HupB,a Nucleoid-Associated Protein of Mycobacterium tuberculosis,Is Modified by Serine/Threonine Protein Kinases In Vivo

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H₃₇Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr⁶⁵ and Thr⁷⁴ in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg⁵⁵ is also identified as an important residue for N-HupB–DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H₃₇Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.     

Rv3080c regulates the rate of inhibition of mycobacteria by isoniazid through FabD

The mycobacterial FASII multi-enzyme complex has been identified to be a target of Ser/Thr protein kinases (STPKs) of Mycobacterium tuberculosis (MTB), with substrates, including the malonyl-CoA:ACP transacylase (FabD) and the β-ketoacyl-ACP synthases KasA and KasB. These proteins are phosphorylated by various kinases in vitro. The present study links the correlation of FASII pathway with serine threonine protein kinase of MTB. In the preliminary finding, we have shown that mycobacterial protein Rv3080c (PknK) phosphorylates FabD and the knockdown of PknK protein in mycobacteria down regulates FabD expression. This event leads to the differential inhibition of mycobacteria in the presence of isoniazid (INH), as the inhibition of growth of mycobacteria in the presence of INH is enhanced in PknK deficient mycobacteria.     

Novel mechanistic insights into physiological signaling pathways mediated by mycobacterial Ser/Thr protein kinases

Protein phosphorylation is known to be one of the keystones of signal sensing and transduction in all living organisms. Once thought to be essentially confined to the eukaryotic kingdoms, reversible phosphorylation on serine, threonine and tyrosine residues, has now been shown to play a major role in many prokaryotes, where the number of Ser/Thr protein kinases (STPKs) equals or even exceeds that of two component systems. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is one of the most studied organisms for the role of STPK-mediated signaling in bacteria. Driven by the interest and tractability of these enzymes as potential therapeutic targets, extensive studies revealed the remarkable conservation of protein kinases and their cognate phosphatases across evolution, and their involvement in bacterial physiology and virulence. Here, we present an overview of the current knowledge of mycobacterial STPKs structures and kinase activation mechanisms, and we then focus on PknB and PknG, two well-characterized STPKs that are essential for the intracellular survival of the bacillus. We summarize the mechanistic evidence that links PknB to the regulation of peptidoglycan synthesis in cell division and morphogenesis, and the major findings that establishes PknG as a master regulator of central carbon and nitrogen metabolism. Two decades after the discovery of STPKs in M. tuberculosis, the emerging landscape of O-phosphosignaling is starting to unveil how eukaryotic-like kinases can be engaged in unique, non-eukaryotic-like, signaling mechanisms in mycobacteria.     

Forkhead-associated Domain-containing Protein Rv0019c and Polyketide-associated Protein PapA5, from Substrates of Serine/Threonine Protein Kinase PknB to Interacting Proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis profoundly exploits protein phosphorylation events carried out by serine/threonine protein kinases (STPKs) for its survival and pathogenicity. Forkhead-associated domains (FHA), the phosphorylation-responsive modules, have emerged as prominent players in STPK mediated signaling. In this study, we demonstrate the association of the previously uncharacterized FHA domain-containing protein Rv0019c with cognate STPK PknB. The consequent phosphorylation of Rv0019c is shown to be dependent on the conserved residues in the Rv0019c FHA domain and activation loop of PknB. Furthermore, by creating deletion mutants we identify Thr³⁶ as the primary phosphorylation site in Rv0019c. During purification of Rv0019c from Escherichia coli, the E. coli protein chloramphenicol acetyltransferase (CAT) specifically and reproducibly copurifies with Rv0019c in a FHA domain-dependent manner. On the basis of structural similarity of E. coli CAT with M. tuberculosis PapA5, a protein involved in phthiocerol dimycocerosate biosynthesis, PapA5 is identified as an interaction partner of Rv0019c. The interaction studies on PapA5, purified as an unphosphorylated protein from E. coli, with Rv0019c deletion mutants reveal that the residues N-terminal to the functional FHA domain of Rv0019c are critical for formation of the Rv0019c-PapA5 complex and thus constitute a previously unidentified phosphoindependent binding motif. Finally, PapA5 is shown to be phosphorylated on threonine residue(s) by PknB, whereas serine/threonine phosphatase Mstp completely reverses the phosphorylation. Thus, our data provides initial clues for a possible regulation of PapA5 and hence the phthiocerol dimycocerosate biosynthesis by PknB, either by direct phosphorylation of PapA5 or indirectly through Rv0019c.     

Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry

In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.     

