RESUMO
Mitogen-activated protein kinase pathways are implicated in the regulation of cell differentiation, although their precise roles in many differentiation programs remain elusive. The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade has been proposed to both promote and inhibit adipogenesis. Here, we titrate expression of the molecular scaffold kinase suppressor of Ras 1 (KSR1) to regulate signaling through the Raf/MEK/ERK/p90 ribosomal S6 kinase (RSK) kinase cascade and show how it determines adipogenic potential. Deletion of KSR1 prevents adipogenesis in vitro, which can be rescued by introduction of low levels of KSR1. Appropriate levels of KSR1 coordinate ERK and RSK activation with C/EBPbeta synthesis leading to the phosphorylation and stabilization of C/EBPbeta at the precise moment it is required within the adipogenic program. Elevated levels of KSR1 expression, previously shown to enhance cell proliferation, promote high, sustained ERK activation that phosphorylates and inhibits peroxisome proliferator-activated receptor gamma, inhibiting adipogenesis. Titration of KSR1 expression reveals how a molecular scaffold can modulate the intensity and duration of signaling emanating from a single pathway to dictate cell fate.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Proteínas Quinases/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proliferação de Células , Tamanho Celular , Células Cultivadas , Ativação Enzimática , Fibroblastos , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Mitose , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Fosforilação , Filogenia , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
While scaffold proteins are thought to be key components of signaling pathways, their exact function is unknown. By preassembling multiple components of signaling cascades, scaffolds are predicted to influence the efficiency and/or specificity of signaling events. Here we analyze a potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal even though ERK kinase activation was attenuated to a degree sufficient to block T-cell activation and inhibit tumor development. Consistent with its role as a scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK were lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that is not required for but enhances signaling via the Ras/MAPK signaling pathway.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular , Ativação Enzimática , Fibroblastos , Citometria de Fluxo , Deleção de Genes , Marcação de Genes , Interferon gama/análise , Interleucina-2/metabolismo , Interleucina-4/análise , Ativação Linfocitária , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Peso Molecular , Fenótipo , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is a small accessory protein that regulates nuclear import of the viral preintegration complex and facilitates infection of nondividing cells, such as macrophages. Studies demonstrated that a fraction of Vpr molecules is phosphorylated in the virions and in HIV-1-infected cells, but the role of phosphorylation in nuclear import activity of Vpr has not been established. We found that Vpr is phosphorylated predominantly on the serine residue in position 79, and mutations affecting Vpr phosphorylation significantly attenuated viral replication in macrophages, but not in activated T lymphocytes or cell lines. The replication defect was mapped by polymerase chain reaction analysis to the step of nuclear import. These results suggest that phosphorylation of Vpr regulates its activity in the nuclear import of the HIV-1 preintegration complex.
Assuntos
Núcleo Celular/virologia , Produtos do Gene vpr/metabolismo , HIV-1/fisiologia , Macrófagos/virologia , Sequência de Aminoácidos , Linhagem Celular , Produtos do Gene vpr/química , Humanos , Dados de Sequência Molecular , Fosforilação , Homologia de Sequência de Aminoácidos , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND: The Adenosine triphosphate (ATP) bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. METHODS: Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface) of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. RESULTS: When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU) for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. CONCLUSIONS: This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event.
Assuntos
Trifosfato de Adenosina/análise , Bacillus anthracis/isolamento & purificação , Medições Luminescentes , Bioterrorismo , Contagem de Colônia Microbiana , Contaminação de Equipamentos , Esporos Bacterianos/isolamento & purificaçãoRESUMO
Kinase suppressor of ras 1 (KSR1) is a molecular scaffold of the Raf/MEK/extracellular signal-regulated kinase (ERK) cascade that enhances oncogenic Ras signaling. Here we show KSR1-dependent, but ERK-independent, regulation of metabolic capacity is mediated through the expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and estrogen-related receptor α (ERRα). This KSR1-regulated pathway is essential for the transformation of cells by oncogenic Ras. In mouse embryo fibroblasts (MEFs) expressing H-Ras(V12), ectopic PGC1α was sufficient to rescue ERRα expression, metabolic capacity, and anchorage-independent growth in the absence of KSR1. The ability of PGC1α to promote anchorage-independent growth required interaction with ERRα, and treatment with an inhibitor of ERRα impeded anchorage-independent growth. In contrast to PGC1α, the expression of constitutively active ERRα (CA-ERRα) was sufficient to enhance metabolic capacity but not anchorage-independent growth in the absence of KSR1. These data reveal KSR1-dependent control of PGC1α- and ERRα-dependent pathways that are necessary and sufficient for signaling by oncogenic H-Ras(V12) to regulate metabolism and anchorage-independent growth, providing novel targets for therapeutic intervention.
Assuntos
Genes ras , Proteínas Quinases/metabolismo , Receptores de Estrogênio/metabolismo , Transativadores/metabolismo , Proteínas ras/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Estrogênio/genética , Tiazóis/farmacologia , Transativadores/genética , Fatores de Transcrição , Proteínas ras/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Kinase suppressors of Ras 1 and 2 (KSR1 and KSR2) function as molecular scaffolds to potently regulate the MAP kinases ERK1/2 and affect multiple cell fates. Here we show that KSR2 interacts with and modulates the activity of AMPK. KSR2 regulates AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryonic fibroblasts and glycolysis in a neuronal cell line. Disruption of KSR2 in vivo impairs AMPK-regulated processes affecting fatty acid oxidation and thermogenesis to cause obesity. Despite their increased adiposity, ksr2(-/-) mice are hypophagic and hyperactive but expend less energy than wild-type mice. In addition, hyperinsulinemic-euglycemic clamp studies reveal that ksr2(-/-) mice are profoundly insulin resistant. The expression of genes mediating oxidative phosphorylation is also downregulated in the adipose tissue of ksr2(-/-) mice. These data demonstrate that ksr2(-/-) mice are highly efficient in conserving energy, revealing a novel role for KSR2 in AMPK-mediated regulation of energy metabolism.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético , Resistência à Insulina , Proteínas Serina-Treonina Quinases/metabolismo , Tecido Adiposo/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Resistência à Insulina/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Obesidade/etiologia , Obesidade/metabolismo , Oxirredução , Fosforilação Oxidativa , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Termogênese/fisiologiaRESUMO
KSR (kinase suppressor of Ras) has been proposed as a molecular scaffold regulating the Raf/MEK/ERK kinase cascade. KSR is phosphorylated on multiple phosphorylation sites by associated kinases. To identify potential mechanisms used by KSR to regulate ERK activation, green fluorescent protein was fused to intact and mutated KSR constructs lacking specific phosphorylation sites, and the subcellular distribution of each construct was observed in live cells. Mutation of a subset of KSR phosphorylation sites caused the redistribution of KSR to the nucleus. To determine whether intact KSR is normally imported to the nucleus, REF-52 fibroblasts expressing KSR were treated with 10 nm leptomycin B, which inhibits Crm1-dependent nuclear export. KSR accumulated in the nucleus within 2 h of treatment with leptomycin B, suggesting that KSR cycles continuously through the nucleus. Nuclear import of KSR was blocked by mutations that inhibit the interaction of KSR with MEK. Coexpression of fluorescent forms of KSR and MEK in cells revealed that each protein promoted the localization of the other in the cytoplasm. These data indicate that the subcellular distribution of KSR is dynamically regulated through phosphorylation and MEK interaction in a manner that may affect signaling through ERK.