Biochemical and Spatial Coincidence in the Provisional Ser/Thr Protein Kinase Interaction Network of Mycobacterium tuberculosis*♦

Background: Ser/Thr protein kinases (STPKs) form multilayered signaling networks that mediate cellular responses in eukaryotes and prokaryotes. Results: A preliminary interaction network for the STPKs in Mycobacterium tuberculosis is described. Conclusion: STPKs that cross-phosphorylate are often co-localized, suggesting multiple activation mechanisms. Significance: The initial map of this prokaryotic STPK network provides a framework for defining the logic of M. tuberculosis signaling pathways. Many Gram-positive bacteria coordinate cellular processes by signaling through Ser/Thr protein kinases (STPKs), but the architecture of these phosphosignaling cascades is unknown. To investigate the network structure of a prokaryotic STPK system, we comprehensively explored the pattern of signal transduction in the Mycobacterium tuberculosis Ser/Thr kinome. Autophosphorylation is the dominant mode of STPK activation, but the 11 M. tuberculosis STPKs also show a specific pattern of efficient cross-phosphorylation in vitro. The biochemical specificity intrinsic to each kinase domain was used to map the provisional signaling network, revealing a three-layer architecture that includes master regulators, signal transducers, and terminal substrates. Fluorescence microscopy revealed that the STPKs are specifically localized in the cell. Master STPKs are concentrated at the same subcellular sites as their substrates, providing additional support for the biochemically defined network. Together, these studies imply a branched functional architecture of the M. tuberculosis Ser/Thr kinome that could enable horizontal signal spreading. This systems-level approach provides a biochemical and spatial framework for understanding Ser/Thr phospho-signaling in M. tuberculosis, which differs fundamentally from previously defined linear histidine kinase cascades.

ning to be defined, the networks that are responsible for integrating cellular signals remain obscure.
Like many eukaryotic protein kinases, the M. tuberculosis STPKs are generally stimulated by phosphorylation of the activation loop, a conserved peptide bordered by the DFG and APE tripeptides (23)(24)(25). Studies of PknD (26) and PknB (27) show that this process can be initiated by intermolecular autophosphorylation reactions. Interactions across two interfaces mediate PknB phosphorylation. An allosteric "back-to-back" N-lobe interface stabilizes the active conformation, and transient "front-to-front" contacts through the G-helix in the kinase C-lobe are required for efficient intermolecular autophosphorylation (26 -28). These two activation mechanisms result in an autophosphorylated active kinase. In contrast, PknG contains phosphorylation sites in the C-terminal domain that may be necessary for activity (30,31). These biochemical and structural insights suggest a general model for activation in which ligand binding to the extracellular domain promotes KD dimerization and intermolecular autophosphorylation (26,32).
In addition to autophosphorylation, inter-kinase cross-phosphorylation of PknA and PknB has been reported in Mycobacterium smegmatis (15). Kinase cross-phosphorylation, such as that observed in the signaling cascade that controls development in Myxococcus xanthus (33), potentially adds a level of regulatory control and integration to the system. However, it is not clear to what extent kinase cross-talk acts as an alternative activation pathway and whether prokaryotic STPK networks share the multiply branched structure that is characteristic of eukaryotic kinase cascades.
The linear structures of bacterial HK pathways have been experimentally defined by determining the intrinsic biochemical substrate specificity of the kinases. The resulting pattern of efficient in vitro phosphorylation of response regulators parallels the physiological activation pathways in vivo (2). This rationale also has been used to identify potential signaling partners of eukaryotic kinases by defining intrinsic biochemical preferences, and this approach has been especially useful in pathways that lack scaffolding proteins that restrict kinase specificity (34). These precedents suggest that the efficiency of kinase reactions in vitro can provide candidates for the physiological interactions that comprise signaling networks in vivo. The regulation of the bacterial STPKs by activation loop phosphorylation (23,24,35) and the discovery of functional kinase cross-phosphorylations in M. tuberculosis and M. xanthus (15,33) suggested that bacterial STPKs may form a hierarchical network that could be defined based on the kinetic efficiency with which each kinase modified the others (36).
