Extensive phosphorylation with overlapping
specificity by Mycobacterium tuberculosis serine/
threonine protein kinases
Sladjana Prisica, Selasi Dankwaa,1, Daniel Schwartzb, Michael F. Choub, Jason W. Locasalec, Choong-Min Kanga,2,
Guy Bemisd, George M. Churchb, Hanno Steene,f, and Robert N. Hussona,3
aDivision of Infectious Diseases, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115;bDepartment of Genetics, Harvard Medical School,
Boston, MA 02115;cDivision of Signal Transduction, Beth Israel Deaconess Medical Center and Department of Systems Biology, Harvard Medical School,
Boston, MA 02115;dVertex Pharmaceuticals, Cambridge, MA 02139;eDepartment of Pathology, Children’s Hospital Boston, Harvard Medical School, Boston,
MA 02115; andfProteomics Center, Children’s Hospital Boston, Boston, MA 02115
Edited* by Barry R. Bloom, Harvard School of Public Health, Boston, MA, and approved March 5, 2010 (received for review November 20, 2009)
The Mycobacterium tuberculosis genome encodes 11 serine/threo-
nine protein kinases (STPKs) that are structurally related to eukary-
otickinases. To gain insightintothe role ofSer/Thrphosphorylation
in this major global pathogen, we used a phosphoproteomic
approach to carry out an extensive analysis of protein phosphory-
lation in M. tuberculosis. We identified more than 500 phosphory-
lation events in 301 proteins that are involved in a broad range of
functions. Bioinformatic analysis of quantitative in vitro kinase
assays on peptides containing a subset of these phosphorylation
sites revealed a dominant motif shared by six of the M. tuberculosis
STPKs. Kinase assays on a second set of peptides incorporating tar-
motif and identified additional residues preferred by individual
kinases. Our data provide insight into processes regulated by STPKs
in M. tuberculosis and create a resource for understanding how
specific phosphorylation events modulate protein activity. The
results further provide the potential to predict likely cognate STPKs
for newly identified phosphoproteins.
signal transduction|phosphorylation motif|phosphoproteomics
propagate through complex signal transduction networks whose
activity is often regulated by reversible protein phosphorylation.
Although Ser/Thr/Tyr protein phosphorylation-based signaling in
eukaryotes has been intensively studied, the extent to which this
mechanism is used in prokaryotes has only recently begun to be
appreciated (1). The number of protein kinases in prokaryotes
varies widely. Although many bacteria have only a few or none of
these enzymes, some cyanobacteria and streptomycetes have doz-
ens of them (2). Bacteria that do possess Ser/Thr or Tyr kinases
often have complex lifestyles and depend on these kinases to reg-
ulate critical processes, such as stress adaptation, development,
and virulence (2).
Mycobacterium tuberculosis is an extraordinarily versatile
pathogen that can exist in distinct states in the host, leading to
asymptomatic latent tuberculosis (TB) infection in which bac-
teria are thought to be dormant, or active TB disease in which
the organisms are actively replicating. To achieve these different
physiologic states M. tuberculosis requires mechanisms to sense a
wide range of signals from the host and to coordinately regulate
multiple cellular processes. In most bacterial pathogens, the
predominant phosphorylation-based signal transduction mecha-
nism is the two-component system. The M. tuberculosis genome,
however, encodes 11 Ser/Thr protein kinases (STPKs) and an
equal number of two-component system sensor kinases, sug-
gesting that these two phospho-based signaling systems are of
comparable importance in this organism (3).
Knowledge of the substrates of each of the M. tuberculosis
STPKs is essential for understanding their function; however,
key feature of all living cells is the ability to sense environ-
mental signals and implement adaptive changes. These inputs
only a small number of kinase-substrate cognate pairs have been
and PknB, which regulate cell shape and cell wall synthesis via
phosphorylation of the cell pole-localized protein Wag31 and the
that has been implicated in TB pathogenesis, PknG, phosphor-
ylates the forkhead-associated (FHA) domain-containing protein
and nitrogen metabolism in a phosphorylation state-specific
Our current limited view of protein phosphorylation in M.
tuberculosis mirrors the relatively sparse phosphorylation data in
prokaryotic organisms more generally. To obtain a more com-
prehensive understanding of in vivo phosphorylation events in M.
tuberculosis, we used a mass spectrometry-based approach to
identify phosphorylation sites in M. tuberculosis proteins. These
results provide the most extensive data on Ser/Thr phosphor-
ylation currently available for any bacterium, more than doubling
the currently known bacterial phosphoproteome, and provide
insight into the range of functions regulated by Ser/Thr phos-
phorylation in M. tuberculosis. Bioinformatic analysis of these in
vivo phosphorylations, and of data from in vitro kinase assays,
enabled us to identify and validate a phosphorylation site motif
shared by several kinases, leading to a model of STPK–substrate
interaction. In addition to providing insights into Ser/Thr phos-
phorylation in M. tuberculosis, these data will serve as an impor-
tant resource for further investigation of these signal transduction
pathways in M. tuberculosis, and in prokaryotes more broadly.
