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.     

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.     

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.     

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.     

Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases

A family of eukaryotic-like Ser/Thr protein kinases occurs in bacteria, but little is known about the structures and functions of these proteins. Here we characterize PknB, a transmembrane signaling kinase from Mycobacterium tuberculosis. The intracellular PknB kinase domain is active autonomously, and the active enzyme is phosphorylated on residues homologous to regulatory phospho-acceptors in eukaryotic Ser/Thr kinases. The crystal structure of the PknB kinase domain in complex with an ATP analog reveals the active conformation. The predicted fold of the PknB extracellular domain matches the proposed targeting domain of penicillin-binding protein 2x. The structural and chemical similarities of PknB to metazoan homologs support a universal activation mechanism of Ser/Thr protein kinases in prokaryotes and eukaryotes.     

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.     

GTPase activity of mycobacterial FtsZ is impaired due to its transphosphorylation by the eukaryotic-type Ser/Thr kinase, PknA

FtsZ, a homolog of eukaryotic tubulin, is involved in the process of cell division, particularly in septum formation in bacteria. The primary amino acid sequences of this protein are fairly conserved in prokaryotes. We observed that a eukaryotic-type Ser/Thr protein kinase, PknA from Mycobacterium tuberculosis, when expressed in Escherichia coli exhibited cell elongation due to a defect in septum formation. We found that FtsZ either from Escherichia coli (eFtsZ) or from M. tuberculosis (mFtsZ) was phosphorylated on co-expression with PknA. Consistent with these observations, solid phase binding and in vitro kinase assays revealed the ability of PknA to interact with mFtsZ protein and also to phosphorylate it. We, therefore, ascertained mFtsZ as a substrate of PknA. Furthermore, the phosphorylated mFtsZ exhibited impairment in its GTP hydrolysis and polymerization abilities. Thus, our results highlighted the ability of PknA to phosphorylate as well as to regulate the functionality of FtsZ, the protein central to cell division throughout the bacterial lineage.     

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.     

Two Ser/Thr protein kinases essential for efficient aggregation and spore morphogenesis in Myxococcus xanthus

Myxococcus xanthus has a complex life cycle that involves vegetative growth and development. Previously, we described the espAB locus that is involved in timing events during the initial stages of fruiting body formation. Deletion of espA caused early aggregation and sporulation, whereas deletion of espB caused delayed aggregation and sporulation resulting in reduced spore yields. In this study, we describe two genes, pktA5 and pktB8, that flank the espAB locus and encode Ser/Thr protein kinase (STPK) homologues. Cells deficient in pktA5 or pktB8 formed translucent mounds and produced low spore yields, similar in many respects to espB mutants. Double mutant analysis revealed that espA was epistatic to pktA5 and pktB8 with respect to aggregation and fruiting body morphology, but that pktA5 and pktB8 were epistatic to espA with respect to sporulation efficiency. Expression profiles of pktA5-lacZ and pktB8-lacZ fusions and Western blot analysis showed that the STPKs are expressed under vegetative and developmental conditions. In vitro kinase assays demonstrated that the RD kinase, PktA5, autophosphorylated on threonine residue(s) and phosphorylated the artificial substrate, myelin basic protein. In contrast, autophosphorylation of the non-RD kinase, PktB8, was not observed in vitro; however, the phenotype of a pktB8 kinase-dead point mutant resembled the pktB8 deletion mutant, indicating that this residue was important for function and that it likely functions as a kinase in vivo. Immunoprecipitation of Tap-tagged PktA5 and PktB8 revealed an interaction with EspA during development in M. xanthus. These results, taken together, suggest that PktA5 and PktB8 are STPKs that function during development by interacting with EspA and EspB to regulate M. xanthus development.     

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.     

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.     

Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase

PknH Ser/Thr protein kinase of Mycobacterium tuberculosis controls the expression of a variety of cell wall related enzymes and regulates the in vivo growth in mice. Therefore, we predicted that the PknH kinase could phosphorylate several substrates controlling different metabolic and physiological pathways. Using a bioinformatic approach, we identified 40 potential substrates. Two substrates were shown to be phosphorylated by recombinant PknH kinase in vitro. Point mutation studies verified that substrates are phosphorylated at the in silico-predicted sites. Kinetic studies revealed a similar relative-phosphorylation rate (V(max)) of PknH towards two new substrates and the only previously known substrate, EmbR. Unlike the EmbR protein, the Rv0681 and DacB1 proteins do not contain an FHA domain and are possible participants of new signaling pathways mediated by the PknH kinase in M. tuberculosis.     

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.     

丝/苏氨酸蛋白激酶Plk1研究进展

Plk1是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶。Plk1与不同的细胞周期检查点的精密调控有关,从而确保了细胞周期事件按照严格的时间和顺序正常进行。Plk1在增殖活跃的细胞中呈高水平表达,Plk1的高度表达和肿瘤患者的低存活率之间具有显著的统计相关性。Plk1可能是非常有效的抗癌药物设计的靶点。     

The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.     

The bacterial HPr kinase/phosphorylase: a new type of Ser/Thr kinase as antimicrobial target

Protein phosphorylation plays a major role in bacterial cellular regulation as in eukaryotes. The HPr Kinase/Phosphorylase (HprK/P) was the first bacterial serine protein kinase to have had its structure determined, establishing that it is unrelated to the eukaryotic kinases. HprK/P belongs to another large structural family, the P-loop containing proteins. Among them, P-loop containing kinases have been assumed to only phosphorylate small molecules, but the example of HprK/P suggests that some may have proteins as substrates, defining novel cellular signal transduction pathways. Another major result of the studies presented here is that HprK/P also catalyses the phosphorolysis of the phosphoserine, yielding serine and pyrophosphate. The two different catalytic activities are carried out at the same active site. The determination of the structure of the complex with the protein substrates HPr and PserHPr allowed us to propose a catalytic mechanism. Since regulation of HPr phosphorylation has been shown to be involved in the virulence process of pathogenic bacteria, a search for specific inhibitors of HprK/P is of clinical interest and the first hit has already been found.