ModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in Escherichia coli. ModE is activated by binding molybdate and acts as both an activator and a repressor. By genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as morA and morB, located on antisense strands of yghW and torY, respectively. The morA gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the morA 5′ untranslated region exhibited an inhibitory influence on colony formation of E. coli K-12.
The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of “constitutive promoters” that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent “inducible promoters”. One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature.
The decrease in overall translation in stationary-phase Escherichia coli is accompanied with the formation of functionally inactive 100S ribosomes mediated by the ribosome modulation factor (RMF). At present, however, little is known regarding the regulation of stationary-phase-coupled RMF expression. In the course of a systematic screening of regulation targets of DNA-binding transcription factors from E. coli, we realized that CRP (cyclic AMP [cAMP] receptor protein), the global regulator for carbon source utilization, participates in regulation of some ribosomal protein genes, including the rmf gene. In this study, we carried out detailed analysis of the regulation of the RMF gene by cAMP-CRP. The cAMP-dependent binding of CRP to the rmf gene promoter was confirmed by gel shift and DNase I footprinting assays. By using a reporter assay system, the expression level of RMF was found to decrease in the crp knockout mutant, indicating the involvement of CRP as an activator of the rmf promoter. In good agreement with the reduction of rmf promoter activity, we observed decreases in RMF production and 100S ribosome dimerization in the absence of CRP. Taken together, we propose that CRP regulates transcription activation of the rmf gene for formation of 100S ribosome dimers. Physiological roles of CRP involvement in RMF production are discussed.
Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (non-initiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.
Parkinson's disease genes PINK1 and parkin encode kinase and ubiquitin ligase, respectively. The gene products PINK1 and Parkin are implicated in mitochondrial autophagy, or mitophagy. Upon the loss of mitochondrial membrane potential (ΔΨm), cytosolic Parkin is recruited to the mitochondria by PINK1 through an uncharacterised mechanism – an initial step triggering sequential events in mitophagy. This study reports that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarisation of ΔΨm. The introduction of mutations at Ser65 suggests that phosphorylation of Ser65 is required not only for the efficient translocation of Parkin, but also for the degradation of mitochondrial proteins in mitophagy. Phosphorylation analysis of Parkin pathogenic mutants also suggests Ser65 phosphorylation is not sufficient for Parkin translocation. Our study partly uncovers the molecular mechanism underlying the PINK1-dependent mitochondrial translocation and activation of Parkin as an initial step of mitophagy.
The FixJ/LuxR family transcription factor CsgD is a master regulator of biofilm formation in Escherichia coli. Previously, we identified more than 10 transcription factors that participate in regulation of the csgD promoter. After genomic SELEX screening of regulation targets, an uncharacterized TetR-type transcription factor YbjK was found to be involved in regulation of the csgD promoter. In addition, a number of stress-response genes were found to be under the direct control of YbjK. Taken together, we propose to rename it to RcdA (regulator of csgD). One unique feature of RcdA is its mode of DNA binding. Gel shift, DNase-I footprinting, and atomic force microscopic (AFM) analyses indicated that RcdA is a DNA-binding protein with a high level of cooperativity, with which it covers the entire surface of probe DNA through protein–protein interaction and moreover it induces the formation of aggregates of DNA–RcdA complexes.
Biofilm formation; csgD promoter; genomic SELEX; regulation network; transcription factor
Showing convergence with budding yeast mitotic exit network signaling, the LATS1/WARTS kinase phosphorylates the MYPT1 phosphatase to control PLK1 at the G2 DNA damage checkpoint.
In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase–targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage–induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.