Identification of 301 Phosphoproteins in M. tuberculosis. We used a
proteomic approach to identify phosphoproteins and their
phosphorylation sites in M. tuberculosis proteins (Fig. 1). To
Author contributions: S.P., H.S., and R.N.H. designed research; S.P., S.D., C.-M.K., and H.S.
performed research; D.S., M.F.C., J.W.L., and G.M.C. contributed new reagents/analytic
tools; S.P., S.D., D.S., M.F.C., J.W.L., C.-M.K., G.B., G.M.C., H.S., and R.N.H. analyzed data;
and S.P., D.S., M.F.C., and R.N.H. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The entire dataset of the chromatography tandem mass spectrometry
results is in an Excel file that is part of the supplemental material. Raw spectral data files
are available at http://www.researchcomputing.org/Husson/Mtb_Phosphoproteome_
1Present address: Department of Immunology and Infectious Diseases, Harvard School of
Public Health, Boston, MA 02115.
2Present address: Department of Biological Sciences, Wayne State University, Detroit,
3To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| April 20, 2010
| vol. 107
| no. 16
increase the number of phosphoproteins identified, protein
extracts were prepared from M. tuberculosis H37Rv cultures
(i) supplied with different carbon sources, (ii) grown to different
growth stages, and (iii) exposed to stresses including NO, per-
oxide, and hypoxia. More than 150 samples were analyzed by
liquid chromatography tandem mass spectrometry (LC-MS/MS)
and phosphopeptides were identified using the ProteinPilot and
Mascot algorithms with high stringency cutoffs.
We identified 301 phosphoproteins containing at least 516
phosphorylation sites demonstrating that at least 7% of M.
tuberculosis proteins are phosphorylated (Table S1). Of these
phosphoproteins, more than 40% contained more than one
phosphorylation site, with some proteins having as many as seven
sites (Tables S1 and S2). Among the 301 phosphoproteins
identified in this study are several of the previously defined M.
tuberculosis phosphoproteins, including four STPKs, GarA,
Rv1422, and FhaA (4, 8, 10). The MS/MS search algorithms that
we used can identify the presence of a phosphorylation site with
high specificity but cannot always determine the precise site
phosphorylation within the peptide backbone. We therefore used
the Ascore algorithm (11) to attempt to identify the specific
phosphoacceptor residue within each phosphopeptide. Using
this approach we identified 215 phosphoacceptor residues with
Phosphorylation in M. tuberculosis was biased toward Thr
compared with Ser (60%:40%),a striking departure from findings
in eukaryotes, where Ser phosphorylation may account for 80–
90% of total phosphorylation sites (12). Among other bacteria,
Bacillus subtilis, Escherichia coli, and Pseudomonas species all
show greater phosphorylation of Ser than Thr, whereas data from
Lactococcus lactis showed 51% and 46% Thr and Ser phosphor-
ylation, respectively (13–16).
To determine whether specific sequences are preferentially
targeted for Ser/Thr phosphorylation, we used the motif-x algo-
rithm (17) to search for sequence motifs surrounding the phos-
phoacceptor for the 215 well-localized phosphorylation sites.
Four statistically significant motifs were identified, all with Thr
as the phosphoacceptor (Fig. S1). This result indicates that the
M. tuberculosis STPKs, in addition to preferentially phosphor-
ylating Thr vs. Ser, target the phosphoacceptor in the context of
Ser/Thr Phosphorylation Regulates a Wide Range of Functions in
M. tuberculosis. The phosphoproteins identified in these experi-
ments belong to all functional classes of proteins (18) (Fig. S2),
with the largest numbers involved in cell wall/cell processes and
intermediary metabolism/respiration. The proportion of phos-
phoproteins in each category is not significantly different from
the distribution of all proteins in the M. tuberculosis H37Rv
genome annotation (18). Closer inspection of these data, how-
ever, provides interesting insights regarding the regulation of cell
physiology by STPKs in M. tuberculosis. Several chaperones, for
example, are phosphorylated, suggesting extensive regulation of
protein turnover by Ser/Thr phosphorylation (Table S1). Multi-
ple phosphorylation events on transporter proteins and lipid
metabolic enzymes suggest modulation of the interface between
the cell and the extracellular environment by the STPKs. Many
cell division proteins are phosphoproteins, indicating a key role
for phosphorylation in regulating this process.
Phosphorylation of the STPKs themselves is of interest for
understanding their regulation. We found that four STPKs
(PknA, PknB, PknD, and PknG) were phosphorylated in vivo. In
addition to activation loop phosphorylation, sites in the intra-
cellular juxtamembrane region, which has been hypothesized to
have a regulatory function, were identified for PknA, PknB, and
PknD (19, 20). Some of these sites were previously shown to be
phosphorylated in vitro (19–21). In contrast to the other protein
kinases, we found that PknG is phosphorylated near its amino
terminus, also consistent with previous in vitro results (8).