Focusing on the kinase domains (KDs), we used this strategy to initially discover all of the efficient in vitro auto-and cross-STPK phosphorylations. The 11 STPKs expressed by M. tuberculosis provide a tractable family with sufficient complexity to enable this comprehensive approach. The interaction pattern was remarkably restricted, dominated by autophosphorylation and a small number of intermolecular phosphorylations. These reactions targeted the conserved Thr residues in the activation loop where phosphorylation stimulates kinase activity. This global network represents a functional map of potential STPK activation. Using STPKs fused to fluorescent tags in M. smeg-matis, we found that each kinase was specifically concentrated in polar, septal, cytoplasmic, or diffuse membrane-associated locations. Remarkably, the interaction map is consistent with the cellular distribution of functionally interacting kinases. This systems level analysis implies a branching network that differs from the linear signaling pathways mediated by HKs.

EXPERIMENTAL PROCEDURES
STPK Cloning and Mutagenesis-KD boundaries were determined using the PHYRE homology prediction server (32). M. tuberculosis H37Rv genomic DNA was used as the PCR template for cloning. KDs with a tobacco etch virus protease cleavage site were cloned into the pHMGWA or the pET28b expression vectors (EMD Millipore) (43). Site-directed mutants were constructed using the two-primer method (Invitrogen). Construct details are listed in Table 1.
Kinase Assays-Reactions were run in size-exclusion chromatography buffer with 25 M substrate protein and 1 M active kinase. The reaction was initiated by the simultaneous addition of 1 Ci of [␥-32 P]ATP (6000 Ci/mmol and 10 mCi/ml; MP Biomedicals), ATP (Sigma), and MnCl 2 to final concentrations of 50 nCi/l, 50 M, and 50 M, respectively. After 30 min at room temperature, reactions were quenched with SDS-PAGE loading dye, separated by SDS-PAGE, and radioactivity was detected with a Typhoon 860 phosphorimager from GE Healthcare.
For the assays of the full-length PknI and PknG constructs, the His 6 -MBP tags were cleaved prior to running the reactions on SDS-PAGE. The reactions were quenched by the simultaneous addition of EDTA to 20 mM and 6 g of tobacco etch virus protease. After 2 h, the reactions were separated by SDS-PAGE and imaged as described above. All autoradiographs were quantified using ImageJ, and intensity values were normalized for each kinase to the activity detected for autophosphorylation.
For Western blots and Pro-Q Diamond phosphoprotein gel stain (Invitrogen), 25 M substrate protein and 1 M active kinase were incubated with 1 mM ATP (Sigma) and 1 mM MnCl 2 . Reactions were separated by SDS-PAGE. Diamond staining was completed according to the manual and imaged on

Map of the M. tuberculosis STPK Phosphorylation Network
a Typhoon 860 imager from GE Healthcare. Samples for Western blotting were transferred to a nitrocellulose membrane. The membranes were blocked for 4 h in 3% (w/v) BSA in PBS with 0.2% Tween (PBST and then incubated with (1:1000) phosphothreonine antibody (9381, Cell Signaling Technology) overnight in 3% (w/v) BSA in PBST. After washing with PBST, Cy3-labeled anti-rabbit secondary antibody (Invitrogen) at 1:5,000 dilution in PBST was added and incubated for 30 min. Blots were imaged on a Typhoon 860 imager from GE Healthcare.
Liquid Chromatography-Mass Spectrometry-Prior to LC-MS analysis, 25 M substrate protein and 1 M active kinase were incubated with 1 mM ATP (Sigma) and 1 mM MnCl 2 for 12-20 h at 4°C. Intact protein constructs were analyzed using an Agilent 1200 series liquid chromatograph (LC) connected in-line with an LTQ Orbitrap XL hybrid mass spectrometer equipped with an Ion Max electrospray ionization source (Thermo Fisher Scientific). The LC was equipped with a C 8 column (Poroshell 300SB-C8, 5 m, analytical 75 mm ϫ 0.5 mm, Agilent). Solvent A was 0.1% formic acid, 99.9% water (v/v), and solvent B was 0.1% formic acid, 99.9% acetonitrile (v/v). For each sample, ϳ100 pmol of analyte was injected onto the column. Following sample injection, analyte trapping was PknB. Phosphoproteins were visualized using Pro-Q Diamond Phosphoprotein Gel Stain. Active STPK autophosphorylation is visible (upper bands) as is trans-phosphorylation on the MyBP substrate; equal protein loading was confirmed by Coomassie stain. D, trans-phosphorylation activity of KD and full-length ICD constructs was compared using GarA as a substrate. The autoradiogram confirms that the PknA ICD is more active than the PknA KD as reported previously (38), but that activity does not differ for the two PknB and PknL constructs.  External mass calibration was performed prior to analysis using the standard LTQ calibration mixture. Mass spectra were recorded in the positive ion mode over the range m/z ϭ 500 -2000. Raw mass spectra were processed using Xcalibur software (version 2.0.7 SP1, Thermo), and measured charge-state distributions of proteins were deconvoluted using ProMass software (version 2.5 SR-1, Novatia).