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

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.     

pETPhos: a customized expression vector designed for further characterization of Ser/Thr/Tyr protein kinases and their substrates

Bacterial genomics revealed the widespread distribution of serine/threonine protein kinases (STPKs), which regulate various cellular processes. However, understanding the role of phosphorylation in prokaryotes has been hampered by the paucity of endogenous substrates identified and the restricted number of tools allowing identification and characterization of the phosphoresidues. Herein, we describe an improved vector, pETPhos, to express proteins harboring a N-terminal His-tag fusion, which can be efficiently removed using the TEV protease. One major advantage of pETPhos relies on the lack of Ser and Thr residues in the fusion tag, representing potential non-specific phosphorylation sites. The usefulness of pETPhos is illustrated by a comparative analysis in which the Mycobacterium tuberculosis protein Rv2175c, a substrate of the STPK PknL, is expressed either in a pET28 derivative or in pETPhos. Following in vitro phosphorylation with PknL, phosphoaminoacid analysis revealed the presence of phosphorylated Ser and Thr in Rv2175c expressed in the pET28 derivative. However, when expressed in pETPhos, only Thr were phosphorylated. These findings indicate that STPKs can phosphorylate Ser-containing His-tag fusions, thus conducting to misleading results. We demonstrate that pETPhos represents a valuable tool for characterization of the phosphoacceptors in bacterial STPKs, and presumably also in Tyr protein kinases, as well as in their substrates.     

Biochemical Analysis of the NAD+-Dependent Malate Dehydrogenase,a Substrate of Several Serine/Threonine Protein Kinases of Mycobacterium tuberculosis

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD⁺-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.     

Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.

A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.     

Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains

The physiologic roles and the substrates of the Mycobacterium tuberculosis (Mtb) serine/threonine kinases are largely unknown. Here, we report six novel interactions of PknB, PknD, PknE, and PknF with the Forkhead-Associated (FHA) domains of Rv0020c and the putative ABC transporter Rv1747. Purified PknB and PknF kinase domains phosphorylated multiple FHA-domain proteins in vitro. Although they remain to be verified in vivo, these reactions suggest a web of interactions between STPKs and FHA domains.     

Proteome and phosphoproteome analysis of the serine/threonine protein kinase E mutant of Mycobacterium tuberculosis

Aims

Serine/threonine protein kinases (STPKs) have prominent roles in the survival mechanisms of Mycobacterium tuberculosis (M. tuberculosis). Previous studies from our laboratory underscored the role of PknE, an STPK in virulence, adaptation and the suppression of host cell apoptosis. In this study, two-dimensional gel electrophoresis was used to study the proteome and phosphoproteome profiles of wild type M. tuberculosis and its isogenic pknE deletion mutant (ΔpknE) during growth in Middlebrook 7H9 and nitric oxide stress.

Main methods

Wild-type M. tuberculosis and its isogenic pknE deletion mutant strain were grown in Middlebrook 7H9 as well as subjected to nitric oxide stress using sodium nitroprusside. Whole cell lysates were prepared and analyzed by 2D-gel electrophoresis. Phosphoproteomes were analyzed using phospho serine and phospho threonine antibodies after subjecting the 2D-gels to western blotting. Proteins of interest were identified using mass spectrometry.

Key findings

Our analysis provides insights into the targets that impose pro-apoptotic as well as altered cellular phenotypes on ΔpknE, revealing novel substrates and functions for PknE.

Significance

For the first time, our proteome and phosphoproteome data decipher the function of PknE in cell division, virulence, dormancy, suppression of sigma factor B and its regulated genes, suppression of two-component systems and in the metabolic activity of M. tuberculosis.     

Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus,a developmental bacterium exhibiting social behavior

Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior. Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum. On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D. discoideum. It was indeed found that M. xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes. This is the first unambiguous demonstration of eukaryotic-like protein serine/threonine kinases in the prokaryotes. © 1993 Wiley-Liss, Inc.     

Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions

In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.     

Substrate binding to Src: A new perspective on tyrosine kinase substrate recognition from NMR and molecular dynamics

Most signal transduction pathways in humans are regulated by protein kinases through phosphorylation of their protein substrates. Typical eukaryotic protein kinases are of two major types: those that phosphorylate‐specific sequences containing tyrosine (~90 kinases) and those that phosphorylate either serine or threonine (~395 kinases). The highly conserved catalytic domain of protein kinases comprises a smaller N lobe and a larger C lobe separated by a cleft region lined by the activation loop. Prior studies find that protein tyrosine kinases recognize peptide substrates by binding the polypeptide chain along the C‐lobe on one side of the activation loop, while serine/threonine kinases bind their substrates in the cleft and on the side of the activation loop opposite to that of the tyrosine kinases. Substrate binding structural studies have been limited to four families of the tyrosine kinase group, and did not include Src tyrosine kinases. We examined peptide‐substrate binding to Src using paramagnetic‐relaxation‐enhancement NMR combined with molecular dynamics simulations. The results suggest Src tyrosine kinase can bind substrate positioning residues C‐terminal to the phosphoacceptor residue in an orientation similar to serine/threonine kinases, and unlike other tyrosine kinases. Mutagenesis corroborates this new perspective on tyrosine kinase substrate recognition. Rather than an evolutionary split between tyrosine and serine/threonine kinases, a change in substrate recognition may have occurred within the TK group of the human kinome. Protein tyrosine kinases have long been therapeutic targets, but many marketed drugs have deleterious off‐target effects. More accurate knowledge of substrate interactions of tyrosine kinases has the potential for improving drug selectivity.     