Dan is a transcription factor that regulates the ttd operon encoding tartrate dehydratase. During anaerobic conditions, its copy number increases by 100-fold, making Dan an abundant nucleoid-associated protein. However, little is known about the mode of Dan–DNA interaction. To understand its cellular functions, we used single-molecule manipulation and imaging techniques to show that Dan binds cooperatively along DNA, resulting in formation of a rigid periodic nucleoprotein filament that strongly restricts accessibility to DNA. Furthermore, in the presence of physiologic levels of magnesium, these filaments interact with each other to cause global DNA condensation. Overall, these results shed light on the architectural role of Dan in the compaction of Escherichia coli chromosomal DNA under anaerobic conditions. Formation of the nucleoprotein filament provides a basis in understanding how Dan may play roles in both chromosomal DNA protection and gene regulation.
After determination of the whole genome sequence, the research frontier of bacterial molecular genetics has shifted to reveal the genome regulation under stressful conditions in nature. The gene selectivity of RNA polymerase is modulated after interaction with two groups of regulatory proteins, 7 sigma factors and 300 transcription factors. For identification of regulation targets of transcription factors in Escherichia coli, we have developed Genomic SELEX system and subjected to screening the binding sites of these factors on the genome. The number of regulation targets by a single transcription factor was more than those hitherto recognized, ranging up to hundreds of promoters. The number of transcription factors involved in regulation of a single promoter also increased to as many as 30 regulators. The multi-target transcription factors and the multi-factor promoters were assembled into complex networks of transcription regulation. The most complex network was identified in the regulation cascades of transcription of two master regulators for planktonic growth and biofilm formation.
transcription regulation; genome regulation; transcription factor; regulation network; genomic SELEX; Escherichia coli
Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-012-0452-2) contains supplementary material, which is available to authorized users.
Metabolomics; CE-MS; Phosphoproteomics; Lung cancer; Prostate cancer; Tumor microenvironment
An intermediate filament protein, Nestin, is known as a neural stem/progenitor cell marker. It was shown to be required for the survival and self-renewal of neural stem cells according to the phenotypes of Nestin knockout mice. Nestin expression has also been reported in vascular endothelial cells, and we recently reported Nestin expression in proliferating endothelial progenitor cells, but not in mature endothelial cells. Using quantitative phosphoproteome analysis, we studied differences in phosphorylation levels between CNS Nestin in adult neural stem cells and vascular Nestin in adult bone-marrow-derived endothelial progenitor cells. We detected 495 phosphopeptides in the cell lysates of adult CNS stem/progenitor cells and identified 11 significant phosphorylated amino acid residues in the Nestin protein. In contrast, endothelial progenitor cells showed no significant phosphorylation of Nestin. We also measured neoplastic endothelial cells of the mouse brain and identified 13 phosphorylated amino acid residues in the Nestin protein. Among the 11 phosphorylated amino acids of adult CNS Nestin, five (S565, S570, S819, S883, and S886) were CNS Nestin-specific phosphorylation sites. Detection of the CNS-specific phosphorylation sites in Nestin, for example, by a phospho-specific Nestin antibody, may allow the expression of CNS Nestin to be distinguished from vascular Nestin.
Our recently developed rice proteogenomics database (OryzaPG-DB) is the first sustainable resource for rice shotgun-based proteogenomics, providing information on peptides identified in rice protein digested peptides measured by means of liquid chromatography–tandem mass spectrometry (LC–MS/MS), and mapping of the peptides to their genomic origins and the genomic novelty of each peptide. The sequences of the peptides, proteins, cDNAs and genes, and the gene annotations are available for download in FASTA and GFF3 formats, respectively. Further, an annotated visualization of the gene models, corresponding peptides, and genomic novelty is available for each gene, and MS/MS spectra are available for each peptide. In this article, we discuss the utilization of OryzaPG-DB and report on its development, recent content expansions, and newly added features in the current version (OryzaPG-DB v1.1).
