RESUMO
Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase.
Assuntos
Quinases raf/química , Quinases raf/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Dimerização , Ativação Enzimática , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Conformação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Alinhamento de Sequência , Triptofano/metabolismo , Quinases raf/genéticaRESUMO
Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here, we adapted a biosensor for glycolysis, HYlight, for use in Caenorhabditis elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons cell-autonomously perform glycolysis and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function and uncovers unique relationships between neuronal identities and metabolic landscapes in vivo.
Assuntos
Glicólise , Neurônios , Animais , Metabolismo Energético , Caenorhabditis elegans , Plasticidade NeuronalRESUMO
Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.
Assuntos
Técnicas Biossensoriais , Frutose , Glicólise , Análise de Célula Única , Fluorescência , Frutose/análise , Frutosedifosfatos/análise , Humanos , Células Secretoras de Insulina/química , Células Secretoras de Insulina/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Célula Única/métodosRESUMO
Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1 have been proposed to mediate this activation. Using HEK293 cells as a model system, we have recently shown that both Ras and Rap1 are required for cAMP signaling to ERKs. However, cAMP-dependent Ras signaling to ERKs is transient and rapidly terminated by PKA phosphorylation of the Raf isoforms C-Raf and B-Raf. In contrast, cAMP-dependent Rap1 signaling to ERKs and Rap1 is potentiated by PKA. We show that this is due to sustained binding of B-Raf to Rap1. One of the targets of PKA is Rap1 itself, directly phosphorylating Rap1a on serine 180 and Rap1b on serine 179. We show that these phosphorylations create potential binding sites for the adaptor protein 14-3-3 that links Rap1 to the scaffold protein KSR. These results suggest that Rap1 activation of ERKs requires PKA phosphorylation and KSR binding. Because KSR and B-Raf exist as heterodimers within the cell, this binding also brings B-Raf to Rap1, allowing Rap1 to couple to ERKs through B-Raf binding to Rap1 independently of its Ras-binding domain.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lisina-tRNA Ligase/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Animais , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Células HEK293 , Humanos , Lisina-tRNA Ligase/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas rap de Ligação ao GTP/genéticaRESUMO
This report summarizes and highlights the fifth International RASopathies Symposium: When Development and Cancer Intersect, held in Orlando, Florida in July 2017. The RASopathies comprise a recognizable pattern of malformation syndromes that are caused by germ line mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. Because of their common underlying pathogenetic etiology, there is significant overlap in their phenotypic features, which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, gastrointestinal and ocular abnormalities, neurological and neurocognitive issues, and a predisposition to cancer. The RAS pathway is a well-known oncogenic pathway that is commonly found to be activated in somatic malignancies. As in somatic cancers, the RASopathies can be caused by various pathogenetic mechanisms that ultimately impact or alter the normal function and regulation of the MAPK pathway. As such, the RASopathies represent an excellent model of study to explore the intersection of the effects of dysregulation and its consequence in both development and oncogenesis.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas ras/genética , Animais , Regulação da Expressão Gênica , Estudos de Associação Genética/métodos , Desenvolvimento Humano , Humanos , Modelos Biológicos , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Organogênese/genética , Transdução de Sinais , Síndrome , Proteínas ras/metabolismoRESUMO
Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1, have been proposed to mediate this activation, with either Ras or Rap1 acting in distinct cell types. Using Hek293 cells, we show that both Ras and Rap1 are required for cAMP signaling to ERKs. The roles of Ras and Rap1 were distinguished by their mechanism of activation, dependence on the cAMP-dependent protein kinase (PKA), and the magnitude and kinetics of their effects on ERKs. Ras was required for the early portion of ERK activation by cAMP and was activated independently of PKA. Ras activation required the Ras/Rap guanine nucleotide exchange factor (GEF) PDZ-GEF1. Importantly, this action of PDZ-GEF1 was disrupted by mutation within its putative cyclic nucleotide-binding domain within PDZ-GEF1. Compared with Ras, Rap1 activation of ERKs was of longer duration. Rap1 activation was dependent on PKA and required Src family kinases and the Rap1 exchanger C3G. This is the first report of a mechanism for the cooperative actions of Ras and Rap1 in cAMP activation of ERKs. One physiological role for the sustained activation of ERKs is the transcription and stabilization of a range of transcription factors, including c-FOS. We show that the induction of c-FOS by cAMP required both the early and sustained phases of ERK activation, requiring Ras and Rap1, as well as for each of the Raf isoforms, B-Raf and C-Raf.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Ligação a Telômeros/metabolismo , Animais , Bovinos , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Complexo Shelterina , Proteínas de Ligação a Telômeros/genéticaRESUMO
The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras-GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras-GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras-GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12-induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras-GTP to stimulate Ras-induced senescence in nontransformed human cells.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Senescência Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas ras/metabolismo , Dimerização , Fibroblastos , Células HCT116 , Humanos , Queratinócitos , Microscopia de Fluorescência , Fosforilação , Plasmídeos/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , AMP Cíclico/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Proteína Oncogênica p21(ras)/genética , Fosforilação/genética , Ligação Proteica/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismoRESUMO
The small G protein Rap1 can mediate "inside-out signaling" by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.
Assuntos
Movimento Celular , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas rap1 de Ligação ao GTP/química , Sequência de Aminoácidos , Animais , Bovinos , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Fosforilação , Transdução de SinaisRESUMO
The duration of signaling through the MAP kinase (or ERK pathway) cascade has been implicated in thymic development, particularly positive and negative selection. In T cells, two isoforms of the MAP kinase kinase kinase Raf function to transmit signals from the T-cell receptor to ERK: C-Raf and B-Raf. In this study, we conditionally ablated B-Raf expression within thymocytes to assess the effects on ERK activation and thymocyte development. The complete loss of B-Raf is accompanied by a dramatic loss of ERK activation in both the double positive (DP) and single positive (SP) thymocytes, as well as peripheral splenocytes. There was a significant decrease in the cellularity of KO thymi, largely due to a loss of pre-selected DP cells, a decrease in DP cells undergoing positive selection, and a defect in SP maturation. B-Raf plays significant roles in survival of DP thymocytes and function of SP cells in the periphery. Surprisingly, we saw no effect of B-Raf deficiency on negative selection of autoreactive SP thymocytes, despite the greatly reduced ERK activation in these cells.
Assuntos
Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciação Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Deleção Clonal/genética , Ativação Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/imunologiaRESUMO
Hormonal stimulation of cyclic adenosine monophosphate (cAMP) and the cAMP-dependent protein kinase PKA regulates cell growth by multiple mechanisms. A hallmark of cAMP is its ability to stimulate cell growth in many cell types while inhibiting cell growth in others. In this review, the cell type-specific effects of cAMP on the mitogen-activated protein (MAP) kinase (also called extracellular signal-regulated kinase, or ERK) cascade and cell proliferation are examined. Two basic themes are discussed. First, the capacity of cAMP for either positive or negative regulation of the ERK cascade accounts for many of the cell type-specific actions of cAMP on cell proliferation. Second, there are several specific mechanisms involved in the inhibition or activation of ERKs by cAMP. Emerging new data suggest that one of these mechanisms might involve the activation of the GTPase Rap1, which can activate or inhibit ERK signaling in a cell-specific manner.
Assuntos
AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Divisão Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptor Cross-Talk , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.
