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: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| 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.).
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