Construction of Fluorescent Strains and Confirmation of Expression-Vectors expressing fluorescent protein-tagged STPKs were constructed using multisite Gateway cloning (Invitrogen). Each reporter fusion was under the control of the P16 promoter and contained an N-terminal FLAG-mVenus tag in the episomal expression vector, pDE43-MEK, or in the integrating vector, pDE43-MEK (kindly provided by D. Schnappinger). Plasmids were transformed into M. smegmatis MC 2 155. To confirm expression, each strain was lysed by beadbeating in PBS with 2% (w/v) SDS. The soluble fraction of the lysate was quantified for total protein content using a BCA assay (Pierce) and separated by SDS-PAGE. Western blots were performed with anti-FLAG M2 primary antibody (Sigma) and Cy-5 antimouse secondary antibody (Invitrogen), and then imaged on a Typhoon 860 imaging system (GE Healthcare).
Fluorescence Microscopy-M. smegmatis strains were grown in 7H9/ADS/Tween medium to an A 600 of 0.8 and imaged on a DeltaVision microscope (Applied Precision) mounted on agar pads. Bacteria were stained with FM4-64 (Invitrogen) at a final concentration of 1 g/ml for 90 min at room temperature before imaging. Images were deconvolved, and statistical analysis was completed using softWoRx (Applied Precision) and Microsoft Excel.

STPK Intermolecular Phosphorylation Follows Specific
Patterns-To explore the intrinsic specificity of the M. tuberculosis STPKs, we expressed and purified a complete set of kinase domains. The catalytic activity of several M. tuberculosis STPK KDs has been demonstrated previously (35,37). We focused on the minimal KD constructs, because these segments contain conserved activation loop Thr residues that must be phosphorylated to elicit activity. Although the STPKs also contain juxta-membrane phosphorylation sites implicated in regulation or binding accessory proteins (38,39), phosphorylation of the activation loop Thr is the primary mechanism of activation. Moreover, the in vitro specificity of a number of KDs has been shown to recapitulate functional substrate phosphorylations in vivo (40). Although other factors such as co-expression, co-localization, and phosphatase activity can influence functional phosphorylations in vivo, the biochemical specificity of the KDs provides a provisional network architecture. tuberculosis STPKs in [␥-32 P]ATP transfer assays. His-MBP tags on the active kinases and tagless inactive kinases enable separation of the proteins in each reaction by SDS-PAGE. Assays were imaged by autoradiography in parallel. Products on the diagonal reflect efficient autophosphorylation. Off-diagonal bands in each column indicate cross-phosphorylation. B, PknA ICD was tested against the 11 Asp-to-Asn mutant kinases. No additional inter-kinase interactions were observed. C, PknG and PknI KDs were not phosphorylated by any of the other kinases. The full-length PknG and PknI constructs are cross-phosphorylated by multiple kinases, presumably on sites outside of the catalytic domain. D, autoradiographs were quantified in ImageJ and normalized to the autophosphorylation signal observed for each kinase. PknA and PknL do not efficiently autophosphorylate; therefore, these two kinases are not included in this graph. The PknH-PknE interaction was the lowest detected by LC-MS and was used as the lower boundary. Low intensity interactions at or below this cutoff are depicted by gray columns.
