Eukaryotic-like Protein Kinases Research Articles

Molecular basis of the final step of cell division in Streptococcus pneumoniae

Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.

Phosphorylation of the Archaeal Holliday Junction Resolvase Hjc Inhibits Its Catalytic Activity and Facilitates DNA Repair in Sulfolobus islandicus REY15A.

Protein phosphorylation is one of the main protein post-translational modifications and regulates DNA repair in eukaryotes. Archaeal genomes encode eukaryotic-like DNA repair proteins and protein kinases (ePKs), and several proteins involved in homologous recombination repair (HRR) including Hjc, a conserved Holliday junction (HJ) resolvase in Archaea, undergo phosphorylation, indicating that phosphorylation plays important roles in HRR. Herein, we performed phosphorylation analysis of Hjc by various ePKs from Sulfolobus islandicus. It was shown that SiRe_0171, SiRe_2030, and SiRe_2056, were able to phosphorylate Hjc in vitro. These ePKs phosphorylated Hjc at different Ser/Thr residues: SiRe_0171 on S34, SiRe_2030 on both S9 and T138, and SiRe_2056 on T138. The HJ cleavage activity of the phosphorylation-mimic mutants was analyzed and the results showed that the cleavage activity of S34E was completely lost and that of S9E had greatly reduced. S. islandicus strain expressing S34E in replacement of the wild type Hjc was resistant to higher doses of DNA damaging agents. Furthermore, SiRe_0171 deletion mutant exhibited higher sensitivity to DNA damaging agents, suggesting that Hjc phosphorylation by SiRe_0171 enhanced the DNA repair capability. Our results revealed that HJ resolvase is regulated by protein phosphorylation, reminiscent of the regulation of eukaryotic HJ resolvases GEN1 and Yen1.

Autophosphorylation and Cross-Phosphorylation of Protein Kinases from the Crenarchaeon Sulfolobus islandicus.

Protein phosphorylation, one of the most important post-translational modifications, regulates almost every cellular process. Although signal transduction by protein phosphorylation is extensively studied in Eukaryotes and Bacteria, the knowledge of this process in archaea is greatly lagging behind, especially for Ser/Thr/Tyr phosphorylation by eukaryotic-like protein kinases (ePKs). So far, only a few studies on archaeal ePKs have been reported, most of which focused on the phosphorylation activities in vitro, but their physiological functions and interacting network are still largely unknown. In this study, we systematically investigated the autophosphorylation and cross-phosphorylation activities of ePKs from Sulfolobus islandicus REY15A using proteins expressed in Escherichia coli or S. islandicus. In vitro kinase assay showed that 7 out of the 11 putative ePKs have autophosphorylation activity. A protein Ser/Thr phosphatase, SiRe_1009, was able to dephosphorylate various autophosphorylated ePKs, confirming that these proteins are Ser/Thr kinases. Two ePKs, SiRe_2030 and SiRe_2056, homologs of typical eukaryotic PKs involved in peptide synthesis in response to various cellular stresses, exhibit highly efficient phosphorylation activities on both themselves and other ePKs. Overexpression of the protein kinases in vivo revealed that elevated level of either SiRe_1531 or SiRe_2056 inhibited the cell growth of S. islandicus cells. Finally, a phosphorylation network of the protein kinases was proposed and their putative physiological roles were discussed.

Characterization of a eukaryotic-like protein kinase, DspB, with an atypical catalytic loop motif from Myxococcus xanthus

Serine (Ser)/threonine (Thr) or tyrosine (Tyr) protein kinases in eukaryotes contain RDxKxxN or RDx(A/R)A(A/R)N sequences, respectively, in the catalytic loop. Myxococcus xanthus DspB is a dual-specificity kinase that contains an atypical sequence, RDVAQKN, in the catalytic loop. The DspB mutant (A165K), which contains the canonical RDxKxxN motif, had an approximate 1.3-fold increase in kinase activity toward myelin basic protein (MBP). Arginine-aspartate (RD) kinases carry a conserved Arg immediately preceding the catalytic Asp that is required for autophosphorylation of the activation loop. DspB belongs to the RD kinase family and contains one Ser residue (Ser-190) and one Thr residue (Thr-194) in the activation loop. Mutation of Ser-190 or Thr-194 to Ala did not significantly affect the kinase activity toward MBP. We previously reported that four M. xanthus eukaryotic-like kinases (EPKs) are autophosphorylated on Tyr residues. These EPKs contain six Tyr residues at homologous positions, and five of those Tyr residues, Y25, Y102, Y145, Y173, and Y205, are conserved in DspB. DspB is mainly autophosphorylated on Y145, and a Y145F mutant has reduced kinase activity, suggesting that autophosphorylation of the Tyr residue of DspB may be required for high-level kinase activity.