In Vitro Phosphorylation of Peptides Containing in Vivo Phosphory-
lation Sites. We designed 336 13-mer biotinylated peptides corre-
sponding to in vivo phosphorylation sites in M. tuberculosis
proteins. The peptides were incubated with the recombinant kin-
ase domain of nine STPKs (we were unable to produce active
PknI and PknJ) in presence of [γ-33P] ATP. After binding peptides
to streptavidin-coated plates, excess radiolabeled ATP was washed
away and incorporated33P signal was measured using a scintilla-
tion counter. Half of the peptides were phosphorylated 2-fold or
more above background by at least one kinase in these experi-
ments. Most peptides that were phosphorylated in vitro were
phosphorylated by more than one kinase, and some were actively
targeted by the majority of kinases (Table S3).
In contrast to the peptides phosphorylated by multiple kinases,
we found 48 peptides that were phosphorylated by a single kin-
ase. The peptides that are uniquely phosphorylated by a single
kinase suggest several interesting STPK–substrate pairs. For
example, PknD uniquely and strongly phosphorylates a peptide
from the amino terminus of SecF, whereas PknH uniquely and
strongly phosphorylates a peptide derived from the chaperone
DnaJ1. These data suggest that PknD may regulate protein
secretion and PknH may regulate protein turnover. These data
thus predict candidate in vivo targets of individual STPKs and
provide a basis for experimental investigation of their role in
regulating cell physiology.
Identification of a Shared Phosphorylation Site Motif. To identify
sequence motifs among the peptides phosphorylated by the M.
tuberculosis STPKs, we analyzed separately the peptides that
were and were not phosphorylated in vitro, using motif-x (Figs.
S3 and S4). We found motifs that were highly similar to those
that we obtained in the analysis of well-localized in vivo phos-
phorylation sites (Fig. S1). Notably, the set of peptides that were
phosphorylated in vitro had markedly different motifs from the
peptides that were not (Figs. S3 and S4).
We next performed analyses to attempt to identify preferred
phosphorylation site sequences for each kinase. We used two
approaches: a threading algorithm that compared the frequency
of residues at each position in sequences of highly phosphory-
lated peptides with the entire set of synthesized peptide
sequences (SI Materials and Methods), and the motif-x algorithm
(17) using the entire M. tuberculosis proteome as the back-
ground. We were able to identify significant motifs for the six
most active kinases—PknA, PknB, PknD, PknE, PknF, and
PknH—using motif-x and for five of these six (excluding PknH)
with the threading algorithm (Fig. 2 and Figs. S5 and S6). For all of
these kinases, major features of the preferred phosphorylation site
motif include Thr as the phosphoacceptor and highly significant
selection for hydrophobic residues at the +3 and +5 positions
M. tuberculosis grown under six conditions
Protein extraction & size fractionation
Reduction, alkylation, trypsin digestion
LC-MS/MS with ProteinPilot, Mascot and Ascore searches
In vitro kinase assays with recombinant STPKss with
336 synthetic peptides3
32 synthetic peptides3
detection and phosphorylation site motif identification and verification in
Flow diagram of approach to Ser/Thr protein phosphorylation
| www.pnas.org/cgi/doi/10.1073/pnas.0913482107 Prisic et al.
(3 and 5 residues carboxyl-terminal to the phosphoacceptor). At
the +3 position, several large hydrophobic residues were highly
selected, whereas at the +5 position Ile was the predominant
residue (Fig. 2 and Fig. S6).
In addition to these major features at +3 and +5, we identi-
fied additional significantly overrepresented residues at other
positions. Most prominent are the preferences of several kinases
for acidic resides at positions N-terminal to the phosphoacceptor
(−1 to −4) and for Pro or Arg at the +4 position (Fig. 2 and Fig.
S6). The only motif identified with Ser as a phosphoacceptor had
borderline significant preference by PknD for acidic residues at
the −5 position (Fig. S5).
To verify that the major features of the phosphorylation motif
identified in the peptide kinase assays are important for substrate
recognition in the context of a full-length protein, we performed
kinase assays with PknB, using WT and substituted forms of the
M. tuberculosis protein GarA as the substrate. GarA has been
shown to be phosphorylated on adjacent residues by PknB
(Thr22) and PknG (Thr21) (8). The sequence surrounding the
PknB-phosphorylated residue (VTVETTSVFRA, Thr22 in bold)
contains the major features of the PknB phosphorylation motif,
including the large hydrophobic residue (Phe) at +3 (Fig. 2) and
an acidic residue (Glu) at −2. Substitution of Phe at +3 with Ala
(F25A) resulted in markedly decreased phosphorylation of GarA
by PknB, comparable to removal of the phosphoacceptor (T22A)
(Fig. S7). Substitution of Glu at −2 (E20V) also severely
decreased phosphorylation. In contrast, substitution of Thr21
(T21A)didnothavea significant effectonPknBphosphorylation,
confirming Thr22 as the PknB phosphoacceptor in these assays.
Validation of the Dominant Motif and Identification of Specificity
Determinants. In addition to the phosphorylation motif shared by
the six most active kinases, our analysis also suggested differences
in the optimal substrate sequences for each kinase (Fig. 2 and Fig.