Assuntos
AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Camundongos , Células PC12 , Transporte Proteico , RNA Interferente Pequeno , Ratos , Células Tumorais CultivadasRESUMO
The Ras superfamily of small G proteins is remarkable for both its diversity and physiological functions. One member, Rap1, has been implicated in a particularly wide range of biological processes, from cell proliferation and differentiation to cell adhesion. But the diversity of Rap1 has lead to contradictory reports of its effects. Originally identified as an antagonist of Ras-induced transformation, Rap1 can oppose other actions of Ras including regulation of cell growth and differentiation, integrin-dependent responses and synaptic plasticity. Furthermore, recent evidence confirms that Rap1, like Ras, can activate the MAP kinase cascade (ERK) in several cell types. These diverse functions of Rap1 underscore that the activation and action of Rap1 are regulated by complex factors that are cell-type specific.
Assuntos
Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Ativação Enzimática , Humanos , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/metabolismoRESUMO
In Drosophila embryos, the Torso receptor tyrosine kinase (RTK) activates the small G protein Ras (D-Ras1) and the protein kinase Raf (D-Raf) to activate ERK to direct differentiation of terminal structures . However, genetic studies have demonstrated that Torso, and by extension other RTKs, can activate Raf and ERK independently of Ras . In mammalian cells, the small G protein Rap1 has been proposed to couple RTKs to ERKs. However, the ability of Rap1 to activate ERKs remains controversial, in part because direct genetic evidence supporting this hypothesis is lacking. Here, we present biochemical and genetic evidence that D-Rap1, the Drosophila homolog of Rap1, can activate D-Raf and ERK. We show that D-Rap1 binds D-Raf and activates ERKs in a GTP- and D-Raf-dependent manner. Targeted disruption of D-Rap1 expression decreased both Torso-dependent ERK activation and the ERK-dependent expression of the zygotic genes tailless and huckebein to levels similar to those achieved in D-Ras1 null embryos. Furthermore, combined deficiencies of D-Ras1 and D-Rap1 completely abolished expression of these genes, mimicking the phenotype observed in embryos lacking D-Raf. These studies provide the first direct genetic evidence of Rap1-mediated activation of the MAP kinase cascade in eukaryotic organisms.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Cromatografia de Afinidade , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Transferência de Genes , Hibridização In Situ , Dados de Sequência Molecular , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Transfecção , Quinases raf/metabolismo , Proteínas ras/metabolismoRESUMO
The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) is activated following engagement of the T-cell receptor and is required for interleukin 2 (IL-2) production and T-cell proliferation. This activation is enhanced by stimulation of the coreceptor CD28 and inhibited by the coreceptor CTLA-4. We show that the small G protein Rap1 is regulated in the opposite manner; it is inhibited by CD28 and activated by CTLA-4. Together, CD3 and CTLA-4 activate Rap1 in a sustained manner. To delineate T-cell function in the absence of Rap1 activity, we generated transgenic mice expressing Rap1GAP1, a Rap1-specific GTPase-activating protein. Transgenic mice showed lymphadenopathy, and transgenic T cells displayed increased ERK activation, proliferation, and IL-2 production. More significantly, the inhibitory effect of CTLA-4 on T-cell function in Rap1GAP1-transgenic T cells was reduced. We demonstrate that CTLA-4 activates Rap1, and we propose that intracellular signals from CTLA-4 antagonize CD28, at least in part, at the level of Rap1.