Characterizing activities of eukaryotic-like protein kinases with atypical catalytic loop motifs from Myxococcus xanthus

Myxococcus xanthus has eukaryotic-like protein kinases (EPKs) with different atypical catalytic loop motifs. Seven out of 14 recombinant M. xanthus EPKs containing atypical motifs in the catalytic loop showed protein kinase activity against myelin basic protein and four autophosphorylated EPKs were detected using anti-phosphotyrosine antibody by western blotting.

Regulation of eukaryotic-like protein kinase activity of DspA from Myxococcus xanthus by autophosphorylation

A Myxococcus xanthus DspA contains 12 subdomains characteristic of eukaryotic-like protein kinases but with an atypical sequence, RDxSPHN, in the catalytic loop, different from the consensus motifs observed in Ser/Thr kinases (RDxKxxN) or Tyr kinases (RDx(A/R)A(A/R)N). DspA phosphorylated myelin basic protein (MBP) on Ser and Thr residues. Mutations of the SPHN motif within the catalytic loop to KPHN or KPEN for Ser/Thr kinases, AARN for Tyr kinases and TPHN or TSHN for Dictyostelium Tyr kinases markedly reduced autophosphorylation and kinase activities. Phosphorylation assays, Western blot analysis and mutational analysis revealed that DspA is a dual-specificity kinase that autophosphorylates on two Thr residues (Thr-199 and Thr-201) in the activation loop and two Tyr residues (Tyr-35 and Tyr-111). RD kinases such as DspA are activated by phosphorylation in the activation loop. Replacement of Thr-199 or/and Thr-201 in the DspA activation loop by alanine also almost abolished autophosphorylation and kinase activities. In addition, mutation of either Tyr-35 or Tyr-111 to phenylalanine decreased kinase activities against MBP, and double mutation abolished kinase activity. These results suggested that DspA is activated by dual autophosphorylation of Thr residues in the activation loop, and autophosphorylation on two Tyr residues of DspA are required for high-level kinase activity.

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

The protein kinase catalytic domain contains several conserved residues of unknown functions. Here, using a combination of computational and experimental approaches, we show that the function of some of these residues is to maintain the backbone geometry of the active site in a strained conformation. Specifically, we find that the backbone geometry of the catalytically important HRD motif deviates from ideality in high-resolution structures and the strained geometry results in favorable hydrogen bonds with conserved noncatalytic residues in the active site. In particular, a conserved aspartate in the F-helix hydrogen bonds to the strained HRD backbone in diverse eukaryotic and eukaryotic-like protein kinase crystal structures. Mutations that alter this hydrogen-bonding interaction impair catalytic activity in Aurora kinase. Although the backbone strain is present in most active conformations, several inactive conformations lack the strain because of a peptide flip in the HRD backbone. The peptide flip is correlated with loss of hydrogen bonds with the F-helix aspartate as well as with other interactions associated with kinase regulation. Within protein kinases that are regulated by activation loop phosphorylation, the strained residue is an arginine, which coordinates with the activation loop phosphate. Based on analysis of strain across the protein kinase superfamily, we propose a model in which backbone strain co-evolved with conserved residues for allosteric control of catalytic activity. Our studies provide new clues for the design of allosteric protein kinase inhibitors.

Change of carbon source causes dramatic effects in the phospho-proteome of the archaeon Sulfolobus solfataricus.