S6). These include the specific hydrophobic residues preferred by
each kinase at the +3 position, the position of acidic residues N-
terminal to the phospho-Thr, and preference for Pro vs. Arg at
+4.Inaddition,therewasapparentselectivity bysomekinases for
residues at other positions, which did not reach statistical sig-
nificance. To validate the motif and test these predictions, we
chose a peptide (ITVAELTGEIPII) that was highly phosphory-
lated by the six most active kinases, and changed selected residues
in a manner predicted to increase or decrease phosphorylation by
incubated with the six kinases (Fig. 3).
Results of phosphorylation of these substituted peptides were
mostly in agreement with predictions from the original in vitro
phosphorylation data (Fig. 3). These experiments confirmed that
large hydrophobic residues at +3 and +5 are dominant com-
ponents of a common phosphorylation motif for PknA, PknB,
PknD, PknE, PknF, and PknH. Acidic residues from −2 to −5
increase phosphorylation by most of these kinases, although
generally they are not as important as the +3 and +5 positions.
Surprisingly, Pro or Arg at +4 was required for optimal phos-
phorylation by all six kinases. It is particularly noteworthy that, as
predicted from the kinase-specific motifs, Ser could not sub-
stitute for Thr as the phosphoacceptor for any kinase.
In addition to these shared components of the phosphor-
ylation site motif, differences in kinase-specific preferences are
evident in these data. For example, the importance of the acidic
residues at the −2 to −5 positions, and whether Asp or Glu is
optimal, varies among the different kinases. We also observed
increased phosphorylation by PknB, PknE, PknF, and especially
PknH when Val is at the +2 position. At the +4 position there
was strong preference for Pro (PknA and PknB) vs. Arg (PknD,
PknE, and PknH) among different STPKs. Also as predicted, Ile
is preferred at position +5, although Leu can substitute without
a significant decrease in phosphorylation by PknD, PknE, and
PknH. Surprisingly, substitution of Gly for Ile at position +6
markedly impaired phosphorylation by PknA, PknB, and PknD.
Combining the results of the initial in vitro phosphorylation with
the refinement provided by the substituted peptides yields the
shared motif XααααTX(X/V)ϕ(P/R)I (where α is an acidic res-
idue and ϕ a large hydrophobic residue), with the potential for
kinase-specific selectivity in the specific residues at positions that
contribute to the motif.
Model of a PknB Peptide–Substrate Complex Suggests a Basis for the
Phosphorylation Site Motif. Several crystal structures of M. tuber-
culosis kinases have been reported; however, in none of these is
median are shown for PknA, PknB, PknD, PknE, PknF, and PknH. The pLOGos show the relative statistical significance (with respect to the M. tuberculosis
proteomic background) of residues within 6 aa of the central Thr phosphorylation site. Residues above the midline are overrepresented, whereas those below
the midline are underrepresented. The red horizontal line indicates the 0.01 significance level (after Bonferroni correction).
Phosphorylation site motif analysis. pLOGos generated using motif-x based on in vitro assays in which peptide phosphorylation was >3-fold above
Prisic et al.PNAS
| April 20, 2010
| vol. 107
| no. 16
the kinase cocrystallized with a substrate, nor do any of these
structures include the activation loop. To search for structural
features of M. tuberculosis STPK active sites that might explain
substrate specificity, we generated a model PknB structure in
complex with an optimal substrate peptide, using rabbit phos-
phorylase kinase (Phk) in complex with its substrate as a refer-
ence structure (Fig. 4A). The peptide substrate in this model
contains residues from −3 to +5 around the phosphoacceptor
Although this model does not identify exact contacts of PknB
with the peptide, it does suggest probable residues that con-
tribute to substrate binding. As shown in Fig. 4B, the central part
of the activation loop is in close contact with substrate residues
hydrogen bonds between the peptide backbone of the substrate
(from −1 to +3) and the kinase active site (hydroxyl group from
Thr179 and peptide bond of Val176 and Gly178). Several resi-
dues in this portion of the activation loop (positions 174–179)
are highly conserved among the M. tuberculosis STPKs, sug-
gesting that it may be a common binding site for substrate resi-
dues at the +2, +3, and +5 positions (Fig. 4C).
As an initial test of the model based on predicted interactions
with the +3 hydrophobic residue in the optimal substrate motif,
we investigated the effect of mutation of PknB at Val176 to
either a negatively (V176D) or positively (V176R) charged res-
idue (Fig. 5). These altered forms of PknB are catalytically
functional on the basis of active autophosphorylation at levels
comparable to WT PknB. Both substitutions resulted in mark-
edly decreased phosphorylation of the “ideal peptide,” which
contains Ile at the +3 position, compared with WT PknB, con-
sistent with the prediction of the structural model.
The model indicates that Pro at the +4 position in the PknB
substrate motif serves to position the +3 and +5 residues for
binding. Arg, a preferred residue at this position for some
kinases, would serve a similar purpose, but might also have
contacts with the active site in kinases that prefer this residue.