KD constructs (Table 1) were designed based on sequence alignments, secondary structure prediction, and available crystal structures (41)(42)(43). His-MBP tags were maintained on the active kinases throughout all experiments to enable size separation of STPKs from untagged or minimally tagged (His 6 ) substrates. In contrast to the other STPKs, the PknG and PknI KDs are not active. Consequently, the full-length PknG and PknI proteins were included in the comprehensive set of active M. tuberculosis STPKs.
Each of the STPKs was found to phosphorylate the M. tuberculosis substrate protein GarA (Fig. 1B). GarA contains a forkhead-associated domain known to bind phosphopeptides and has previously been reported as a PknB and PknG substrate (19). As PknF and PknH displayed reduced activity on GarA, we confirmed that these two STPKs were active on myelin basic protein (MyBP), a model substrate previously reported to be phosphorylated by these kinases (44,45). PknF and PknH display equal trans-phosphorylation activity on MyBP in comparison with PknB when imaged by Pro-Q Diamond phosphoprotein gel stain (Invitrogen) (Fig. 1C).
The PknA kinase domain also displayed reduced activity on GarA. As the full-length intracellular domain (ICD) of PknA has been previously reported to be necessary for full activity (38), we tested the activity of the PknA KD and PknA ICD constructs on GarA (Fig. 1D). The two PknA constructs display significantly different activity; therefore, both versions of this kinase were used in later experiments. We also tested the relative activity of KD and ICD constructs for PknB and PknL as these two STPKs are closely related to PknA by sequence homology (46). No significant difference in activity was noted Inter-kinase phosphorylation occurs in a substrate-specific pattern and is not due to differences in enzyme activity. PknB, PknD, and PknH, three STPKs with differing activity, were incubated with the PknB and PknD Asp-to-Asn mutant kinases. Time points taken at 30, 60, and 120 min and 24 h after reaction initiation display the same relative inter-kinase phosphorylation patterns (lower band) when Western-blotted with a phosphothreonine antibody. Active STPK autophosphorylation was similar at each time point (upper bands). Coomassie-stained gels demonstrate equal substrate protein concentrations in all reactions.
for PknB and PknL (Fig. 1D); thus the PknB and PknL KD constructs were used for all assays. Substrates for intermolecular cross-phosphorylation assays were created by generating inactive variants of all 11 kinase constructs. To eliminate activity in each kinase, an Asp-to-Asn substitution was introduced in the catalytic HRD motif (17).
Using this complete set of active and inactive kinase constructs, we assayed all pairwise combinations for autophosphorylation (i.e. modification of homologous inactive STPK) and cross-phosphorylation (i.e. modification of heterologous inactive STPK). Each substrate was trans-phosphorylated by a distinctive subset of the active kinase constructs (Fig. 2A). Seven of the STPKs catalyzed intermolecular autophosphorylation. The PknA and PknL KDs did not efficiently autophosphorylate. Likewise, the PknA ICD does not efficiently phosphorylate the PknA KD (Fig. 2B). Instead, PknA and PknL were only efficiently phosphorylated by heterologous kinases. Notably, the PknA ICD construct does not display increased inter-kinase phosphorylation compared with PknA KD, indicating that the kinase-kinase interactions are specific and not promoted by increased enzymatic activity. In addition, strong and specific inter-STPK preferences were apparent. Quantification of the autoradiographs reveals that each active kinase phosphorylated 0 -3 heterologous KDs (Fig. 2D). To confirm the specificity of the observed inter-STPK phosphorylation pattern, we assayed specific interactions at a series of time points (Fig. 3). Despite reaction times of up to 24 h in the presence of excess ATP and MnCl 2 , promiscuous phosphorylation was not observed. The small number of efficient inter-STPK phosphorylations suggested the presence of a signaling network defined at least in part by the substrate specificity inherent in the kinase domains.
The PknG and PknI KDs were not substrates for trans-phosphorylation ( Fig. 2A). We tested catalytically inactive, fulllength mutants of these two kinases as substrates and discovered widespread trans-phosphorylation by a number of the STPKs (Fig. 2C). Given that PknG and PknI lack threonines in the activation loop and are therefore activated through an alternative mechanism other than STPKs, it is possible that the phosphorylation on other regulatory domains alters activity.
