Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates

Searching the genome sequence of Streptococcus pneumoniae revealed the presence of a single Ser/Thr protein kinase gene stkP linked to protein phosphatase phpP. Biochemical studies performed with recombinant StkP suggest that this protein is a functional eukaryotic-type Ser/Thr protein kinase. In vitro kinase assays and Western blots of S. pneumoniae subcellular fractions revealed that StkP is a membrane protein. PhpP is a soluble protein with manganese-dependent phosphatase activity in vitro against a synthetic substrate RRA(pT)VA. Mutations in the invariant aspartate residues implicated in the metal binding completely abolished PhpP activity. Autophosphorylated form of StkP was shown to be a substrate for PhpP. These results suggest that StkP and PhpP could operate as a functional pair in vivo. Analysis of phosphoproteome maps of both wild-type and stkP null mutant strains labeled in vivo and subsequent phosphoprotein identification by peptide mass fingerprinting revealed two possible substrates for StkP. The evidence is presented that StkP can phosphorylate in vitro phosphoglucosamine mutase GlmM which catalyzes the first step in the biosynthetic pathway leading to the formation of UDP-N-acetylglucosamine, an essential common precursor to cell envelope components.     

Structural model of the full-length Ser/Thr protein kinase StkP from S. pneumoniae and its recognition of peptidoglycan fragments

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.     

Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae

The eukaryotic-type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal-transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C-terminal domain of StkP kinase (C-StkP), elaborated a three-dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C-StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to β-lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA-containing kinase, suggesting that non-crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for β-lactam antibiotics.     

Interaction of Penicillin‐Binding Protein 2x and Ser/Thr protein kinase StkP,two key players in Streptococcus pneumoniae R6 morphogenesis

Bacterial cell growth and division require the co‐ordinated action of peptidoglycan biosynthetic enzymes and cell morphogenesis proteins. However, the regulatory mechanisms that allow generating proper bacterial shape and thus preserving cell integrity remain largely uncharacterized, especially in ovococci. Recently, the conserved eukaryotic‐like Ser/Thr protein kinase of Streptococcus pneumoniae (StkP) was demonstrated to play a major role in cell shape and division. Here, we investigate the molecular mechanisms underlying the regulatory function(s) of StkP and show that it involves one of the essential actors of septal peptidoglycan synthesis, Penicillin‐Binding Protein 2x (PBP2x). We demonstrate that StkP and PBP2x interact directly and are present in the same membrane‐associated complex in S. pneumoniae. We further show that they both display a late‐division localization pattern at the division site and that the positioning of PBP2x depends on the presence of the extracellular PASTA domains of StkP. We demonstrate that StkP and PBP2x interaction is mediated by their extracellular regions and that the complex formation is inhibited in vitro in the presence of cell wall fragments. These data suggest that the role of StkP in cell division is modulated by an interaction with PBP2x.     

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.     

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.     

Cell Wall Stress Stimulates the Activity of the Protein Kinase StkP of Streptococcus pneumoniae,Leading to Multiple Phosphorylation

Streptococcus pneumoniae is an opportunistic human pathogen that encodes a single eukaryotic-type Ser/Thr protein kinase StkP and its functional counterpart, the protein phosphatase PhpP. These signaling enzymes play critical roles in coordinating cell division and growth in pneumococci. In this study, we determined the proteome and phosphoproteome profiles of relevant mutants. Comparison of those with the wild-type provided a representative dataset of novel phosphoacceptor sites and StkP-dependent substrates. StkP phosphorylates key proteins involved in cell division and cell wall biosynthesis in both the unencapsulated laboratory strain Rx1 and the encapsulated virulent strain D39. Furthermore, we show that StkP plays an important role in triggering an adaptive response induced by a cell wall-directed antibiotic. Phosphorylation of the sensor histidine kinase WalK and downregulation of proteins of the WalRK core regulon suggest crosstalk between StkP and the WalRK two-component system. Analysis of proteomic profiles led to the identification of gene clusters regulated by catabolite control mechanisms, indicating a tight coupling of carbon metabolism and cell wall homeostasis. The imbalance of steady-state protein phosphorylation in the mutants as well as after antibiotic treatment is accompanied by an accumulation of the global Spx regulator, indicating a Spx-mediated envelope stress response. In summary, StkP relays the perceived signal of cell wall status to key cell division and regulatory proteins, controlling the cell cycle and cell wall homeostasis.     

