A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.     

Mammalian NDR protein kinases: from regulation to a role in centrosome duplication

The NDR (nuclear Dbf2-related) family of kinases is highly conserved from yeast to human, and has been classified as a subgroup of the AGC group of protein kinases based on the sequence of the catalytic domain. Like all other members of the AGC class of protein kinases, NDR kinases require the phosphorylation of conserved Ser/Thr residues for activation. Importantly, NDR family members have two unique stretches of primary sequence: an N-terminal regulatory (NTR) domain and an insert of several residues between subdomains VII and VIII of the kinase domain. The kinase domain insert functions as an auto-inhibitory sequence (AIS), while binding of the co-activator MOB (Mps-one binder) proteins to the NTR domain releases NDR kinases from inhibition of autophosphorylation. However, despite such advances in our understanding of the molecular activation mechanism(s) and physiological functions of NDR kinases in yeast and invertebrates, most biological NDR substrates still remain to be identified. Nevertheless, by showing that the centrosomal subpopulation of human NDR1/2 is required for proper centrosome duplication, the first biological role of human NDR1/2 kinases has been defined recently. How far NDR-driven centrosome overduplication could actually contribute to cellular transformation will also be discussed.     

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.     

Regulation of yeast Yak1 kinase by PKA and autophosphorylation-dependent 14-3-3 binding

Yak1 is a member of an evolutionarily conserved family of Ser/Thr protein kinases known as dual-specificity Tyr phosphorylation-regulated kinases (DYRKs). Yak1 was originally identified as a growth antagonist, which functions downstream of Ras/PKA signalling pathway. It has been known that Yak1 is phosphorylated by PKA in vitro and is translocated to the nucleus upon nutrient deprivation. However, the regulatory mechanisms for Yak1 activity and localization are largely unknown. In the present study, we investigated the role of PKA and Bmh1, a yeast 14-3-3 protein, in regulation of Yak1. We demonstrate that PKA-dependent phosphorylation of Yak1 on Ser295 and two minor sites inhibits nuclear localization of Yak1. We also show that intramolecular autophosphorylation on at least four Ser/Thr residues in the non-catalytic N-terminal domain is required for full kinase activity of Yak1. The most potent autophosphorylation site, Thr335, plays an essential role for Bmh1 binding in collaboration with a yet unidentified second binding site in the N-terminal domain. Bmh1 binding decreases the catalytic activity of Yak1 without affecting its subcellular localization. Since the binding of 14-3-3 proteins to Yak1 coincides with PKA activity, such regulatory mechanisms might allow cytoplasmic retention of an inactive form of Yak1 under high glucose conditions.     

Cloning, Characterization, and Chromosome Mapping of RPS6KC1, a Novel Putative Member of the Ribosome Protein S6 Kinase Family, to Chromosome 12q12–q13.1

A novel cDNA encoding a putative Ser/Thr protein kinase was isolated from a human skeletal muscle cDNA library. It contains an open reading frame that extends from nt 104 to 1510 and codes for a protein of 469 amino acids. A catalytic domain containing the conserved residues of the Ser/Thr protein kinase, especially human ribosome protein S6 kinase (RSK), was found to be located in the C-terminal end of the deduced protein. The gene was mapped to human chromosome 12q12-q13.1 by fluorescence in situ hybridization, and this result was confirmed with the Radiation Hybrid GB4 panel. Northern hybridization showed that the novel gene is expressed in all 16 human tissues tested with especially strong expression in testis, skeletal muscle, and brain, whereas weak expression was detected in kidney, thymus, small intestine, liver, lung, heart, and colon.     

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.     

Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 7120.

Eukaryotic-like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes. In most cases, their biochemical properties have been poorly characterized. The nitrogen-fixing and heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 houses a family of eukaryotic-like Ser/Thr kinases. Some of these enzymes are required for cell growth or development under certain conditions. None of them, however, has been shown experimentally to possess Ser/Thr kinase activity. A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp. PCC 7120. The recombinant PknC was shown to be phosphorylated on a Thr residue. This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC. PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates. PknC is thus a Ser/Thr kinase with broad substrate specificity. The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.     

Characterization of a eukaryotic-like tyrosine protein kinase expressed by the Shiga toxin-encoding bacteriophage 933W

The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.     