Protein phosphorylation is known to occur in Archaea. However, knowledge of phosphorylation in the third domain of life is rather scarce. Homology-based searches of archaeal genome sequences reveals the absence of two-component systems in crenarchaeal genomes but the presence of eukaryotic-like protein kinases and protein phosphatases. Here, the influence of the offered carbon source (glucose versus tryptone) on the phospho-proteome of Sulfolobus solfataricus P2 was studied by precursor acquisition independent from ion count (PAcIFIC). In comparison to previous phospho-proteome studies, a high number of phosphorylation sites (1318) located on 690 phospho-peptides from 540 unique phospho-proteins were detected, thus increasing the number of currently known archaeal phospho-proteins from 80 to 621. Furthermore, a 25.8/20.6/53.6 Ser/Thr/Tyr percentage ratio with an unexpectedly high predominance of tyrosine phosphorylation was detected. Phospho-proteins in most functional classes (21 out of 26 arCOGs) were identified, suggesting an important regulatory role in S. solfataricus. Focusing on the central carbohydrate metabolism in response to the offered carbon source, significant changes were observed. The observed complex phosphorylation pattern hints at an important physiological function of protein phosphorylation in control of the central carbohydrate metabolism, which might particularly operate in channeling carbon flux into the respective metabolic pathways.

Insights from the molecular docking of withanolide derivatives to the target protein PknG from Mycobacterium tuberculosis.

A crucial virulence factor for intracellular Mycobacterium tuberculosis survival is Protein kinase G (PknG), a eukaryotic-like serinethreonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG results in mycobacterial transfer to lysosomes. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show various pharmacological activities. We describe the docking of 26 withanferin and 14 withanolides from Withania somnifera into the three dimensional structure of PknG of M. tuberculosis using GLIDE. The inhibitor binding positions and affinity were evaluated using scoring functions- Glidescore. The withanolide E, F and D and Withaferin - diacetate 2 phenoxy ethyl carbonate were identified as potential inhibitors of PknG. The available drug molecules and the ligand AX20017 showed hydrogen bond interaction with the aminoacid residues Glu233 and Val235. In addition to Val235 the other amino acids, Gly237, Gln238 and Ser239 are important for withanolide inhibitor recognition via hydrogen bonding mechanisms.

Two-Component Systems and Their Co-Option for Eukaryotic Signal Transduction

Two-component signaling pathways involve histidine kinases, response regulators, and sometimes histidine-containing phosphotransfer proteins. Prevalent in prokaryotes, these signaling elements have also been co-opted to meet the needs of signal transduction in eukaryotes such as fungi and plants. Here we consider the evolution of such regulatory systems, with a particular emphasis on the roles they play in signaling by the plant hormones cytokinin and ethylene, in phytochrome-mediated perception of light, and as integral components of the circadian clock.

Myxococcus xanthus Pph2 Is a Manganese-dependent Protein Phosphatase Involved in Energy Metabolism

The multicellular behavior of the myxobacterium Myxococcus xanthus requires the participation of an elevated number of signal-transduction mechanisms to coordinate the cell movements and the sequential changes in gene expression patterns that lead to the morphogenetic and differentiation events. These signal-transduction mechanisms are mainly based on two-component systems and on the reversible phosphorylation of protein targets mediated by eukaryotic-like protein kinases and phosphatases. Among all these factors, protein phosphatases are the elements that remain less characterized. Hence, we have studied in this work the physiological role and biochemical activity of the protein phosphatase of the family PPP (phosphoprotein phosphatases) designated as Pph2, which is forming part of the same operon as the two-component system phoPR1. We have demonstrated that this operon is induced upon starvation in response to the depletion of the cell energy levels. The increase in the expression of the operon contributes to an efficient use of the scarce energy resources available for developing cells to ensure the completion of the life cycle. In fact, a Deltapph2 mutant is defective in aggregation, sporulation yield, morphology of the myxospores, and germination efficiency. The yeast two-hybrid technology has shown that Pph2 interacts with the gene products of MXAN_1875 and 5630, which encode a hypothetical protein and a glutamine synthetase, respectively. Because Pph2 exhibits Ser/Thr, and to some extent Tyr, Mn(2+)-dependent protein phosphatase activity, it is expected that this function is accomplished by dephosphorylation of the specific protein substrates.

Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome

Ser/Thr/Tyr kinases, which together comprise a major class of regulatory proteins in eukaryotes, were not believed to play an important role in prokaryotes until recently. However, our analysis of 626 prokaryotic genomes reveals that eukaryotic-like protein kinases (ELKs) are found in nearly two-thirds of the sequenced strains. We have identified 2697 ELKs, most of which are encoded by multicellular strains of the phyla Proteobacteria (Myxococcales), Actinobacteria, Cyanobacteria, and Chloroflexi, and 2 Acidobacteria and 1 Planctomycetes. Astonishingly, 7 myxobacterial strains together encode 892 ELKs, with 4 of the strains exhibiting a genomic ELK density similar to that observed in eukaryotes. Most myxobacterial ELKs show a modular organization in which the kinase domain is located at the N terminus. The C-terminal portion of the ELKs is highly diverse and often contains sequences with similarity to characterized domains, most of them involved in signaling mechanisms or in protein-protein interactions. However, many of these architectures are unique to the myxobacteria, an observation that suggests that this group exploits sophisticated and novel signal transduction systems. Phylogenetic reconstruction using the kinase domains revealed many orthologous sequence pairs and a huge number of gene duplications that probably occurred after speciation. Furthermore, studies of the microsynteny in the ELK-encoding regions reveal only low levels of synteny among Myxococcus xanthus, Plesiocystis pacifica, and Sorangium cellulosum. However, extensive similarities between M. xanthus, Stigmatella aurantiaca, and 3 Anaeromyxobacter strains were observed, indicating that they share regulatory pathways involving various ELKs.

Transcription in the prpC-yloQ region in Bacillus subtilis

The prpC and prkC genes encode two proteins with opposing activities, eukaryotic-like protein phosphatase and protein kinase involved in the sporulation and biofilm formation processes. The prpC gene overlaps 3 bp of the prkC gene; prkC is followed by the essential gene, yloQ. The organisation of the prpC, prkC and yloQ genes is conserved among several Gram-positive bacteria. In this work, we have found two promoters which function within the region of these genes and we also described conditions in which these promoters become activated. One promoter appears to be located upstream of prpC resulting in transcription of both prpC and prkC, as well as the yloQ gene. A second promoter is located 7 bp upstream of the start codon of yloQ. The yloQ promoter was activated during ethanol stress, but was sigmaB-independent. We also showed that prpC, prkC and yloQ genes form an operon.

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.

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

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.

PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis.

Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles. Here we focus on the catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis. PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth.

Protein PknE, a novel transmembrane eukaryotic-like serine/threonine kinase from Mycobacterium tuberculosis

Protein PknE from Mycobacterium tuberculosis has been overproduced and purified, and its biochemical properties have been analyzed. This protein is shown to be a eukaryotic-like (Hanks’-type) protein kinase with a structural organization similar to that of membrane-bound eukaryotic sensor serine/threonine kinases. It consists of a N-terminal catalytic domain located in the cytoplasm, linked via a single transmembrane-spanning region to an extracellular C-terminal domain. The full-length enzyme, as well as the cytosolic domain alone, can autophosphorylate on serine and threonine residues. Such autokinase activity requires the presence of a lysine residue at position 45 in subdomain II, which is known to be essential also for eukaryotic kinase activity. Involvement of PknE in the transduction of external signals into the cytosol of bacteria is proposed.

Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes.

PrkC was shown to be a eukaryotic-like (Hanks-type) protein kinase from Bacillus subtilis with a structural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF beta or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane-spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co-transcribed with prkC, is also required for normal biofilm and spore formation.

Components of Eukaryotic-like Protein Signaling Pathways inMycobacterium tuberculosis

Eukaryotic-like protein kinases have been identified recently in several prokaryotes by comparative studies of DNA sequences and Western blotting techniques using antiphosphoprotein antibodies. Examination of the Mycobacterium tuberculosis genome by means of PCR amplification with consensus primers, Southern hybridization, and comparative analysis of DNA sequences with genomic databases revealed the existence of at least seven eukaryotic-like protein kinases in this pathogen. In addition, we report the biochemical identification of phosphorylated proteins in M. tuberculosis. Taken together, these findings show that M. tuberculosis possesses elements of cell signaling similar to those observed in eukaryotic organisms. We suggest that some of these processes may play roles in the pathogenesis of M. tuberculosis.