This model also locates the basic residues Arg101 and Lys140 of
PknB close to the acidic residues present at the −2 to −4 posi-
tions of the substrate (Fig. 4 B and C). Lys140 is conserved in all
M. tuberculosis STPKs except PknI, whereas Arg101 is less con-
served (Fig. 4C).
The presence of 11 STPK genes in the M. tuberculosis genome
suggests that Ser/Thr phosphorylation is an important mecha-
nism for signal transduction in this organism but that the total
phosphoproteome is likely to be substantially smaller than that of
most eukaryotes. By searching for phosphoproteins in M. tuber-
culosis grown under several conditions, we identified 516 phos-
phorylation events, occurring in 301 M. tuberculosis proteins,
several-fold more than any prior study in prokaryotes. On the
basis of our identification of some but not all previously descri-
bed phosphoproteins, however, there are likely many phospho-
proteins that have not been identified.
the six kinases that share a substrate phosphorylation site motif. A peptide
from Rv0497 that has features of the shared phosphorylation site motif was
synthesized, together with 31 additional peptides incorporating sub-
stitutions predicted to enhance or diminish phosphorylation. The phos-
phoacceptor is shown in bold. Substituted residues are underlined.
Phosphorylation of each peptide is expressed as a ratio relative to phos-
phorylation of the original peptide (WT). Substitutions that increase or
decrease phosphorylation by 50% or more are shaded in green or red,
respectively. Mean values of two independent experiments are shown.
In vitro phosphorylation of ideal (WT) and substituted peptides by
PknA (101) EPLNSVLKRT--GRLSLRHALDMLEQTGRALQIAHAAGLVHRDVKPGNI
PknL (102) GTLRELLIER--GPMPPHAVVAVLRPVLGGLAAAHRAGLVHRDVKPENI
PknG (238) QSLKRSKGQK---LP-VAEAIAYLLEILPALSYLHSIGLVYNDLKPENI
PknK (109) NSLETLIRRH--GPLDWRETLSIGVKLAGALEAAHRVGTLHRDVKPGNI
PknA (148) LIT----PTGQVKITDFGIAKAVDAAP--VTQTGMVMGTAQYIAPE
PknB (145) MIS----ATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPE
PknL (149) LIS----DDGDVKLADFGLVRAVAAAS--ITSTGVILGTAAYLSPE
PknG (183) MLT-----EEQLKLIDLGAVSRIN-------SFGYLYGTPGFQAPE
PknD (145) LVT----ASDFAYLVDFGIARAAS-DPG-LTQTGTAVGTYNYMAPE
PknE (146) LVS----ADDFAYLVDFGIASATT-DEK-LTQLGNTVGTLYYMAPE
PknH (146) LIT----RDDFAYLVDFGIASATT-DEK-LTQLGTAVGTWKYMAPE
PknF (144) LIANPDSPDRRIMLADFGIAGWVDDPSG-LTATNMTVGTVSYAAPE
PknI (144) VLTSQSAGDQRILLADFGIASQP--------S---------YPAPE
PknJ (143) LLSRAAGGDERVLLSDFGIARALG-DTG-LTSTGSVLATLAYAAPE
PknK (156) LLT----DYGEPQLTDFGIARIAG---GFETATGVIAGSPAFTAPE
strate. (A) The PknB kinase domain in complex with an ideal peptide sub-
strate (AELTGEIPI) was modeled using x-ray crystal structures of the
M. tuberculosis PknB kinase domain [Protein Data Bank (PDB) no. 1o6y] and of
a Phk-peptide substrate complex (PDB no. 2phk). ATP is in red, and the peptide
in blue. (B) Likely contacts between PknB active site residues and the peptide,
within 4 Å, are shown. Colors of PknB residues correspond to highlighted
amino acids shown in C. The peptide is in blue except for the phosphoacceptor
Thr, which is yellow. Hydrogen atoms and the main chain atoms were omitted,
except for hydrogen bond contacts. (C) Alignment of the kinase domains of all
M. tuberculosis STPKs, highlighting residues that are predicted to be in close
contact with the peptide. The numbers in the color code indicate the position
in the peptide substrate with which the residues shaded in that color may
interact. In addition to the interactions indicated by the color code, kinase
residues labeled with “*” are predicted to interact with the −2 position, “#”
with the +2 position, and “&” with +2 and +5 positions.
Model of PknB structure in a complex with an ideal peptide sub-
| www.pnas.org/cgi/doi/10.1073/pnas.0913482107Prisic et al.
Comparing our results to studies of Ser/Thr phosphorylation in
other bacteria shows interesting similarities. Of 41 phosphopro-
teins identified in the soil actinomycete Corynebacterium gluta-
in M. tuberculosis. This finding suggests that the M. tuberculosis
phosphoproteome identified here may provide insight into pro-
teins targeted by STPKs in other Actinomycetes, and possibly in
other bacteria. Phosphorylation of ribosomal proteins has been
found in Pseudomonas putida (16), L. lactis (13), and E. coli (15).