proteomics; proteogenomics; bioinformatics; rice; LC–MS/MS; database
Endothelin plays important roles in various physiological functions including vascular constriction. Recent studies reported that the endothelin receptors ETA and ETB are highly expressed in lung and skin tumor tissues. In contrast, there are few reports on endothelin signalling in the proliferation of head and neck cancer. We found that both ETA and ETB endothelin receptors were overexpressed in tumor cells of tongue cancer samples by immunohistochemistry. ETA and ETB were expressed in cultured lingual and esophageal squamous cell carcinoma (SCCs) cell lines. When both cultured cell lines were treated with an ETA selective antagonist (BQ123) or an ETB selective antagonist (BQ788), inhibition of cell growth was observed. Similar results were observed when SCCs were treated with specific siRNA for the suppression of ETA or ETB. Furthermore, inhibition of the mitogen-activated protein (MAP) kinase pathway by the treatments with ET receptor antagonists and siRNA was also observed. These results indicate that endothelin signalling may, in part, play important roles in cell growth in SCCs through the MAP kinase pathway.
squamous cell carcinoma; tongue; cellular proliferation; endothelin receptor; MAP kinase signaling pathway
CsgD, the master regulator of biofilm formation, activates the synthesis of curli fimbriae and extracellular polysaccharides in Escherichia coli. To obtain insights into its regulatory role, we have identified a total of 20 novel regulation target genes on the E. coli genome by using chromatin immunoprecipitation (ChIP)-on-chip analysis with a high-density DNA microarray. By DNase I footprinting, the consensus CsgD-binding sequence predicted from a total of 18 target sites was found to include AAAAGNG(N2)AAAWW. After a promoter-lacZ fusion assay, the CsgD targets were classified into two groups: group I genes, such as fliE and yhbT, are repressed by CsgD, while group II genes, including yccT and adrA, are activated by CsgD. The fliE and fliEFGH operons for flagellum formation are directly repressed by CsgD, while CsgD activates the adrA gene, which encodes an enzyme for synthesis of cyclic di-GMP, a bacterial second messenger, which in turn inhibits flagellum production and rotation. Taking these findings together, we propose that the cell motility for planktonic growth is repressed by CsgD, thereby promoting the switch to biofilm formation.
Escherichia coli MntR protein is the Mn2+-responsive transcriptional repressor of the MntH manganese transporter. We have used chromatin immunoprecipitation to determine the distribution of Mn2+-MntR across the entire E. coli chromosome in vivo, and we report that MntR binds to only four targets, adjacent to the mntH, mntR, yebN, and dps genes. Unexpectedly, we found that dps expression is directly repressed by Mn2+-MntR.
Cra (catabolite repressor activator) is a global regulator of the genes for carbon metabolism in Escherichia coli. To gain insights into the regulatory roles of Cra, attempts were made to identify the whole set of regulation targets using an improved genomic SELEX (systematic evolution of ligands by exponential enrichment) system. Surprisingly, a total of 164 binding sites were identified for Cra, 144 (88%) of which were newly identified. The majority of known targets were included in the SELEX chip pattern. The promoters examined by the lacZ reporter assay in vivo were all regulated by Cra. These two lines of evidence indicate that a total of as many as 178 promoters are under the control of Cra. The majority of Cra targets are the genes coding for the enzymes involved in central carbon metabolism, covering all the genes for the enzymes involved in glycolysis and metabolism downstream of glycolysis, including the tricarboxylic acid (TCA) cycle and aerobic respiration. Taken together, we propose that Cra plays a key role in balancing the levels of the enzymes for carbon metabolism.
CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.
Proteogenomics aims to utilize experimental proteome information for refinement of genome annotation. Since mass spectrometry-based shotgun proteomics approaches provide large-scale peptide sequencing data with high throughput, a data repository for shotgun proteogenomics would represent a valuable source of gene expression evidence at the translational level for genome re-annotation.