Inter-STPK Phosphorylation Targets Regulatory Activation Loop Thr Residues-To investigate the functional significance of cross-phosphorylation, we first determined whether the activation loop Thr residues are involved in activating each STPK. Activation loop Thr residues essential for kinase activity in PknB and PknH are conserved in all of the M. tuberculosis STPKs except PknG and PknI (Fig. 4A) (23,25,35,47). In all nine active KDs, alanine substitutions of the conserved pair of threonines abolished kinase activity on GarA (Fig. 4B). These results suggest that activation loop Thr phosphorylation is necessary for function of the nine canonical STPKs and that PknG and PknI are activated by alternative mechanisms. As a result, the conserved threonines were candidates for the sites of modification observed in the auto-and cross-phosphorylation reactions of the active kinases.
To survey the importance of these sites, we measured the phosphorylation of representative inactive substrate kinases containing the Asp-to-Asn HRD mutation in which the activation loop threonines were also mutated to alanine. The activation loop mutations significantly reduced or abolished intermolecular phosphorylation (Fig. 4C). These results suggest that most intermolecular phosphorylation of KDs occurs specifically on the activation loop residues that are essential for STPK activity.
LC-MS measurements of several cross-phosphorylated STPKs revealed that these reactions produced residue-specific modifications with distinct stoichiometries. Under the defined conditions used in our assay, for example, the inactive PknD substrate kinase was phosphorylated on up to two sites by active PknB and PknD (Fig. 5B). In contrast, the active PknD KD purified from E. coli was a heterologous mixture modified from 3 to 11 phosphoryl groups, likely due to robust autophosphorylation during expression of the protein (Fig. 5A). Like PknD, PknK exhibited similarly limited patterns of in vitro crossphosphorylation. PknB and PknJ phosphorylated only a single residue on the inactive PknK substrate KD, whereas PknK autophosphorylation involved the addition of two to three phosphates (Fig. 5C). Consistent with biochemical results, the PknA KD did not autophosphorylate but was phosphorylated on one site by PknB (Fig. 5D). PknE efficiently autophosphorylated, with the addition of a single phosphoryl group (Fig. 5D). These examples illustrate that under defined conditions, STPK crossphosphorylation involves the addition of a limited number of phosphates on the substrate STPK. Because the Thr mutations in the substrate kinase abolished phosphorylation, these interactions appear to specifically target the activation loop.
Provisional Kinase Network Is Consistent with the Cellular Distribution of STPKs-To explore whether the observed specificity of in vitro STPK KD interactions was recapitulated in vivo, we investigated whether the STPKs shared the same subcellular localization with their preferred substrates. N-terminal mVenus STPK fusions were expressed in M. smegmatis under the control of a relatively weak promoter. Reporter fusions for PknA and PknB were constructed using catalytically inactive Asp-to-Asn mutants to reduce the toxic effects of constitutive expression (15). Expression of four fusion proteins (PknF, PknG, PknI, and PknK) could not be detected by Western blot- ting or fluorescence microscopy. The other seven STPK fusions, however, were expressed at equivalent levels (Fig. 6A).
To control for the possible effects of gene copy number, expression was compared using either a low copy number episome or a single copy integrating plasmid. Fusions that failed to express in one vector also failed to express in the other. PknA provides a representative example of the effects of switching vectors. The level of the PknA fusion was reduced using the integrating vector (Fig. 6B). The differences in expression, however, did not change the localization pattern of any of the STPK fusions (e.g. Fig. 6B). The results indicate that in the ranges tested, expression levels did not influence localization. Based on these controls, we conclude that intrinsic localization signals determine the observed cellular distributions of the mVenus-STPK fusions.
The seven expressed STPK fusions were differentially localized in three distinct patterns. PknA and PknB were found exclusively at the poles and the septa, along with the kinase most similar in primary sequence, PknL (Fig. 7A) (46). PknD, PknE, and PknH were more evenly distributed along the cell membrane (Fig. 7B). Finally, PknJ was only discretely localized at the midplane of a small percentage of cells (Fig. 7C). Subtle differences were apparent within these three groups. For example, although all of the kinases were localized to the midplane of some dividing bacteria, the percentage of cells with fluorescent STPK located at this site varied from 6.0% (PknJ) to 65.2% (PknA) (Fig. 7). These results suggested that STPK localization was temporally regulated during cell division.