Evolution of transmembrane protein kinases implicated in coordinating remodeling of gram-positive peptidoglycan: inside versus outside

Members of a family of serine/threonine protein kinases (STPKs), unique to gram-positive bacteria, comprise an intracellular kinase domain and reiterated extracellular PASTA (for "penicillin-binding protein and serine/threonine kinase associated") domains. PASTA domains exhibit low affinity for beta-lactam antibiotics that are structurally similar to their likely normal ligands: stem peptides of unlinked peptidoglycan. The PASTA-domain STPKs are found in the actinobacteria and firmicutes and, as exemplified by PknB of Mycobacterium tuberculosis, they are functionally implicated in aspects of growth, cell division, and development. Whereas the kinase domains are well conserved, there is a wide divergence in the sequences of the multiple PASTA domains. Closer inspection reveals position-dependent evolution of individual PASTA domains: a domain at one position within a gene has a close phylogenetic relationship with a domain at a similar position in an orthologous gene, whereas neighboring domains have clearly diverged one from one another. A similar position-dependent relationship is demonstrated in the second family of proteins with multiple PASTA domains: the high-molecular-weight type II penicillin-binding protein (PBP2x) family. These transpeptidases are recruited to the division site by a localized pool of unlinked peptidoglycan. We infer that protein localization is guided by low-affinity interactions between structurally different unlinked peptidoglycan stem peptides and individual PASTA domains. The STPKs possess a greater multiplicity and diversity of PASTA domains, allowing interactions with a wider range of stem-peptide ligands. These interactions are believed to activate the intracellular kinase domain, allowing an STPK to coordinate peptidoglycan remodeling and reproduction of a complex cell wall structure.     

Identification of a potential physiological substrate for oncogenic Ras-activated protein kinases in activated Xenopus egg extracts: Correlation with oncogenic RAS-induced cell cycle arrest

Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these activated extracts, we previously showed that purified, bacterially expressed oncogenic human RasH protein arrests cell cycle progression. Because oncogenic Ras activates many serine/threonine protein kinases in Xenopus oocytes and egg extracts, it is possible that induction of cell cycle arrest involves the action of oncogenic Ras-activated kinases. Thus, the identification of the physiological substrates for oncogenic Ras-activated kinases is important for elucidating the molecular mechanism underlying oncogenic Ras-induced cell cycle arrest. We used ³²P-orthophosphate as a label to identify the potential substrates. Our results demonstrated that the ³²P-labeling of both a 32 and a 33 kDa protein were greatly enhanced by oncogenic Ras during the incubation of activated Xenopus egg extracts. The enhanced labeling correlated with the induced cell cycle arrest and was contributed by serine phosphorylation. Moreover, the 33 kDa protein was detected only in the presence of oncogenic Ras and was a serine-hyperphosphorylated form of the 32 kDa protein. Furthermore, new protein synthesis was not required for the enhanced labeling, consistent with the concept that the enhanced serine phosphorylation of the 32 kDa protein is by oncogenic Ras-activated protein kinases. In addition to serine phosphorylation, our results also suggested that an as yet unidentified modification of the 32 kDa protein might also be induced by oncogenic Ras. Our results suggest that the 32 kDa protein is a potential physiological substrate for oncogenic Ras-activated protein kinases. © 1996 Wiley-Liss, Inc.     

A modified immunoblot method to identify substrates of protein kinases

While protein kinases are key components in multiple cellular processes, efficient identification of cognate in vivo substrates remains challenging. Here we describe a powerful method to screen potential substrates of protein kinases by partial transfer of proteins from a 2D-PAGE gel to a Western blot membrane. This approach allowed precise pinpointing of candidate substrate spots in the 2D gel, and identifying physiological substrates of protein kinases in Mycobacterium tuberculosis.     

From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division

Corynebacterium glutamicum contains four serine/threonine protein kinases (STPKs) named PknA, PknB, PknG, and PknL. Here we present the first biochemical and comparative analysis of all four C. glutamicum STPKs and investigate their potential role in cell shape control and peptidoglycan synthesis during cell division. In vitro assays demonstrated that, except for PknG, all STPKs exhibited autokinase activity. We provide evidence that activation of PknG is part of a phosphorylation cascade mechanism that relies on PknA activity. Following phosphorylation by PknA, PknG could transphosphorylate its specific substrate OdhI in vitro. A mass spectrometry profiling approach was also used to identify the phosphoresidues in all four STPKs. The results indicate that the nature, number, and localization of the phosphoacceptors varies from one kinase to the other. Disruption of either pknL or pknG in C. glutamicum resulted in viable mutants presenting a typical cell morphology and growth rate. In contrast, we failed to obtain null mutants of pknA or pknB, supporting the notion that these genes are essential. Conditional mutants of pknA or pknB were therefore created, leading to partial depletion of PknA or PknB. This resulted in elongated cells, indicative of a cell division defect. Moreover, overexpression of PknA or PknB in C. glutamicum resulted in a lack of apical growth and therefore a coccoid-like morphology. These findings indicate that pknA and pknB are key players in signal transduction pathways for the regulation of the cell shape and both are essential for sustaining corynebacterial growth.