Here, we present OryzaPG-DB, a rice proteome database based on shotgun proteogenomics, which incorporates the genomic features of experimental shotgun proteomics data. This version of the database was created from the results of 27 nanoLC-MS/MS runs on a hybrid ion trap-orbitrap mass spectrometer, which offers high accuracy for analyzing tryptic digests from undifferentiated cultured rice cells. Peptides were identified by searching the product ion spectra against the protein, cDNA, transcript and genome databases from Michigan State University, and were mapped to the rice genome. Approximately 3200 genes were covered by these peptides and 40 of them contained novel genomic features. Users can search, download or navigate the database per chromosome, gene, protein, cDNA or transcript and download the updated annotations in standard GFF3 format, with visualization in PNG format. In addition, the database scheme of OryzaPG was designed to be generic and can be reused to host similar proteogenomic information for other species. OryzaPG is the first proteogenomics-based database of the rice proteome, providing peptide-based expression profiles, together with the corresponding genomic origin, including the annotation of novelty for each peptide.
The OryzaPG database was constructed and is freely available at http://oryzapg.iab.keio.ac.jp/.
Following recent advances in high-throughput mass spectrometry (MS)–based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-omics data. First, new phosphoproteome data on yeast cells were obtained by MS-based proteomics and unified with publicly available yeast phosphoproteome data. This revealed that nearly 60% of ∼6,000 yeast genes encode phosphoproteins. We mapped these unified phosphoproteome data on a yeast protein–protein interaction (PPI) network with other yeast multi-omics datasets containing information about proteome abundance, proteome disorders, literature-derived signaling reactomes, and in vitro substratomes of kinases. In the phospho-PPI, phosphoproteins had more interacting partners than nonphosphoproteins, implying that a large fraction of intracellular protein interaction patterns (including those of protein complex formation) is affected by reversible and alternative phosphorylation reactions. Although highly abundant or unstructured proteins have a high chance of both interacting with other proteins and being phosphorylated within cells, the difference between the number counts of interacting partners of phosphoproteins and nonphosphoproteins was significant independently of protein abundance and disorder level. Moreover, analysis of the phospho-PPI and yeast signaling reactome data suggested that co-phosphorylation of interacting proteins by single kinases is common within cells. These multi-omics analyses illuminate how wide-ranging intracellular phosphorylation events and the diversity of physical protein interactions are largely affected by each other.
To date, high-throughput proteome technologies have revealed that hundreds to thousands of proteins in each of many organisms are phosphorylated under the appropriate environmental conditions. A critical role of phosphorylation is control of protein signaling. However, only a fraction of the identified phosphoproteins participate in currently known protein signaling pathways, and the biological relevance of the remainder is unclear. This has raised the question of whether phosphorylation has other major roles. In this study, we identified new phosphoproteins in budding yeast by mass spectrometry and unified these new data with publicly available phosphoprotein data. We then performed an integrative data-mining of large-scale yeast phosphoproteins and protein–protein interactions (complex formation) by an exhaustive analysis that incorporated yeast protein information from several other sources. The phosphoproteome data integration surprisingly showed that nearly 60% of yeast genes encode phosphoproteins, and the subsequent data-mining analysis derived two models interpreting the mutual intracellular effects of large-scale protein phosphorylation and binding interaction. Biological interpretations of both large-scale intracellular phosphorylation and the topology of protein interaction networks are highly relevant to modern biology. This study sheds light on how in vivo protein pathways are supported by a combination of protein modification and molecular dynamics.
The histone‐modifying enzymes histone deacetylase (HDAC) and histone acetyltransferase (HAT) control gene transcriptional activation and repression in human malignancies.
To analyse the expression of HDAC/HAT‐associated molecules such as HDAC1, CREB‐binding protein (CBP) and p300 in human colorectal carcinomas, and investigate the relationship between their expression levels and clinicopathological parameters.
Expression levels of HDAC1, CBP, and p300 in human colorectal cancer were investigated by immunohistochemistry. In situ hybridisation (ISH) and reverse transcription (RT)‐PCR analyses were also carried out to confirm mRNA expression levels of these genes. Immunoreactivity was evaluated semi‐quantitatively using a staining index (SI). The relationships between the SIs and clinicopathological findings were analysed and survival curves were calculated using the Kaplan–Meier method and log‐rank tests.