We found that five ribosomal and a ribosome-associated protein
are phosphorylated in M. tuberculosis, indicating regulation of
translation by Ser/Thr phosphorylation in this pathogen. Together
with phosphorylation of proteins involved in DNA and RNA
turnover, and in essential metabolic pathways, these data suggest
that Ser/Thr phosphorylation provides a mechanism for the coor-
dinate regulation of M. tuberculosis physiology during infection.
We previously determined that PknA and PknB both prefer-
entially phosphorylate peptides that have a large hydrophobic
residue at the +3 position (4). Here, motif extraction from
quantitative phosphorylation of more than 300 peptides, fol-
lowed by validation using peptides with targeted substitutions,
allowed us to define an extensive motif shared by these two
kinases and by PknD, PknE, PknF, and PknH. The conservation
of the dominant components of the motif among these six
kinases, although initially surprising, is consistent with the sim-
ilarity of the predicted substrate binding sites of these STPKs
(Fig. 4). Beyond the shared components of the motif, this
approach provided insight into potential differences in the
sequences that each of these kinases optimally target. In con-
trast, the peptides phosphorylated by PknG and PknK do not
match this common motif, consistent with their kinase domains
being markedly dissimilar to those of the other STPKs (Fig. S8).
This common motif has at least two potential implications for
substrate targeting by M. tuberculosis STPKs. One is that multi-
ple kinases may target a single protein, which has been shown
previously for at least two M. tuberculosis STPK substrates, GarA
and Rv1422 (4, 23). The other implication is that other factors
must contribute to in vivo substrate specificity of the individual
STPKs. These factors are likely to include coordinated expres-
sion and colocalization of kinase and substrate, and protein–
protein interactions both between the substrate and a cognate
STPK, and in many cases with other members of larger protein
complexes. In addition, less dominant kinase-specific phosphor-
ylation site sequence preferences may be important determinants
of specificity. Although the shared motif of the six STPKs limits
direct mapping of individual substrates to a cognate kinase, these
secondary motif characteristics should allow prediction in some
cases of the STPK(s) most likely to phosphorylate newly iden-
tified M. tuberculosis phosphoproteins, and potentially to predict
de novo protein substrates of individual STPKs.
The importance of the +3 position in the common motif, which
is typically a critical position for FHA recognition (24), suggests
that phospho-dependent interactions of STPK substrates with
FHA domain proteins may also contribute to specific downstream
signaling mediated by these kinases. In support of this idea, the
GarA FHA domain was previously shown to prefer hydrophobic
residues at the +3 position relative to phospho-Thr(25). As noted
above, the sequence adjacent to the Thr22 residue in GarA that is
phosphorylated by PknB, and that is also bound by its FHA
domain, matches remarkably well the preferred phosphorylation
site motifofPknB,includingthehydrophobicresidue (Phe) at +3.
In summary, we have identified extensive Ser/Thr phosphor-
ylation of M. tuberculosis proteins, demonstrating that the STPKs
of this organism regulate a broad range of cellular processes and
indicating that this regulatory mechanism is likely to be impor-
tant for the virulence of this major global pathogen. We defined
common and specific components of a preferred substrate motif
shared by six STPKs. These data provide insight into signal
transduction in prokaryotes, and provide a resource for inves-
tigation of the regulation of M. tuberculosis physiology and
pathogenesis by Ser/Thr phosphorylation.
Materials and Methods
M. tuberculosis Cultures, Protein/Peptide Preparation, and Analysis. M. tuber-
culosis H37Rv was grown in broth culture under several conditions (NO
stress, oxidative stress, hypoxia, and glucose or acetate as a carbon source)
and harvested at different growth stages. Proteins were extracted using
TRIzol, separated by SDS/PAGE, reduced, alkylated, and trypsin-digested,
followed by phosphopeptide enrichment (see SI Materials and Methods). A
total of 152 samples were run on a Thermo Finnigan LTQ instrument.
Phosphopeptides were identified using the ProteinPilot 2.0 (Applied Bio-
systems) and Mascot (Matrix Science) software. Spectra were analyzed with
the Ascore algorithm to attempt to unambiguously localize the phos-
phoacceptor in each tryptic phosphopeptide (11).
Recombinant Protein Kinase Expression and in Vitro Kinase Assays. Kinase
domains of all 11 STPK genes were cloned from M. tuberculosis genomic DNA
PknG, PknI, and PknJ were not successfully obtained using this approach;
therefore, full-length proteins were expressed in Mycobacterium smegmatis.
A total of 336 biotinylated 13-mer peptides containing phosphorylation
sites identified from M. tuberculosis protein lysates were synthesized as
crude product (BioTides; JPT Peptide Technologies). Kinase assays were
performed in 50 μL containing the following components: recombinant
kinase (10–200 ng/μL), ATP (10 μM), [γ-33P] ATP (EasyTide; PerkinElmer) (0.5
μCi), and peptide substrate (5 μM) in 50 mM Mops (pH 7.4), 10 mM MgCl2,
and 10 mM MnCl2. The reaction was incubated at least 4 h at 30 °C, stopped,
and transferred to streptavidin-coated FlashPlates (Perkin-Elmer). After
overnight incubation, plates were washed and read in a TopCount instru-
ment (Perkin-Elmer). For motif validation, 32 peptides were synthesized and
purified to at least 70% purity, and kinase assays were performed as
described above, except that incubation times were shorter (1.5–2 h).
