The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.     

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.     

The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c

Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. 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, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.     

Structure–sequence features based prediction of phosphosites of serine/threonine protein kinases of Mycobacterium tuberculosis

Elucidation of signaling events in a pathogen is potentially important to tackle the infection caused by it. Such events mediated by protein phosphorylation play important roles in infection, and therefore, to predict the phosphosites and substrates of the serine/threonine protein kinases, we have developed a Machine learning-based approach for Mycobacterium tuberculosis serine/threonine protein kinases using kinase-peptide structure–sequence data. This approach utilizes features derived from kinase three-dimensional-structure environment and known phosphosite sequences to generate support vector machine (SVM)-based kinase-specific predictions of phosphosites of serine/threonine protein kinases (STPKs) with no or scarce data of their substrates. SVM outperformed the four machine learning algorithms we tried (random forest, logistic regression, SVM, and k-nearest neighbors) with an area under the curve receiver-operating characteristic value of 0.88 on the independent testing dataset and a 10-fold cross-validation accuracy of ~81.6% for the final model. Our predicted phosphosites of M. tuberculosis STPKs form a useful resource for experimental biologists enabling elucidation of STPK mediated posttranslational regulation of important cellular processes.     

Multiple phosphorylation of membrane-associated calcium-dependent protein serine/threonine kinase in Streptomyces fradiae

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate aerial mycelium formation and sporulation. Using in vitro labeling, we demonstrate that in S. fradiae in the late exponential growth phosphorylation of 65-kDa membrane-associated protein is also influenced by Ca(2+) added exogenously. Calcium ions at physiological concentration stimulate intensive Ca(2+)-dependent phosphorylation of 65-kDa protein at multiple sites on serine, threonine, and tyrosine residues. Assay of protein kinases in situ demonstrated in the fraction of membrane-associated proteins the presence of two autophosphorylating protein serine/threonine kinases with molecular masses of 127 kDa and 65 kDa. Autophosphorylation of both proteins is also Ca(2+)-dependent.     

Mycobacterium tuberculosis Maltosyltransferase GlgE,a Genetically Validated Antituberculosis Target,Is Negatively Regulated by Ser/Thr Phosphorylation

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.     

Down-regulation of c-FLIP increases reactive oxygen species,induces phosphorylation of serine/threonine kinase Akt,and impairs motility of cancer cells

The role of c-FLIP in cell motility was investigated using RNA interference. Down-regulation of c-FLIP increased amounts of reactive oxygen species (ROS), while over-expression of c-FLIP produced opposite effect. ROS induced phosphorylation of Akt and impaired cell motility.     

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor‐like kinases

The Arabidopsis thaliana somatic embryogenesis receptor‐like kinase (SERK) family consists of five leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)‐mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC‐MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C‐terminally located residue Ser‐562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr‐462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP‐tagged SERK1 from plant extracts followed by MS/MS identified Ser‐303, Thr‐337, Thr‐459, Thr‐462, Thr‐463, Thr‐468, and Ser‐612 or Thr‐613 or Tyr‐614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser‐299 and Thr‐462. This suggests both intra‐ and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser‐887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.     

Allele-specific suppression of aSaccharomyces cerevisiae prp20 mutation by overexpression of a nuclear serine/threonine protein kinase

The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search forprp20-10 allele-specific high-copy-number suppressors, theKSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.     

Signatures of the ATP-binding pocket as a basis for structural classification of the serine/threonine protein kinases of gram-positive bacteria

Eukaryotic-like serine/threonine protein kinases (ESTPKs) are widely spread throughout the bacterial genomes. These enzymes can be potential targets of new antibacterial drugs useful for the treatment of socially important diseases such as tuberculosis. In this study, ESTPKs of pathogenic, probiotic, and antibiotic-producing Gram-positive bacteria were classified according to the physicochemical properties of amino acid residues in the ATP-binding site of the enzyme. Nine residues were identified that line the surface of the adenine-binding pocket, and ESTPKs were classified based on these signatures. Twenty groups were discovered, five of them containing >10 representatives. The two most abundant groups contained >150 protein kinases that belong to the various branches of the phylogenetic tree, whereas certain groups are genus- or even species-specific. Homology modeling of the typical representatives of each group revealed that the classification is reliable, and the differences between the protein kinase ATP-binding pockets predicted based on their signatures are apparent in their structure. The classification is expected to be useful for the selection of targets for new anti-infective drugs.     

