The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.     

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB

Background

The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs.

Principal Findings

PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn²⁺-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr¹³⁷, Thr¹⁴¹, Thr¹⁷⁴ and Thr²⁹⁰ being the target residues of PknB while Thr¹³⁷ and Thr¹⁷⁴ are phosphorylated by PknA. The Mn²⁺-ion binding residues Asp³⁸ and Asp²²⁹ are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP_c ^D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn²⁺/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.

Conclusions and Significance

This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.     

Interdomain interaction reconstitutes the functionality of PknA, a eukaryotic type Ser/Thr kinase from Mycobacterium tuberculosis

Eukaryotic type Ser/Thr protein kinases have recently been shown to regulate a variety of cellular functions in bacteria. PknA, a transmembrane Ser/Thr protein kinase from Mycobacterium tuberculosis, when constitutively expressed in Escherichia coli resulted in cell elongation and therefore has been thought to be regulating morphological changes associated with cell division. Bioinformatic analysis revealed that PknA has N-terminal catalytic, juxtamembrane, transmembrane, and C-terminal extracellular domains, like known eukaryotic type Ser/Thr protein kinases from other bacteria. To identify the minimum region capable of exhibiting phosphorylation activity of PknA, we created several deletion mutants. Surprisingly, we found that the catalytic domain itself was not sufficient for exhibiting phosphorylation ability of PknA. However, the juxtamembrane region together with the kinase domain was necessary for the enzymatic activity and thus constitutes the catalytic core of PknA. Utilizing this core, we deduce that the autophosphorylation of PknA is an intermolecular event. Interestingly, the core itself was unable to restore the cell elongation phenotype as manifested by the full-length protein in E. coli; however, its co-expression along with the C-terminal region of PknA can associate them in trans to reconstitute a functional protein in vivo. Therefore, these findings argue that the transmembrane and extracellular domains of PknA, although dispensable for phosphorylation activities, are crucial in responding to signals. Thus, our results for the first time establish the significance of different domains in a bacterial eukaryotic type Ser/Thr kinase for reconstitution of its functionality.     

Allosteric activation mechanism of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknB

The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate > 30 ? from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how "back-to-back" N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.     

Regulation of chemosensory receptor expression and sensory signaling by the KIN-29 Ser/Thr kinase

Sensory signals regulate multiple developmental and behavioral circuits in C. elegans, providing a genetically tractable system in which to investigate the mechanisms underlying the acquisition and integration of sensory information. kin-29 mutants are defective in the expression of a set of chemoreceptor genes, and exhibit characteristics associated with altered sensory signaling, including increased lifespan, decreased body size, and deregulated entry into the dauer developmental stage. kin-29 encodes a Ser/Thr kinase with similarity to the MARK and AMPK/SNF1 family of kinases. We show that KIN-29 acts cell-autonomously and non-cell-autonomously in sensory neurons to regulate chemoreceptor expression, body size, and the dauer decision, suggesting that kin-29 function is essential for the correct acquisition and transduction of sensory information.     

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

The Myxococcus xanthus gene, pkn9 , encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus . This region also shows 29, 25 and 29% identity with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I–III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two-dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.     

Allosteric activation by dimerization of the PknD receptor Ser/Thr protein kinase from Mycobacterium tuberculosis

To define how extracellular signals activate bacterial receptor Ser/Thr protein kinases, we characterized the regulatory functions of a weak dimer interface identified in the Mycobacterium tuberculosis PknB and PknE receptor kinases. Sequence comparisons revealed that the analogous interface is conserved in PknD orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed M. tuberculosis PknD kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated M. tuberculosis PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation, and altered substrate specificity. In contrast, an inactive catalytic site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The phosphorylated PknD KD was fully active even in the absence of dimerization, suggesting that phosphorylation provides an additional regulatory mechanism. The conservation of analogous dimers in diverse prokaryotic and eukaryotic Ser/Thr protein kinases implies that this mechanism of protein kinase regulation is ancient and broadly distributed.     

Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the α isoform of human PP2C (PP2Cα), which folds into an α/β domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn²⁺-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.     

The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration

The ftsZ gene is essential for cell division in both Escherichia coli and Bacillus subtilis. In E. coli FtsZ forms a cytokinetic ring at the division site whose formation is under cell-cycle control. In addition, the FtsZ from E. coli has a GTPase activity that shows an unusual lag in vitro. In this study we show that FtsZ in Bacillus subtilis forms a ring that is at the tip of the invaginating septum. The FtsZ ring is dynamic since it is formed as division is initiated, changes diameter during septation, and disperses upon completion of septation. In vitro the purified FtsZ from B. subtilis exhibits a GTPase activity without a demonstrable lag, but the GTPase activity is markedly dependent upon the FtsZ concentration, suggesting that the FtsZ protein must oligomerize to express the GTPase activity.     