Mutagenesis of PknB and GarA and Phosphorylation Analysis. The WT
GST-tagged PknB kinase domain was mutated using the QuikChange kit
(Stratagene), expressed in E. coli, and purified using the B-PER GST kit
(Pierce Biotechnology). GarA was cloned into the pENTR vector (Invitrogen),
mutated using QuikChange (Stratagene), and transferred to pDEST17
(Invitrogen) to express His-tagged proteins in E. coli. HisPur (Pierce Bio-
technology) was used for purification of WT and mutant GarA proteins.
y = 1.6x + 42.6; R2 = 0.970
y = 28.3x - 26.1; R2 = 0.997
y = 179.3x - 63.5; R2 = 0.998
0 20 4060 80100120
relative rate (%)
PknB was mutated to Asp or Arg, and in vitro phosphorylation of an ideal
peptide (peptide 1 in Fig. 3) by WT and substituted PknB proteins was per-
formed and quantified by33P incorporation at serial time points. (B) Auto-
radiography of WT and substituted PknB after incubation with γ-P33-ATP
followed by SDS/PAGE. (C) GelCode Blue (Pierce) stained gel showing
approximately equal amounts of protein loaded on the gel.
Kinase activity of WT and substituted forms of PknB. (A) Val176 of
Prisic et al.PNAS
| April 20, 2010
| vol. 107
| no. 16
PknB mutants were analyzed for ideal peptide phosphorylation (peptide 1
in Fig. 3) using FlashPlates as described above. Kinase assays in the presence
of [γ-33P] ATP were performed to test autophosphorylation of PknB mutants
and phosphorylation of GarA. Reactions were run on SDS/PAGE, and radio-
active signal was detected and quantified using a Storm phosphorimager
Phosphorylation Site Motif Analysis. Two complementary approaches were
used to identify preferred phosphorylation site motifs, an internal working
version of the motif-x algorithm (17), and a threading algorithm. Motif-x
was used to analyze in vivo phosphorylated sequences, and sequences of
peptides that were phosphorylated in vitro at least 3-fold over the median
for all peptides in two replicates (SI Materials and Methods). Probability log-
based logos (pLOGos) were generated for all phosphorylated and non-
phosphrylated peptides, and for the peptides phosphorylated by each kin-
ase. All analyses were performed using the M. tuberculosis proteome as
background. Residues that had values over the 0.01 significance level (after
Bonferroni correction) were deemed statistically significant and used by the
motif-x algorithm to fix motif positions in Fig. 2 and Figs. S1, S3, and S4.
For the threading algorithm, peptides that were phosphorylated in vitro
by a kinase 5-fold over the median value for all peptides were analyzed, using
the whole peptide library for background correction (SI Materials and
Methods). Results of this analysis were used as input into the weblogo
application (weblogo.berkeley.edu) for computing motifs using sequence
entropies (26, 27).
Model of PknB Structure in Complex with an Ideal Peptide Substrate. M.
tuberculosis PknB kinase domain (1o6y) (28) and phosphorylase kinase (Phk)-
peptide substrate complex (2phk) (29) crystal structures were aligned using
PyMOL (DeLano Scientific) to add missing PknB–peptide contact residues in
the activation loop. Activation loop residues from Phk and its substrate were
changed to the corresponding PknB and peptide substrate residues so that
this model should represent the conformation of the active form of PknB.
The Maestro molecular modeling package was used to minimize protein and
substrate side chain conformations, and the protein/substrate H-bond con-
straints with PknB atoms were frozen. Possible contacts between PknB active
site residues and the substrate peptide were predicted using a 4 Å radius
around peptide residues.
Supplemental Data. Detailed methods descriptions, supplemental figures,
mass spectra of phosphopeptides, lists of peptides, and in vitro phosphor-
ylation data are available as supplemental files.
ACKNOWLEDGMENTS. We thank Yin Yin Lin and Nurhan Ozlu for help with
mass spectrometry; Wiebke Timm and Flavio Monigatti for help with data
analysis and management; Mauricio Anaya for initial production of recombi-
nant kinases; and Mark Fleming and Christopher Locher for helpful discus-
sions. This work was funded by research grants from Vertex Pharmaceuticals
Incorporated, the National Institutes of Health, and the Potts Memorial
Foundation (to R.N.H.), and from the US Department of Energy Genomic
Sciences Program and the Bill and Melinda Gates Foundation (to G.M.C.).
1. Deutscher J, Saier MH, Jr (2005) Ser/Thr/Tyr protein phosphorylation in bacteria—for
long time neglected, now well established. J Mol Microbiol Biotechnol 9:125–131.