The mean SIs for HDAC1, CBP, and p300 in this series of tumours were much higher than those in normal colonic mucosa. The presence of HDAC1 and CBP mRNAs on colorectal carcinoma cells as well as normal epithelial cells was confirmed by ISH analysis. A marked increase in p300 mRNA levels was detected in a majority of cases by RT‐PCR. Among the patients with colorectal cancer, overexpression of p300 (SI>11.9) correlated with a poor prognosis, whereas high CBP expression levels (SI>16.6) indicated long‐term survival.
Results showed the up‐regulation of these three histone‐modifying molecules in this series of colorectal cancers and suggested that monitoring of CBP and p300 may assist prediction of the prognosis in patients with colorectal adenocarcinoma.
Compared to other health-care workers, dental health-care workers come in close contact with patients and use a variety of sharp and high-speed rotating instruments. It is important to understand the characteristics of the occupational accidents that occur. We reviewed incident reports from April 1, 2005, to March 31, 2010, at Matsumoto Dental University Hospital. In addition, questionnaires dealing with identification of occupational safety issues, especially splash exposures, were conducted for dentists, dental hygienists, and nurses. Thirty-two occupational injuries were reported during the study period, including 23 sharp instrument injuries (71.9%), 6 splash exposures (18.8%), and 3 others. Of the six splash exposures, only two cases involved potential contamination with blood or other potentially infectious patient material. Of the 66 workers who experienced sharps injuries, 20 workers (30.3%, 20/66) reported them to the hospital work safety team. The questionnaire revealed high incident of splash exposures and conjunctiva exposures: 87.9% (51/58) and 60.3% (35/58) in dentists and 88.6% (39/44) and 61.4% (27/44) in dental hygienists. The compliance rate for routine use of protective eyewear was 60.3% (35/58) for dentists and 34.1% (15/44) for hygienists. Of the presented informational items included in the questionnaire, those that strongly persuaded respondents to use protective eyewear were ‘splatters from the patient’s mouth contain blood’ (90%, 99/110) and ‘dental operations at our clinic are performed based only on a questionnaire without serious examinations for HBV, HCV, and HIV’ (71.8%, 79/110). The reason of low compliance of protective eyewear among dentists might relate to fine dental procedures. Appropriate information is important for the motive of wearing personal protective equipment, and an early educational program may have a potential to increase compliance with the use of that equipment.
splash exposure; occupational safety; sharps injury; personal protective equipment
Plant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable.
To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response.
These results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.
Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches.
We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC) method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF). We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm) tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR) signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled.
The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics-based network, our method will be a complementary strategy to explore new components of protein signaling pathways in combination with previous methods (including software) of predicting direct kinase-substrate relationships.
Bone formation below the crown of mandibular horizontal incompletely impacted third molar is frequently seen in the middle-aged and elderly. The phenomenon shows lamina dura loss without radiolucency and we hypothesized the participation of mature enamel without any influence on the environmental oral status. In order to investigate the characteristics of the phenomenon based on the presence/absence of the lamina dura and radiolucency below the crown, we studied the relationship between 58 men and 43 women with a lamina dura without radiolucency, 12 men and 8 women without a lamina dura with radiolucency, 34 men and 16 women without a lamina dura without radiolucency, and the status of teeth in the ipsilateral mandible. Subjects without a lamina dura without radiolucency were significantly older than those with a lamina dura without radiolucency in both men (P < 0.0001) and women (P <0.01), indicating different chronological causes. Men without lamina dura with radiolucency showed significantly more tooth loss than those with a lamina dura without radiolucency (P < 0.00001) and those without a lamina dura without radiolucency (P < 0.0001), indicating the influence of poor oral health. Thus, the phenomenon without a lamina dura without radiolucency may show the clinical importance of bone formation in the elderly.
bone formation; lamina dura; enamel; elderly