Discovery of Unique Lanthionine Synthetases Reveals New Mechanistic and Evolutionary Insights

Lantibiotic synthetases are remarkable biocatalysts generating conformationally constrained peptides with a variety of biological activities by repeatedly utilizing two simple posttranslational modification reactions: dehydration of Ser/Thr residues and intramolecular addition of Cys thiols to the resulting dehydro amino acids. Since previously reported lantibiotic synthetases show no apparent homology with any other known protein families, the molecular mechanisms and evolutionary origin of these enzymes are unknown. In this study, we present a novel class of lanthionine synthetases, termed LanL, that consist of three distinct catalytic domains and demonstrate in vitro enzyme activity of a family member from Streptomyces venezuelae. Analysis of individually expressed and purified domains shows that LanL enzymes install dehydroamino acids via phosphorylation of Ser/Thr residues by a protein kinase domain and subsequent elimination of the phosphate by a phosphoSer/Thr lyase domain. The latter has sequence homology with the phosphothreonine lyases found in various pathogenic bacteria that inactivate host mitogen activated protein kinases. A LanC-like cyclase domain then catalyzes the addition of Cys residues to the dehydro amino acids to form the characteristic thioether rings. We propose that LanL enzymes have evolved from stand-alone protein Ser/Thr kinases, phosphoSer/Thr lyases, and enzymes catalyzing thiol alkylation. We also demonstrate that the genes for all three pathways to lanthionine-containing peptides are widespread in Nature. Given the remarkable efficiency of formation of lanthionine-containing polycyclic peptides and the latter''s high degree of specificity for their cognate cellular targets, it is perhaps not surprising that (at least) three distinct families of polypeptide sequences have evolved to access this structurally and functionally diverse class of compounds.     

Evolution of domain combinations in protein kinases and its implications for functional diversity

Protein kinases phosphorylating Ser/Thr/Tyr residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Protein kinases classified based on sequence similarity in their catalytic domains, cluster into subfamilies, which share gross functional properties. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules, aiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their functional and molecular properties. Analysis of genomic repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation across various subfamilies of kinases. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by protein kinases in order to regulate variety of biological processes. In addition, domain architecture of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. In this review we discuss the repertoire of non-kinase domains tethered to multi-domain kinases in the metazoans. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization.     

FYVE-DSP1, a dual-specificity protein phosphatase containing an FYVE domain

Dual-specificity protein phosphatases (DSPs) dephosphorylate proteins at Ser/Thr and Tyr. FYVE domain is a double zinc finger motif which specifically binds phosphatidylinositol(3)-phosphate. Here, we report a novel dual specificity phosphatase that contains a FYVE domain at the C-terminus. We designate the protein FYVE-DSP1. Molecular cloning yielded three isoforms of the enzyme presumably derived from alternate RNA splicing. Sequence alignment revealed that the catalytic phosphatase domain of FYVE-DSP1 closely resembled that of myotubularin, while its FYVE domain has all the conserved amino acid residues found in other proteins of the same family. Recombinant FYVE-DSP1 is partitioned in both cytosolic and membrane fractions. It dephosphorylates proteins phosphorylated on Ser, Thr, and Tyr residues and low molecular weight phosphatase substrate para-nitrophenylphosphate. It shows typical characteristics of other DSPs and protein tyrosine phosphatases (PTPs). These include inhibition by sodium vanadate and pervanadate, pH dependency, and inactivation by mutation of the key cysteinyl residue at the phosphatase signature motif. Finally, PCR analyses demonstrated that FYVE-DSP1 is widely distributed in human tissues but different spliced forms expressed differently.     

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.     

Protein kinase associated with ribosomes of streptomycetes

Protein kinases can be classified into two main superfamilies on the basis of their sequence similarity and substrate specificity. The protein His kinase superfamily which autophosphorylate a His residue, and superfamily Ser/Thr and Tyr protein kinases, which phosphorylate Ser, Thr or Tyr residues. During the last years genes encoding Ser/Thr protein kinases have been identified in several microorganisms. Phosphorylation of proteins on Ser/Thr residues can be involved in many functions of prokaryotic cells including cell differentiation, signal transduction and protein biosynthesis. Phosphorylation of prokaryotic protein-synthesizing systems showed that the phosphorylation of initiation and elongation factors is subject to alteration during cell differentiation or bacteriophage infection. Protein kinase associated with ribosomes of streptomycetes phosphorylate the elongation factor Tu and 11 ribosomal proteins even in bacteriophage-uninfected cells. After phosphorylation of ribosomal proteins, ribosomes lose about 30% of their activity at the translation of poly(U). Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