2. Pérez J, Castañeda-García A, Jenke-Kodama H, Müller R, Muñoz-Dorado J (2008)
Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome.
Proc Natl Acad Sci USA 105:15950–15955.
3. Wehenkel A, et al. (2008) Mycobacterial Ser/Thr protein kinases and phosphatases:
Physiological roles and therapeutic potential. Biochim Biophys Acta 1784:193–202.
4. Kang CM, et al. (2005) The Mycobacterium tuberculosis serine/threonine kinases PknA
and PknB: Substrate identification and regulation of cell shape. Genes Dev 19:
5. Kang CM, Nyayapathy S, Lee JY, Suh JW, Husson RN (2008) Wag31, a homologue of
the cell division protein DivIVA, regulates growth, morphology and polar cell wall
synthesis in mycobacteria. Microbiology 154:725–735.
6. Dasgupta A, Datta P, Kundu M, Basu J (2006) The serine/threonine kinase PknB of
Mycobacterium tuberculosis phosphorylates PBPA, a penicillin-binding protein
required for cell division. Microbiology 152:493–504.
7. Cowley S, et al. (2004) The Mycobacterium tuberculosis protein serine/threonine
kinase PknG is linked to cellular glutamate/glutamine levels and is important for
growth in vivo. Mol Microbiol 52:1691–1702.
8. O’Hare HM, et al. (2008) Regulation of glutamate metabolism by protein kinases in
mycobacteria. Mol Microbiol 70:1408–1423.
9. Nott TJ, et al. (2009) An intramolecular switch regulates phosphoindependent FHA
domain interactions in Mycobacterium tuberculosis. Sci Signal 2:ra12.
10. Grundner C, Gay LM, Alber T (2005) Mycobacterium tuberculosis serine/threonine
kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains. Protein Sci
11. Beausoleil SA, Villén J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach
for high-throughput protein phosphorylation analysis and site localization. Nat
12. Ubersax JA, Ferrell JE, Jr (2007) Mechanisms of specificity in protein phosphorylation.
Nat Rev Mol Cell Biol 8:530–541.
13. Soufi B, et al. (2008) The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403
reveals multiply phosphorylated proteins. Proteomics 8:3486–3493.
14. Macek B, et al. (2007) The serine/threonine/tyrosine phosphoproteome of the model
bacterium Bacillus subtilis. Mol Cell Proteomics 6:697–707.
15. Macek B, et al. (2008) Phosphoproteome analysis of E. coli reveals evolutionary
conservation of bacterial Ser/Thr/Tyr phosphorylation. Mol Cell Proteomics 7:299–307.
16. Ravichandran A, Sugiyama N, Tomita M, Swarup S, Ishihama Y (2009) Ser/Thr/Tyr
phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species.
17. Schwartz D, Gygi SP (2005) An iterative statistical approach to the identification of
protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 23:
18. Camus JC, Pryor MJ, Médigue C, Cole ST (2002) Re-annotation of the genome
sequence of Mycobacterium tuberculosis H37Rv. Microbiology 148:2967–2973.
19. Boitel B, et al. (2003) PknB kinase activity is regulated by phosphorylation in two Thr
residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase,
in Mycobacterium tuberculosis. Mol Microbiol 49:1493–1508.
20. Durán R, et al. (2005) Conserved autophosphorylation pattern in activation loops and
juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases.
Biochem Biophys Res Commun 333:858–867.
21. Molle V, et al. (2006) Characterization of the phosphorylation sites of Mycobacterium
tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass
spectrometry. Proteomics 6:3754–3766.
22. Bendt AK, et al. (2003) Towards a phosphoproteome map of Corynebacterium
glutamicum. Proteomics 3:1637–1646.
23. Villarino A, et al. (2005) Proteomic identification of M. tuberculosis protein kinase
substrates: PknB recruits GarA, a FHA domain-containing protein, through activation
loop-mediated interactions. J Mol Biol 350:953–963.
24. Liang X, Van Doren SR (2008) Mechanistic insights into phosphoprotein-binding FHA
domains. Acc Chem Res 41:991–999.
25. Durocher D, et al. (2000) The molecular basis of FHA domain:phosphopeptide binding
specificity and implications for phospho-dependent signaling mechanisms. Mol Cell 6:
26. Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) WebLogo: A sequence logo
generator. Genome Res 14:1188–1190.
27. Schneider TD, Stephens RM (1990) Sequence logos: A new way to display consensus
sequences. Nucleic Acids Res 18:6097–6100.
28. Ortiz-Lombardía M, Pompeo F, Boitel B, Alzari PM (2003) Crystal structure of the
catalytic domain of the PknB serine/threonine kinase from Mycobacterium
tuberculosis. J Biol Chem 278:13094–13100.
29. Lowe ED, et al. (1997) The crystal structure of a phosphorylase kinase peptide
substrate complex: Kinase substrate recognition. EMBO J 16:6646–6658.
| www.pnas.org/cgi/doi/10.1073/pnas.0913482107Prisic et al.