Mycobacterium smegmatis biofilm formation and sliding motility are affected by the serine/threonine protein kinase PknF

Eighteen 'eukaryotic-like' serine/threonine kinases are present in the Mycobacterium smegmatis genome. One of them encoded by the ORF 3677 demonstrates high similarity to the Mycobacterium tuberculosis protein kinase PknF. A merodiploid strain was generated, which showed reduced growth associated with irregular cell structure. The merodiploid strain displayed altered colony morphology, defective sliding motility and biofilm formation. These data indicate a role for PknF in biofilm formation, possibly associated with alterations in glycopeptidolipid composition.     

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.     

Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases

Fluorescent chemosensors of protein kinase activity provide a continuous, high-throughput sensing format for the study of the roles of these enzymes, which are crucial for regulating cellular function. Specifically, chemosensors using the nonnatural amino acid, Sox, and physiological Mg(2+) levels report phosphorylation with dramatic fluorescence changes that are amenable to real-time and high-throughput analysis. In this article, we report 15 probes for a total of six distinct serine/threonine kinases with large fluorescence increases and good reactivity toward the target kinase. The sensing mechanism is detailed, and the optimal sensing motif is determined. These versatile and powerful sensors provide tools for researchers studying the roles of the targeted kinases in signal transduction, and the design principles provide guidelines for the generation of future fluorescent chemosensors for any serine/threonine kinase.     

丝氨酸/苏氨酸蛋白激酶在结核分枝杆菌致病机制中的作用

丝氨酸/苏氨酸蛋白激酶(STPK)是一类真核细胞样的蛋白激酶,是分枝杆菌生长和代谢的重要调节因子,参与其多种细胞活动(如细胞和菌落形态、葡萄糖和谷氨酰胺转运、吞噬体-溶酶体融合、转录因子活性等)的调节.结核分枝杆菌编码产生11种STPK( PknA、PknB、PknD～PknL),可分为5群(ABL群、HED群、FIJ...     

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.     

Identification of a new in vivo phosphorylation site in the cytoplasmic carboxyl terminus of EBV-LMP1 by tandem mass spectrometry

Latent membrane protein 1 (LMP1), an oncogenic protein encoded by Epstein-Barr virus (EBV), has been verified to be phosphorylated in vitro by protein casein kinase 2 (CK2). In this study, we characterized the phosphorylation of the carboxyl terminus of LMP1 fused with glutathione-S-transferase (GST-LMP1c) and the FLAG-epitope-tagged LMP1 (F-LMP1) proteins expressed in HEK293T cells. Using a combination of chemical modification and tandem mass spectrometry, we detected the phosphorylation of a tryptic peptide, 191-223 amino acids, in both GST-LMP1c catalysed by CK2 and F-LMP1-expressing cell lines. Serine residues at positions 211 and 215 were determined to be the substrates of CK2 in vitro. Most importantly, the S215 phosphorylation was also detected in F-LMP1-expressing human cell lines. The phosphorylation of S215, which is located in the carboxyl-terminus activation region 1 of LMP1, provides a new insight for investigating the role and modulation of the phosphorylation of LMP1.     

Differential expression of two functional serine/threonine protein kinases from soyabean that have an unusual acidic domain at the carboxy terminus

Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized. Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis. These kinases are encoded by one- or two-copy genes in the soybean genome. Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues. SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants. In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants. Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different. The two genes also differed in their response to abscisic acid (ABA). SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid. In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::β-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA. Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins. Kinase assays using affinity-purified SPK-4/antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues. Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s). Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses. Received: 6 January 1997 / Accepted: 19 March 1997     

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.