RESUMO
Pseudomonas aeruginosa causes acute and chronic human infections and is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. We previously determined that the sn-glycerol-3-phosphate dehydrogenase encoded by glpD plays a larger role in P. aeruginosa physiology beyond its role in glycerol metabolism. To better understand the effect of a glpD mutation on P. aeruginosa physiology we compared the transcriptomes of P. aeruginosa strain PAO1 and the PAO1ΔglpD mutant using RNA-seq analysis. We determined that a null mutation of glpD significantly altered amino acid metabolism in P. aeruginosa and affected the production of intermediates that are channelled into the tricarboxylic acid cycle. Moreover, the loss of glpD induced a general stress response mediated by RpoS in P. aeruginosa. Several other phenotypes observed for the P. aeruginosa glpD mutant include increased persister cell formation, reduced extracellular ATP accumulation and increased heat output. Taken together, these findings implicate sn-glycerol-3-phosphate dehydrogenase as a key player in energy metabolism in P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Metabolismo Energético/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Glicerolfosfato Desidrogenase/metabolismo , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Deleção de Genes , Glicerolfosfato Desidrogenase/genética , Redes e Vias Metabólicas/genética , Fenótipo , Pseudomonas aeruginosa/metabolismo , Estresse Fisiológico/genéticaRESUMO
Signaling through Ras GTPases controls the activity of many transcription factors including CCAAT/enhancer-binding protein (C/EBPbeta), which regulates oncogenic H-Ras(V12)-induced senescence and growth arrest. Here we report that C/EBPbeta (LAP) DNA binding is inhibited by N-terminal sequences and derepressed by oncogenic Ras signaling. Sequence and mutational analyses showed that auto-repression involves two LXXLF (phiXXphiphi)-like motifs (LX1 and LX2) and a third element, auto-inhibitory domain (AID), located within conserved region CR5. LX1 is a critical component of the transactivation domain and has been shown to mediate C/EBPbeta binding to the TAZ2 region of p300/CREB-binding protein coactivators. C/EBPbeta auto-repression also involves a C-terminal regulatory domain (CRD) adjacent to the leucine zipper. CRD contains a third phiXXphiphi motif (LX3) and a short sequence, KQL, which has similarity to a region in the protein-binding site of TAZ2. The C/EBPbeta N- and C-terminal domains physically associate in a manner that requires the basic region and CRD. We propose a model in which the regulatory sequences form a hydrophobic core that reciprocally inhibits DNA binding and transactivation. We also suggest a mechanism for C/EBPbeta derepression involving several recently identified modifications within AID and CRD. Finally, we show that association of activated C/EBPbeta with p300/CREB-binding protein requires the LX2 and AID auto-inhibitory elements. Thus, the N-terminal regulatory elements have dual roles in auto-inhibition and coactivator binding.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , DNA/química , Elementos Facilitadores Genéticos , Fatores de Transcrição de p300-CBP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais , Transcrição GênicaRESUMO
CCAAT/enhancer binding protein beta (C/EBPbeta) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPbeta is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPbeta in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPbeta activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPbeta, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPbeta as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPbeta on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular/fisiologia , Transformação Celular Neoplásica , Genes ras , Transdução de Sinais , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Quinases relacionadas a CDC2 e CDC28/metabolismo , Quinase 2 Dependente de Ciclina , Inibidores Enzimáticos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Alinhamento de Sequência , Serina/metabolismo , Treonina/metabolismo , Proteínas ras/genéticaRESUMO
Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fase S , Células 3T3 , Animais , Bromodesoxiuridina/farmacologia , Proteínas de Ciclo Celular , Divisão Celular , Células Cultivadas , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Deleção de Genes , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Mutação , Proteínas Nucleares , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Proteína de Replicação C , Fatores de Tempo , Fatores de Transcrição , TransfecçãoRESUMO
We developed an effective and rapid assay to detect both bio-energetic and envelope permeability (BEEP) alterations of Pseudomonas aeruginosa. The assay is based on quantification of extracellular ATP in bacterial cultures using luciferase as a reporter. To demonstrate the validity of our assay we conducted a biased screen of a transposon insertion library in P. aeruginosa strain PAO1 in order to expedite the isolation of mutants with defects in bioenergetic pathways. We successfully isolated insertion mutants that were reduced for extracellular ATP accumulation and identified the corresponding mutations that caused the phenotype. Most of the genes identified from this analysis were associated with energy metabolism and several appeared to be potentially novel bioenergetic targets. In addition, we show that treatment of P. aeruginosa strain PAO1 with antibiotics that disrupt the bacterial cell envelope leads to greater extracellular ATP accumulation. In summary, increases in extracellular ATP accumulation above wild type levels indicated a perturbation of membrane permeability while decreases in extracellular ATP accumulation indicated defects in bioenergetics.