The tricornered Ser/Thr protein kinase is regulated by phosphorylation and interacts with furry during Drosophila wing hair development

The Trc/Ndr/Sax1/Cbk1 family of ser/thr kinases plays a key role in the morphogenesis of polarized cell structures in flies, worms, and yeast. Tricornered (Trc), the Drosophila nuclear Dbf2-related (Ndr) serine/threonine protein kinase, is required for the normal morphogenesis of epidermal hairs, bristles, laterals, and dendrites. We obtained in vivo evidence that Trc function was regulated by phosphorylation and that mutations in key regulatory sites resulted in dominant negative alleles. We found that wild-type, but not mutant Trc, is found in growing hairs, and we failed to detect Trc in pupal wing nuclei, implying that in this developmental context Trc functions in the cytoplasm. The furry gene and its homologues in yeast and Caenorhabditis elegans have previously been implicated as being essential for the function of the Ndr kinase family. We found that Drosophila furry (Fry) also is found in growing hairs, that its subcellular localization is dependent on Trc function, and that it can be coimmunoprecipitated with Trc. Our data suggest a feedback mechanism involving Trc activity regulates the accumulation of Fry in developing hairs.     

Inhibition of phosphatidylinositol 4,5-bisphosphate-stimulated phospholipase D2 activity by Ser/Thr phosphorylation

Treatment of HeLa cells overexpressing PLD2 with the Ser/Thr-specific protein phosphatase inhibitor, okadaic acid, augmented spontaneous phosphorylation of PLD2 with concomitant inhibition of phosphatidylinositol 4,5-bisphosphate (PIP(2))-stimulated PLD2 activity. Dephosphorylation of the immunoprecipitated, spontaneously phosphorylated PLD2 in COS-7 cells by catalytic subunit of protein phosphatase 1gamma1 resulted in the stimulation of the PLD2 catalytic activity. These observations suggest that Ser/Thr phosphorylation regulates PLD2 activity.     

Auto‐activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase

Many Ser/Thr protein kinases are activated by autophosphorylation, but the mechanism of this process has not been defined. We determined the crystal structure of a mutant of the Ser/Thr kinase domain (KD) of the mycobacterial sensor kinase PknB in complex with an ATP competitive inhibitor and discovered features consistent with an activation complex. The complex formed an asymmetric dimer, with the G helix and the ordered activation loop of one KD in contact with the G helix of the other. The activation loop of this putative ‘substrate’ KD was disordered, with the ends positioned at the entrance to the partner KD active site. Single amino‐acid substitutions in the G‐helix interface reduced activation‐loop phosphorylation, and multiple replacements abolished KD phosphorylation and kinase activation. Phosphorylation of an inactive mutant KD was reduced by G‐helix substitutions in both active and inactive KDs, as predicted by the idea that the asymmetric dimer mimics a trans‐autophosphorylation complex. These results support a model in which a structurally and functionally asymmetric, ‘front‐to‐front’ association mediates autophosphorylation of PknB and homologous kinases.     

Akt phosphorylation at Thr308 and Ser473 is required for CHIP-mediated ubiquitination of the kinase

Phosphorylation at Thr308 and Ser473 is known to activate Akt, a major kinase in mammalian cells. Once activated to turn on downstream signaling pathways, Akt returns to an inactive pool via PP2A-mediated dephosphorylation. We show here that Thr308 and Ser473 phosphorylations prompt Akt to enter the CHIP-mediated ubiquitin-proteasome pathway. Mutation at either Thr308 or Ser473 dampened its ability to bind to the U-box E3 ligase CHIP (C-terminal Hsp70 -interacting protein), and the Akt mutants revealed decreased rate of ubiquitination by CHIP. Our study unveils that the well-known phosphorylations responsible for Akt activation turn out to transduce recognition signals for Akt-CHIP binding and ensuing degradation.     

Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis

Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role. The present study concerns one of these enzymes, the serine/threonine protein kinase PknF from Mycobacterium tuberculosis. It is shown that, in addition to its autokinase activity, PknF is able to phosphorylate Rv1747, a newly described ABC transporter. This reaction appears to involve two FHA domains of Rv1747. It is suggested that recruitment and phosphorylation of Rv1747 depend on the interaction between its two non-redundant FHA domains and the autophosphorylated form of PknF.     

Insights into the catalytic mechanism of PPM Ser/Thr phosphatases from the atomic resolution structures of a mycobacterial enzyme

Serine/threonine-specific phosphatases (PPs) represent, after protein tyrosine phosphatases, the second major class of enzymes that catalyze the dephosphorylation of proteins. They are classed in two large families, known as PPP and PPM, on the basis of sequence similarities, metal ion dependence, and inhibitor sensitivity. Despite their wide species distribution and broad physiological roles, the catalytic mechanism of PPM phosphatases has been primarily inferred from studies of a single enzyme, human PP2Calpha. Here, we report the biochemical characterization and the atomic resolution structures of a soluble PPM phosphatase from the saprophyte Mycobacterium smegmatis in complex with different ligands. The structures provide putative snapshots along the catalytic cycle, which support an associative reaction mechanism that differs in some important aspects from the currently accepted model and reinforces the hypothesis of convergent evolution in PPs.     

The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.     

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with (32)P release and a point mutant of Thr 38.     

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC,a Ser/Thr kinase from Bacillus subtilis

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.