The extended conformation of the 2.9-Å crystal structure of the three-PASTA domain of a Ser/Thr kinase from the human pathogen Staphylococcus aureus

PASTA (penicillin-binding protein and serine/threonine kinase associated) modules are found in penicillin-binding proteins and bacterial serine/threonine kinases mainly from Gram-positive Firmicutes and Actinobacteria. They may act as extracellular sensors by binding peptidoglycan fragments. We report here the first crystal structure of a multiple-PASTA domain from Ser/Thr kinase, that of the protein serine/threonine kinase 1 (Stk1) from the Firmicute Staphylococcus aureus. The extended conformation of the three PASTA subunits differs strongly from the compact conformation observed in the two-PASTA domain of penicillin-binding protein PBP2x, whereas linear conformations were also reported for two-subunit fragments of the four-PASTA domain of the Actinobacteria Mycobacterium tuberculosis studied by liquid NMR. Thus, a stretched organization appears to be the signature of modular PASTA domains in Ser/Thr kinases. Signal transduction to the kinase domain is supposed to occur via dimerization and ligand binding. A conserved X-shaped crystallographic dimer stabilized by intermolecular interactions between the second PASTA subunits of each monomer is observed in the two crystal forms of Stk1 that we managed to crystallize. Extracellular PASTA domains are composed of at least two subunits, and this molecular assembly is a plausible candidate for the biological dimer. We have also performed docking experiments, which predict that the hinge regions of the PASTA domain can accommodate peptidoglycan. Finally, a three-dimensional homology molecular model of full-length Stk1 was generated, suggesting an interaction between the kinase domain and the cytoplasmic face of the plasma membrane via a eukaryotic-like juxtamembrane domain. A comprehensive activation mechanism for bacterial Ser/Thr kinases is proposed with the support of these structural data.     

Molecular characterisation and regulation of a Nicotiana tabacum S-domain receptor-like kinase gene induced during an early rapid response to lipopolysaccharides

The isolation, characterization and regulation of the first lipopolysaccharide (LPS)-responsive S-domain receptor-like kinase (RLK) in Nicotiana tabacum are reported. The gene, corresponding to a differentially expressed LPS-responsive EST, was fully characterised to investigate its involvement in LPS-induced responses. The full genomic sequence, designated Nt-Sd-RLK, encodes for a S-domain RLK protein containing conserved modules (B-lectin-, S- and PAN-domains) reported to function in mediating protein-protein and protein-carbohydrate interactions in its extracellular domain, as well as the molecular architecture to transduce signals intracellularly through a Ser/Thr kinase domain. Phylogenetic analysis clustered Nt-Sd-RLK with S-domain RLKs induced by bacteria, wounding and salicylic acid. Perception of LPS induced a rapid, bi-phasic response in Nt-Sd-RLK expression with a 17-fold up-regulation at 3 and 9h. A defence-related W-box cis element was found in the promoter region of Nt-Sd-RLK and the transient induction of Nt-Sd-RLK in cultured cells by LPS exhibited a pattern typical of early response defence genes. Nt-Sd-RLK was also responsive to salicylic acid induction and was expressed in differentiated leaf tissue, where LPS elicited local as well as systemic up-regulation. The results contribute new knowledge about the potential role that S-domain RLKs may play within interactive signal transduction pathways associated with immunity and defence.     

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.     

PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.     

The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 is involved in the perception of insect feeding

The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 (LecRK1) has been recently identified as a component of the mechanism used by plants to suppress the Manduca sexta-triggered accumulation of salicylic acid (SA). The suppression of the SA burst by LecRK1 allows for the unfettered induction of jasmonic acid (JA)-mediated defense responses against M. sexta herbivory. LecRK1 contains a multi-domain extracellular region composed of a G-type Lectin domain and a PAN-AP domain separated by a variable sequence with low similarity to an EGF domain. The LecRK1 intracellular region is composed of a single domain structure with predicted Ser/Thr protein kinase activity. The multi-domain structure of the extracellular region of LecRK1 adds a level of complexity in terms of the potential ligands that this receptor protein could recognize.     