To control for the possible effects of STPK expression on the cell cycle, the reporter strains were stained with FM4-64 membrane dye to enable visualization of the septum. Only the PknA Asp-to-Asn inactive construct caused an increase in cells with discernible septa (Fig. 7A). When expressed as a ratio of the FM4-64-positive septa, STPK localization to this site differed from 15% for PknJ to 121% for PknA. Thus, PknA appears to mark the midplane before membrane deposition at the nascent septum, whereas the other STPKs occupy this location for a shorter and variable portion of the cell cycle. Importantly, these data indicate that the STPK signaling pathways are not distributed randomly in the cell. Remarkably, the STPKs that crossphosphorylate were concentrated at similar subcellular sites, indicating that these preferred biochemical reactions have the opportunity to occur in vivo.

DISCUSSION
Bacterial STPKs were first identified over 2 decades ago (48), but the overall architecture of a prokaryotic Ser/Thr signaling network has not been defined (18). Here, we used a complete set of M. tuberculosis STPK constructs to comprehensively map the preferred substrates and cellular localizations that comprise the relatively complex M. tuberculosis signaling network. At the biochemical level, we found that the pattern of preferred STPK phosphorylation is very limited. Most STPK KDs showed in vitro auto-and cross-phosphorylation reactions of similar efficiencies, as expected for functionally cognate donor-acceptor pairs. The activation loop threonines, which are required for activation, were the specific targets of auto-and cross-phosphorylation reactions for the majority of the intermolecular interactions mapped. The biochemical specificity for these sites matches the expectation for functional interactions. The provisional map of STPK cross-talk recapitulates reported interactions in vivo between PknA and PknB in M. smegmatis (15) and the autophosphorylation of PknD in M. tuberculosis (17).
At the cellular level, the STPKs fused to mVenus were distributed in M. smegmatis in three patterns as follows: at the pole and midplane (PknA, PknB, and PknL), in the cell body membrane and at the midplane (PknD, PknE, and PknH), and solely at the midplane (PknJ). The cellular location of several kinases is also consistent with their functions. PknA and PknB are essential for cell growth and are found exclusively at the pole and septum, sites of new cell wall synthesis for elongation and division (13). It is at these sites where the peptidoglycan-binding extracellular domain of PknB is likely to receive its activating signal (32). Conversely, PknD, PknE, and PknH are broadly distributed along the cell membrane and are not required for cell growth, suggesting that these kinases may respond to condition-specific environmental cues. The convergence of the STPKs at the poles and septum suggest that Ser/Thr phosphosignaling plays a particularly important role at these sites. and microscopy (bottom) that compare protein expression levels for the FLAG-Venus-PknA D-N reporter construct in an episomal (left) and integrating vector (right). Western blots were exposed in parallel. Differences in expression level do not change the apparent localization at the poles and septum. DIC, differential interference contrast.
Rather than being spread uniformly throughout the cell, the STPK network is spatially segregated indicating that kinase localization could play a role in substrate selection.
This concept is consistent with the biochemical specificity of the kinases. PknB, for example, co-localizes with its substrates, PknA and PknL. This pattern provides a potential mechanism to activate PknA and PknL, because these kinases do not efficiently autophosphorylate. Likewise, PknH co-localizes with its substrates. The consistency between the experimentally defined biochemical network and the subcellular distribution of the STPKs indicates that the cognate kinase-substrate pairs are present at the same site. Therefore, these efficient phosphorylation events have the opportunity to occur in vivo.
The provisional STPK signaling network shows three striking features. First, autophosphorylation is the dominant reaction. Seven of the STPKs (excluding PknA, PknG, PknI, and PknL) efficiently phosphorylate otherwise identical inactive KD constructs. Autophosphorylation is a mechanism of efficient signal amplification. A single activated STPK can rapidly phosphorylate identical molecules (26,28).
Second, each STPK phosphorylates a different subset of the provisional kinase network (Fig. 2D). These data suggest that different signals produce distinct phosphorylation states of the kinome, leading to the activation or potentiation of different response pathways in vivo. The coordinated phosphorylation of multiple STPKs allows an integrated response to external signals. This multilevel response may complicate substrate identification as downstream proteins can be phosphorylated in vivo not in response to direct activation of the immediate upstream kinase, but instead through an alternative STPK-STPK activation reaction driven by the integration of multiple signals.