Assuntos
Membrana Celular/fisiologia , Metabolismo Energético , Pseudomonas aeruginosa/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Membrana Celular/química , Elementos de DNA Transponíveis , Luciferases , Mutagênese , Permeabilidade , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologiaRESUMO
The bZIP transcription factor C/EBPbeta is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPbeta signaling, we investigated C/EBPbeta activation by oncogenic Ras. We show that C/EBPbeta DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK-p90(RSK) cascade. Inducible phosphorylation by RSK on Ser273 in the leucine zipper was required for DNA binding. In addition, three other modifications (phosphorylation on Tyr109 [p-Tyr109], p-Ser111, and monomethylation of Arg114 [me-Arg114]) within an N-terminal autoinhibitory domain were important for Ras-induced C/EBPbeta activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g<-->e' salt bridge with Lys268 that increases attractive electrostatic interactions between paired leucine zippers and promotes homodimerization. Mutating Ser273 to Ala or Lys268 to Glu decreased C/EBPbeta homodimer formation, whereas heterodimerization with C/EBPgamma was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPbeta in Ras(V12)-expressing fibroblasts and decreased binding to target cell cycle genes, while a phosphomimetic substitution (S273D) maintained growth arrest function. Our findings identify four novel C/EBPbeta-activating modifications, including RSK-mediated phosphorylation of a bifunctional residue in the leucine zipper that regulates DNA binding and homodimerization and thereby promotes cell cycle arrest.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular/fisiologia , DNA/metabolismo , Zíper de Leucina , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular , DNA/genética , Substâncias de Crescimento/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Alinhamento de Sequência , Ativação Transcricional , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
C/EBPbeta is a member of the CCAAT/enhancer binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of various proinflammatory genes, including IL-6 and MCP-1. Serine 64 in the transactivation domain of C/EBPbeta has recently been identified as a Ras-induced phosphoacceptor site. The integrity of serine 64 along with threonine 189 is important for the Ha-ras(V12)-induced transformation of NIH3T3 cells, however no target genes dependent upon serine 64 for their expression have been reported. In order to evaluate a potential role of serine 64 in C/EBPbeta-regulated cytokine expression, we expressed a form of C/EBPbeta with an alanine substitution at serine 64 (C/EBPbeta(S64A)) in P388 murine B lymphoblasts, which lack endogenous C/EBPbeta expression and are normally unresponsive to LPS for expression of IL-6 and MCP-1. In comparison to wild type C/EBPbeta, which robustly supports the LPS-induced expression of IL-6 and MCP-1, C/EBPbeta(S64A) was severely impaired in its ability to support the LPS-induced transcription of IL-6 and MCP-1. Furthermore, LPS stimulation increased the level of phosphorylation detected at serine 64. Thus, serine 64, probably through its phosphorylation, is a critical determinant of C/EBPbeta activity in the transcription of IL-6 and MCP-1.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Quimiocina CCL2/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/imunologia , Serina/metabolismo , Transcrição Gênica , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular , Quimiocina CCL2/genética , Genes Reporter , Interleucina-6/genética , Camundongos , Fosforilação , Regiões Promotoras GenéticasRESUMO
Transcription factor CCAAT/enhancer-binding protein-beta (C/EBP-beta) regulates a variety of cellular functions in response to exogenous stimuli. We have reported earlier that C/EBP-beta induces gene transcription through a novel interferon (IFN)-response element called gamma-IFN-activated transcriptional element. We show here that IFN-gamma-induced, C/EBP-beta/gamma-IFN-activated transcriptional element-dependent gene expression is regulated by mixed lineage kinases (MLKs), members of the mitogen-activated protein kinase kinase kinase family. MLK3 appears to activate C/EBP-beta in response to IFN-gamma by a mechanism involving decreased phosphorylation of a specific phosphoacceptor residue, Ser(64), within the transactivation domain. Decreased phosphorylation of Ser(64) was independent of IFN-gamma-stimulated ERK1/2 activation and did not require the ERK phosphorylation site Thr(189) located in regulatory domain 2 of C/EBP-beta. Together these studies provide the first evidence that MLK3 is involved in IFN-gamma signaling and identify a novel mechanism of transcriptional activation by IFN-gamma.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação Enzimológica da Expressão Gênica , Interferon gama/metabolismo , MAP Quinase Quinase Quinases/química , MAP Quinase Quinase Quinases/fisiologia , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Ativação Enzimática , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Macrófagos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Modelos Genéticos , Mutação , Fosforilação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Serina/química , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
CCAAT/enhancer-binding proteins (C/EBPs) are basic region leucine zipper (bZIP) transcription factors that regulate cell differentiation, growth, survival, and inflammation. To understand the molecular basis of DNA recognition by the C/EBP family we determined the x-ray structure of a C/EBPalpha bZIP polypeptide bound to its cognate DNA site (A(-5)T(-4)T(-3)G(-2)C(-1)G(1)C(2)A(3)A(4)T(5)) and characterized several basic region mutants. Binding specificity is provided by interactions of basic region residues Arg(289), Asn(292), Ala(295), Val(296), Ser(299), and Arg(300) with DNA bases. A striking feature of the C/EBPalpha protein-DNA interface that distinguishes it from known bZIP-DNA complexes is the central role of Arg(289), which is hydrogen-bonded to base A(3), phosphate, Asn(292) (invariant in bZIPs), and Asn(293). The conformation of Arg(289) is also restricted by Tyr(285). In accordance with the structural model, mutation of Arg(289) or a pair of its interacting partners (Tyr(285) and Asn(293)) abolished C/EBPalpha binding activity. Val(296) (Ala in most other bZIPs) contributes to C/EBPalpha specificity by discriminating against purines at position -3 and imposing steric restraints on the invariant Arg(300). Mutating Val(296) to Ala strongly enhanced C/EBPalpha binding to cAMP response element (CRE) sites while retaining affinity for C/EBP sites. Thus, Arg(289) is essential for formation of the complementary protein-DNA interface, whereas Val(296) functions primarily to restrict interactions with related sequences such as CRE sites rather than specifying binding to C/EBP sites. Our studies also help to explain the phenotypes of mice carrying targeted mutations in the C/EBPalpha bZIP region.