In vitro characterization of the homogalacturonan-binding domain of the wall-associated kinase WAK1 using site-directed mutagenesis

Wall-associated kinase 1--WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell wall in Arabidopsis thaliana (L.) HEYNH. In a previous paper [Decreux, A., Messiaen, J., 2005. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol. 46, 268-278], we showed that a recombinant peptide expressed in yeast corresponding to amino acids 67-254 of the extracellular domain of WAK1 specifically interacts with commercial non-methylesterified homogalacturonic acid, purified homogalacturonans from Arabidopsis and oligogalacturonides in a calcium-induced conformation. In this report, we used a receptor binding domain sequence-based prediction method to identify four putative binding sites in the extracellular domain of WAK1, in which cationic amino acids were selected for substitution by site-directed mutagenesis. Interaction studies between mutated forms of WAK1 and homogalacturonans allowed us to identify and confirm at least five specific amino acids involved in the interaction with homogalacturonan dimers and multimers. The presence of this homogalacturonan-binding domain within the extracellular domain of WAK1 is discussed in terms of cell wall architecture and signal transduction.     

Regulation of p90RSK phosphorylation by SARS-CoV infection in Vero E6 cells

The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.     

A calmodulin-dependent protein kinase from lower eukaryote Physarum polycephalum

A full-length cDNA coding a calmodulin (CaM)-dependent protein kinase gene was cloned from Physarum plasmodia poly(A)-RNA by polymerase chain reaction with the oligonucleotide primers that were designed after the amino acid sequence of highly conserved regions of myosin light-chain kinase. Sequence analysis of the cDNA revealed that this Physarum kinase was a 42,519-Da protein with an ATP-binding domain, Ser/Thr kinase active site signature, and CaM-binding domain. Expression of the cDNA in Escherichia coli demonstrated that the Physarum kinase in the presence of Ca2+ and CaM phosphorylated the recombinant phosphorylatable light chain (PLc) of Physarum myosin II. The peptide analysis after proteolysis of the phosphorylated PLc indicated that Ser 18 was phosphorylated. The site was confirmed by the failure of phosphorylation of PLc, the Ser 18 of which was replaced by Ala. The physiological role of the kinase will be discussed with special reference to the 55-kDa kinase, which had been previously purified from Physarum plasmodia for phosphorylated PLc.     

The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins

Background  

The serine/threonine kinase StkP of Streptococcus pneumoniae is a major virulence factor in the mouse model of infection. StkP is a modular protein with a N-terminal kinase domain a C-terminal PASTA domain carrying the signature of penicillin-binding protein (PBP) and prokaryotic serine threonine kinase. In laboratory cultures, one target of StkP is the phosphoglucosamine mutase GlmM involved in the first steps of peptidoglycan biosynthesis. In order to further elucidate the importance of StkP in S. pneumoniae, its role in resistance to β-lactams has been assessed by mutational analysis in laboratory cultures and its genetic conservation has been investigated in isolates from infected sites (virulent), asymptomatic carriers, susceptible and non-susceptible to β-lactams.     

Autophosphorylated residues involved in the regulation of human chk2 in vitro

Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.     

Phosphorylation-mediated regulation of a rice ABA responsive element binding factor

OREB1 is a rice ABRE binding factor characterized by the presence of multiple highly-conserved phosphorylation domains (C1, C2, C3, and C4) and two kinase recognition motifs, RXXS/T and S/TXXE/D, within different functional domains. An in vitro kinase assay showed that OREB1 is phosphorylated not only by the SnRK2 kinase, but also by other Ser/Thr protein kinases, such as CaMKII, CKII, and SnRK3. Furthermore, the N-terminal phosphorylation domain C1 was found to be differentially phosphorylated by the SnRK2/SnRK3 kinase and by hyperosmotic/cold stress, suggesting that the C1 domain may function in decoding different signals. The phosphorylation-mediated regulation of OREB1 activity was investigated through mutation of the SnRK2 recognition motif RXXS/T within each phosphorylation module. OREB1 contains a crucial nine-amino acid transactivation domain located near the phosphorylation module C1. Deletion of the C1 domain increased OREB1 activity, whereas mutation of Ser 44, Ser 45, and Ser 48 of the C1 domain to aspartates decreased OREB1 activity. In the C2 domain, a double mutation of Ser 118 and Ser 120 to alanines suppressed OREB1 activity. These findings strongly suggest that selective phosphorylation of the C1 or C2 modules may positively or negatively regulate OREB1 transactivation. In addition, mutation of Ser 385 of the C4 domain to alanines completely abolished the interaction between OREB1 and a rice 14-3-3 protein, GF14d, suggesting that SnRK2-mediated phosphorylation may regulate this interaction. These results indicate that phosphorylation domains of OREB1 are not functionally redundant and regulate at least three different functions, including transactivation activity, DNA binding, and protein interactions. The multisite phosphorylation of OREB1 is likely a key for the fine control of its activity and signal integration in the complex stress signaling network of plant cells.