StoPK-1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response

The glycopeptide antibiotic-producing bacterium, Streptomyces toyocaensis NRRL 15009, has proteins phosphorylated on Ser, Thr, Tyr and His, implying the presence of a battery of associated kinases. We have identified the Ser/Thr protein kinase gene fragments stoPK-1, stoPK-2, stoPK-3 and stoPK-4 from S. toyocaensis NRRL 15009 by a polymerase chain reaction (PCR) strategy using oligonucleotide primers based on eukaryotic Ser/Thr and Tyr kinase sequences. One of these (stoPK-1) was subsequently cloned in its entirety from a 3.2 kb genomic BamHI fragment. stoPK-1 encodes a 642-amino-acid protein with a predicted N-terminal Ser/Thr kinase domain and a C-terminal coiled-coil region divided by a membrane-spanning region. Expression of StoPK-1 in Escherichia coli yielded a protein confined to the membrane fraction, which was found to be phosphorylated exclusively on Thr residues and could transfer phosphate to the model substrates myelin basic protein and histone H1. Both autophosphorylation and phosphoryl transfer could be inhibited by the flavanoid apigenin. Disruption of stoPK-1 with the apramycin resistance gene in the S. toyo-caensis chromosome resulted in changes in mycelial morphology and an increased sensitivity to the redox cycling agents paraquat and nitrofurantoin on glucose-containing media. Supplying stoPK-1 or the S. coelicolor homologue pkaF in trans could reverse this sensitivity, whereas a catalytically inactive mutant of stoPK-1 could not, indicating that kinase activity is essential for this phenotype. This suggests a link between this membrane-bound protein kinase in signalling pathways sensitive to oxidative stress and/or glucose metabolism. These results broaden the roles of Ser/Thr protein kinases in bacteria and underscore the diversity of signal transduction mechanisms available to respond to various stimuli.     

The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src‐like autoinhibited conformation

Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic‐like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand‐free kinase domain shows an auto‐inhibited conformation similar to that observed in human Tyr kinases of the Src‐family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases. Proteins 2015; 83:982–988. © 2015 Wiley Periodicals, Inc.     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.     

Analysis of the protein kinase activity of moss phytochrome expressed in fibroblast cell culture

In the moss Ceratodon purpureus a phytochrome gene encodes a phytochrome type (PhyCer) which has a C-terminal domain homologous to the catalytic domain of eukaryotic protein kinases (PKs). PhyCer exhibits sequence conservation to serine/ threonine as well to tyrosine kinases. Since PhyCer is expressed very weakly in moss cells, to investigate the proposed PK activity of PhyCer, we overexpressed PhyCer transiently in fibroblast cells. For this purpose we made a chimeric receptor, EC-R, which consists of the extracellular, the membrane-spanning and the juxtamembrane domains of the human epidermal growth-factor receptor (EGF-R) linked to the PK catalytic domain of PhyCer (CerKin). The expression of EC-R in transiently transfected cells was confirmed with antibodies directed against the extracellular domain of EGF-R or against CerKin. Both EGF-R and EC-R were immunoprecipitated from lysates of overexpressing cells with antibodies against the extracellular domain of EGF-R. Phosphorylation experiments were performed with the immunoprecipitates and the phosphorylation products were subjected to phosphoamino acid analysis. Phosphorylation products specifically obtained with EC-R-transfected cells exhibit phosphorylation on serine and threonine residues. In EC-R transfected cells the endogenous EGF-R showed enhanced phosphorylation of serine and threonine residues compared to EGF-R immuno-precipitated from control cells. Although CerKin is closest to the catalytic domain of a protein tyrosine kinase from Dictyostelium discoideum, EC-R does not appear to phosphorylate tyrosine residues in vitro. From our data we conclude that PhyCer carries an active PK domain capable of phosphorylating serine and threonine residues.Abbreviations CerKin protein kinase catalytic domain of PhyCer - EC-R chimeric receptor consisting of the extracellular, the membrane spanning and the juxtamembrane domains of the human epidermal growth factor receptor (EGF-R) linked to the protein kinase catalytic domain of PhyCer - EGF-R epidermal growth factor receptor - mAb monoclonal antibody - PhyCer phytochrome gene in Ceratodon encoding a phytochrome type which has a C-terminal domain homologous to the catalytic domain of eucaryotic protein kinases - PK protein kinase - PVDF polyvinyl difluoride - Ser serine - Thr threonine - Tyr tyrosine Dr. Patricia Algarra was supported by the Alexander von Humboldt Foundation, Germany. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.     

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.     

Genomic analysis of protein kinases,protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell-cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.