Third, the M. tuberculosis STPKs fall into three distinct functional classes based on the pattern of in vitro phosphorylation as follows: master regulator kinases, signal transduction kinases, and substrate kinases (Fig. 8). Two kinases, PknB and PknH, exclusively undergo autophosphorylation. This requirement for an autoactivating signal suggests that PknB and PknH occupy the role of master regulators. As expected for kinases that sit atop the signaling hierarchy, both STPKs contain folded extracellular sensor domains (ECDs). Importantly, other kinases with folded ECDs, PknD, PknE, and PknJ, are targets of cross-phosphorylation, indicating that domain architecture alone is insufficient to assign network position. By activating additional STPKs, the signals that control the master regulators can coordinate more complex cellular processes than signals that activate downstream kinases.
The second tier of the provisional STPK hierarchy consists of signal transduction kinases that undergo auto-and cross-phosphorylation and also phosphorylate downstream kinases. PknH cross-phosphorylates PknE and PknJ, which in turn phosphorylate PknD, PknF, PknK, and PknL. This pattern of cross-phosphorylation may allow signals to propagate through specific STPKs that regulate distinct sets of downstream cellular proteins. These interactions could function as part of regulatory circuits that spread extracellular signals to intracellular substrates of multiple kinases.
The base of the hierarchy is occupied by the substrate STPKs PknA, PknD, PknF, and PknL. These kinases do not transfer phosphates to any other STPK catalytic domains. Notably, PknA and PknF contain extracellular domains that are predicted to be unfolded, and PknL lacks an ECD altogether. Thus, these kinases apparently lack the machinery to respond directly to an extracellular signal, and the master kinases spread physiological adaptations through cross-phosphorylation of these STPKs (Fig. 8). In contrast, PknD, which contains a ␤-propeller ECD, not only senses osmolarity but also is positioned in the network to regulate the stressosome in response to upstream STPKs (17,22,49).
The pattern of intermolecular in vitro STPK phosphorylation and cellular localization suggests new hypotheses for the spread of signals through the M. tuberculosis kinome. It must be stressed that other factors, such as molecular scaffolding and co-expression, may influence the cross-phosphorylation patterns in vivo. Moreover, the constructs used for in vitro biochemistry lacked possible regulatory sites in the juxtamembrane segments (24,38), potentially masking additional functional relationships. In addition, the network architecture may be modified by the temporally restricted activity of the STPKs mediated, in part by localization or regulation of the antagonistic phosphatase (50). Nonetheless, the exquisite biochemical specificity of each KD and the distinct cellular compartmentalization that coincides with the phosphorylation of signaling partners imposes a fundamental structure on the M. tuberculosis STPK network onto which these additional complexities will be layered. The distinct architecture of HK and STPK cascades that occur in this single organism indicate that these systems integrate signals and outputs through different mechanisms. Linear HK pathways rely on downstream transcriptional changes to achieve a coordinated response, whereas the hierarchical structure of the STPK network may allow the integration of multiple signals into distinct phosphorylation patterns that post-translationally coordinate the activities of disparate cellular enzymes and transcriptional regulators. STPKs with polar and midplane localization are depicted in green. STPKs with membrane and midplane localization are colored blue. PknJ, localizing only transiently to the septa, is colored orange. PknF and PknK, depicted in gray, were characterized biochemically, but expression of the mVenus fusions in M. smegmatis was not detected. The master STPKs, PknB and PknH, only undergo autophosphorylation and contain folded extracellular domains that receive environmental signals. In contrast, PknA and PknL not only fail to autophosphorylate, they also lack folded extracellular receptor domains. However, PknB efficiently phosphorylates the PknL and PknA KDs, and these three kinases co-localize in vivo. PknH localizes along the cell membrane and also initiates a cascade of phosphorylation of other STPKs, PknE and PknD, that also reside along the cell membrane. PknD contains a folded extracellular domain, but phosphorylation by other kinases suggests that it may be activated by other kinases as well as external signals that trigger autophosphorylation.