba-ted 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 dephos-phorylated 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 be-tween PknH and its cognate DNA-binding transcrip-tion regulator, EmbR, triggers the regulatranscrip-tion of mycobacterial embCAB genes and consequently influ-ences ethambutol resistance and the LAM⁄ LM ratio The present study shows that EmbR serves as a sub-strate 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 exempli-fied by the PhoR⁄ PhoM ⁄ PhoB system in Escheri-chia 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 phosphory-lation-dependent ATPase activity despite the absence
of any conserved nucleotide-binding motifs in its amino acid sequence The enhancement of DNA-bind-ing 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
Trang 7OmpR 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
Con-versely, 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 More-over, Mstp-mediated dephosphorylation of kinases abrogates their interaction with EmbR, thus emphasi-zing the antagonistic role played by Mstp in the EmbR–STPK signaling cascade
In conclusion, by demonstrating multiple STPK-mediated phosphorylation and Mstp-STPK-mediated de-phosphorylation of EmbR, our findings add other upstream effectors to the EmbR-mediated signaling
embA -189/+135
P-Emb
A
0 0.3 1 2 5 (µg)
embB -194/+128
0 0.3 1 2 5 (µg)
embC 205/+112
0 0.3 1 2 5 (µg)
B
Glutathione Sepharose + + + + +
-Prephosphorylated PknA + - - - +
-Dephosphorylated PknA - + - - -
-EmbR
EmbR Anti His Blot
Fig 5 (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 32 P-labeled upstream regions of embC, embA and embB genes at 4 C for 30 min After incubation, complexes and free DNA were separated by nonde-naturing 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 dephospho-rylation 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 1 represents the molecular weight marker.
Trang 8network 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)
Collec-tively, 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
characteriza-tion to comprehend the role of these merging
path-ways in mycobacterial pathogenicity are in progress
and are expected to provide intriguing insights into
the significance of corresponding signaling events in
M tuberculosis
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 37C 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
EXTRA CELLU LAR
INTRAC ELLULAR
P
P
RNA Polymerase
P
RNA Polymerase
P
PknA PknB PknH
(Sensor Kinases)
SIGNAL X SIGNAL Y SIGNAL Z
EmbR
Mstp
Autophosphorylation
ATP hydrolysis
EmbR
Dephosphorylation Dephosphorylation
SIGNAL A
Closed complex
Open complex
Increased transcription
of embCAB genes
LAM / LM Ratio
EMB Resistance
Arabinan Metabolism
Fig 6 A hypothetical scheme for the regulation of arabinan metabolism by the Mstp serine ⁄ threonine protein kinases (STPKs) ⁄ EmbR ⁄ emb-CAB system in Mycobacterium tuberculosis By analogy with eukaryotic signal transduction, we speculate that multiple STPKs autophospho-rylate 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.
Trang 9denaturing conditions using Ni-nitrilotriacetic acid resin, as
per the manufacturer’s instructions and as described
previ-ously [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
prin-cipal sigma factor, sigA, was kindly provided by Prof Anil
K Tyagi Purified Holo-RNAP was coated on a 96-well
ELISA plate (100 ngÆwell)1) His–EmbR or His–EmbRDN
fusion proteins were incubated, at three concentrations,
with the coated protein overnight at 4C in buffer
compri-sing 10 mm Tris HCl, 150 mm NaCl, pH 7.5, 0.5% Tween
20 (TBS-T), after which the plates were washed and
devel-oped 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, 1 mm dithiothreitol) with 1 lg of EmbR
and 5 lCi of [32P]ATP[cP] and incubated for 30 min at
37C The reactions were stopped by the addition of SDS
sample buffer, and proteins were separated by 1D gel
elec-trophoresis, electroblotted onto nitrocellulose membranes
and visualized by autoradiography
For resin-bound kinase assays, purified EmbR was
phos-phorylated by PknH in the kinase buffer, as described
pre-viously [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
autophos-phorylated 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, 1 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,
1 mm dithiothreitol (TMD) buffer and 50 mm sodium
fluor-ide (Ser⁄ Thr phosphatase inhibitor) for 30 min at room
tem-perature 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
resin-bound EmbR The effect of kinase inhibitor was investigated
by pre-incubating the whole cell lysate with 1 mm
staurospo-rine The effect of anti-PknH Ig was examined by
pre-incuba-ting the whole cell lysate with anti-PknH Ig (1 : 500 dilution)
for 20 min before incubation with resin-bound EmbR
In vitro phosphatase assay
For resin-bound assays, dephosphorylation of phosphoryl-ated PknH and EmbR by Mstp was examined by measur-ing 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 liberated32Pi and the proteins were eluted at 65C 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 Tris-HCl, pH 8.0, 5 mm MnCl2 and 0.5 mm dithiothreitol, for
30 min at 30C, 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 5 mgÆmL)1BSA, 4 lL of 100 mm ATP⁄ GTP and
71 lL of H2O Five microlitres of purified
dephosphorylat-ed or phosphorylatdephosphorylat-ed EmbR (1 mgÆmL)1) was added to the reaction buffer and incubated at 37C 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 4 m HCl and 0.02 volume of 1% Triton X-100] After 1 min 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 photometri-cally 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 previ-ously, by a filter binding assay [32]
Gel mobility shift assay
The protein–DNA binding assay was performed as des-cribed previously [18] 32P-labeled probe DNA was prepared by end labeling using polynucleotide kinase, and
Trang 10labeled PCR products, representing different promoter
regions, were incubated with various amounts of
phosphor-ylated and dephosphorphosphor-ylated EmbR at 4C for 30 min
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
pull-down 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
pro-tein) of E coli cells expressing EmbR, at 25C for 4 h
in 1 mL of NaCl⁄ Pi buffer The protein–resin complex
was washed six times with 1 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, 5 mm MgCl2,
1 mm dithiothreitol, 15 mm glutathione) Eluted fractions
were precipitated with trichloroacetic acid, resuspended in
30 lL of SDS-loading buffer and boiled for 3 min The
proteins were then resolved on a 10%
SDS–polyacryla-mide gel, electroblotted onto a poly(vinylidene difluoride)
membrane and probed with anti-His Ig conjugated to
horseradish peroxidase (HRP) to detect the poly
histi-dine-tagged EmbR fusion protein As a control, 5 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
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