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1.
Microb Physiol ; 31(2): 146-162, 2021.
Article in English | MEDLINE | ID: mdl-34058747

ABSTRACT

Heterotrophic Proteobacteria are versatile opportunists that have been extensively studied as model organisms in the laboratory, as both pathogens and beneficial symbionts of plants and animals, and as ubiquitous organisms found free-living in many environments. Succeeding in these niches requires an ability to persist for potentially long periods of time in growth-arrested states when essential nutrients become limiting. The tendency of these bacteria to grow in dense biofilm communities frequently leads to the development of steep nutrient gradients and deprivation of interior cells even when the environment is nutrient rich. Surviving within host environments also likely requires tolerating growth arrest due to the host limiting access to nutrients and transitioning between hosts may require a period of survival in a nutrient-poor environment. Interventions to maximise plant-beneficial activities and minimise infections by bacteria will require a better understanding of metabolic and regulatory networks that contribute to starvation survival, and how these networks function in diverse organisms. Here we focus on carbon starvation as a growth-arresting condition that limits availability not only of substrates for biosynthesis but also of energy for ongoing maintenance of the electrochemical gradient across the cell envelope and cellular integrity. We first review models for studying bacterial starvation and known strategies that contribute to starvation survival. We then present the results of a survey of carbon starvation survival strategies and outcomes in ten bacterial strains, including representatives from the orders Enterobacterales and Pseudomonadales (both Gammaproteobacteria) and Burkholderiales (Betaproteobacteria). Finally, we examine differences in gene content between the highest and lowest survivors to identify metabolic and regulatory adaptations that may contribute to differences in starvation survival.


Subject(s)
Gammaproteobacteria , Proteobacteria , Adaptation, Physiological , Animals , Bacteria , Heterotrophic Processes
2.
EMBO Rep ; 16(1): 71-8, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25452588

ABSTRACT

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process.


Subject(s)
Caenorhabditis elegans/genetics , Carrier Proteins/metabolism , Drosophila melanogaster/genetics , GTP-Binding Proteins/metabolism , Nonsense Mediated mRNA Decay/physiology , Nuclear Proteins/metabolism , Animals , Animals, Genetically Modified , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Egg Proteins/genetics , Egg Proteins/metabolism , Embryo, Nonmammalian , Evolution, Molecular , GTP-Binding Proteins/genetics , Gene Knockdown Techniques , HeLa Cells , Humans , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Nuclear Proteins/genetics , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
3.
Proc Natl Acad Sci U S A ; 111(51): 18267-72, 2014 Dec 23.
Article in English | MEDLINE | ID: mdl-25489104

ABSTRACT

Ectopic expression of dual-specificity phosphatase 5 (DUSP5), an inducible mitogen-activated protein (MAP) kinase phosphatase, specifically inactivates and anchors extracellular signal-regulated kinase (ERK)1/2 in the nucleus. However, the role of endogenous DUSP5 in regulating the outcome of Ras/ERK kinase signaling under normal and pathological conditions is unknown. Here we report that mice lacking DUSP5 show a greatly increased sensitivity to mutant Harvey-Ras (HRas(Q61L))-driven papilloma formation in the 7,12-Dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) model of skin carcinogenesis. Furthermore, mouse embryo fibroblasts (MEFs) from DUSP5(-/-) mice show increased levels of nuclear phospho-ERK immediately after TPA stimulation and fail to accumulate total ERK in the nucleus compared with DUSP5(+/+) cells. Surprisingly, a microarray analysis reveals that only a small number of Ras/ERK-dependent TPA-responsive transcripts are up-regulated on deletion of DUSP5 in MEFs and mouse skin. The most up-regulated gene on DUSP5 loss encodes SerpinB2, an inhibitor of extracellular urokinase plasminogen activator and deletion of DUSP5 acts synergistically with mutant HRas(Q61L) and TPA to activate ERK-dependent SerpinB2 expression at the transcriptional level. SerpinB2 has previously been implicated as a mediator of DMBA/TPA-induced skin carcinogenesis. By analyzing DUSP5(-/-), SerpinB2(-/-) double knockout mice, we demonstrate that deletion of SerpinB2 abrogates the increased sensitivity to papilloma formation seen on DUSP5 deletion. We conclude that DUSP5 performs a key nonredundant role in regulating nuclear ERK activation, localization, and gene expression. Furthermore, our results suggest an in vivo role for DUSP5 as a tumor suppressor by modulating the oncogenic potential of activated Ras in the epidermis.


Subject(s)
Cell Nucleus/enzymology , Dual-Specificity Phosphatases/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Genes, ras , Plasminogen Activator Inhibitor 2/metabolism , Skin Neoplasms/prevention & control , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Dual-Specificity Phosphatases/genetics , Mice , Mice, Knockout , Signal Transduction , Tetradecanoylphorbol Acetate/toxicity
4.
J Biol Chem ; 286(44): 38018-38026, 2011 Nov 04.
Article in English | MEDLINE | ID: mdl-21908610

ABSTRACT

MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Dual-Specificity Phosphatases/metabolism , Mitogen-Activated Protein Kinase Phosphatases/metabolism , Amino Acid Motifs , Animals , COS Cells , Catalysis , Catalytic Domain , Chlorocebus aethiops , Humans , Mice , Phosphorylation , Recombinant Proteins/chemistry , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
5.
Biochem J ; 412(2): 287-98, 2008 Jun 01.
Article in English | MEDLINE | ID: mdl-18321244

ABSTRACT

DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling.


Subject(s)
Dual Specificity Phosphatase 6/metabolism , Fibroblast Growth Factors/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Promoter Regions, Genetic , Proto-Oncogene Proteins c-ets/metabolism , Signal Transduction/physiology , Animals , Base Sequence , Binding Sites , Cell Line , Dual Specificity Phosphatase 6/genetics , Enzyme Activation , Feedback, Physiological/physiology , Gene Expression Regulation , Humans , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-ets/genetics , Sequence Alignment , Transgenes
6.
Oncogene ; 23(30): 5138-50, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15133492

ABSTRACT

The E2F family of transcription factors play an important role in regulating cell cycle progression. We report here the characterization and functional properties of a new member of the human E2F family, referred to as E2F-7. E2F-7 has two separate DNA-binding domains, a feature that distinguishes E2F-7 from other mammalian E2F proteins, but resembling the organization of recently isolated E2F-like proteins from Arabidopsis. E2F-7 binds to DNA independently of a DP partner and delays cell cycle progression. Interestingly, E2F-7 modulates the transcription properties of other E2F proteins. A mutational analysis indicates that the integrity of both DNA-binding domains is required for cell cycle delay and transcriptional modulation. Biochemical results and protein modelling studies suggest that in binding to DNA interactions occur between the two DNA-binding domains, most probably as a homodimer, thereby mimicking the organization of an E2F/DP heterodimer. These structural and functional properties of E2F-7 imply a unique role in regulating cellular proliferation.


Subject(s)
DNA-Binding Proteins/genetics , Protein Structure, Tertiary , Repressor Proteins/genetics , Transcription Factors/genetics , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Cell Cycle , Cell Line, Tumor , Cell Nucleus/chemistry , Conserved Sequence , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , E2F7 Transcription Factor , Genes, Reporter , HeLa Cells , Humans , Luciferases/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Precipitin Tests , Protein Binding , Repressor Proteins/chemistry , Repressor Proteins/isolation & purification , Repressor Proteins/metabolism , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/isolation & purification , Transcription Factors/metabolism
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