Assuntos
Antígenos de Diferenciação/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD , Antígeno CTLA-4 , Adesão Celular , Linhagem Celular , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras) , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transgenes/genéticaAssuntos
Fator de Crescimento Neural/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Simulação por Computador , Fator de Crescimento Epidérmico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cinética , Modelos Biológicos , Células PC12 , Ratos , Transdução de Sinais/fisiologia , Proteínas ras/metabolismoRESUMO
The activation of Raf kinases by the small GTPase Ras requires two major sets of phosphorylations. One set lies within the activation loop, and the other lies within the N-terminal acidic region (N region). In the most abundant isoform of Raf, C-Raf, N-region phosphorylations occur on serine 338 (S338) and tyrosine 341 (Y341) and are thought to provide allosteric activation of the Raf dimer. We show that the phosphorylations of these N-region sites does not require C-Raf dimerization, but rather, they precede dimerization. One of these phosphorylations (phospho-Y341) is required for C-Raf dimerization, and this action can be replicated by phosphomimetic mutants both in vivo and in vitro The role of the phosphorylation of Y341 in promoting Raf dimerization is distinct from its well-known function in facilitating S338 phosphorylation. In Ras mutant pancreatic cancer cell lines, the phosphorylation and dimerization of C-Raf are basally elevated. Dimerization is thought to contribute to their elevated growth rate through their activation of the mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase [ERK]) signaling cascade. Blocking the tyrosine phosphorylation of C-Raf with Src family inhibitors blocks growth, basal dimerization, and ERK activation in these cells. We suggest that the kinases mediating C-Raf Y341 phosphorylation are potential candidate drug targets in selected Ras-dependent cancers.
RESUMO
BACKGROUND: Mutations of the human K-Ras 4B (K-Ras) G protein are associated with a significant proportion of all human cancers. Despite this fact, a comprehensive analysis of K-Ras interactions is lacking. Our investigations focus on characterization of the K-Ras interaction network. MATERIALS AND METHODS: We employed a biotin ligase-tagging approach, in which tagged K-Ras proteins biotinylate neighbor proteins in a proximity-dependent fashion, and proteins are identified via mass spectrometry (MS) sequencing. RESULTS: In transfected cells, a total of 748 biotinylated proteins were identified from cells expressing biotin ligase-tagged K-Ras variants. Significant differences were observed between membrane-associated variants and a farnesylation-defective mutant. In pancreatic cancer cells, 56 K-Ras interaction partners were identified. Most of these were cytoskeletal or plasma membrane proteins, and many have been identified previously as potential cancer biomarkers. CONCLUSION: Biotin ligase tagging offers a rapid and convenient approach to the characterization of K-Ras interaction networks.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Repressoras/metabolismo , Animais , Biotinilação , Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Camundongos , Mutação , Células NIH 3T3 , Proteína Oncogênica p21(ras)/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genéticaRESUMO
Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.
Assuntos
Membrana Celular/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Ácido Mirístico/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Citosol/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Plasticidade Neuronal , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Ratos , Especificidade por Substrato , Sinapses/metabolismoRESUMO
Extracellular signal-regulated kinases (ERKs) play important roles in proliferation, differentiation and gene expression. In our previous study, we demonstrated that both ERK5 and ERK1/2 were responsible for neurite outgrowth and tyrosine hydroxylase (TH) expression in rat pheochromocytoma cells (PC12) (J Biol Chem 284, 23,564-23,573, 2009). However, the functional differences between ERK5 and ERK1/2 signaling in neural differentiation remain unclear. In the present study, we show that ERK5, but not ERK1/2 regulates TH levels in rat sympathetic neurons. Furthermore, microarray analysis performed in PC12 cells using ERK5 and ERK1/2-specific inhibitors, identified ankyrin repeat domain 1 (ankrd1) as an ERK5-dependent and ERK1/2-independent gene. Here, we report a novel role of the ERK5/ankrd1 signaling in regulating TH levels and catecholamine biosynthesis. Ankrd1 mRNA was induced by nerve growth factor in time- and concentration-dependent manners. TH levels were reduced by ankrd1 knockdown with no changes in the mRNA levels, suggesting that ankrd1 was involved in stabilization of TH protein. Interestingly, ubiquitination of TH was enhanced and catecholamine biosynthesis was reduced by ankrd1 knockdown. Finally, we examined the relationship of ERK5 to TH levels in human adrenal pheochromocytomas. Whereas TH levels were correlated with ERK5 levels in normal adrenal medullas, ERK5 was down-regulated and TH was up-regulated in pheochromocytomas, indicating that TH levels are regulated by alternative mechanisms in tumors. Taken together, ERK5 signaling is required for catecholamine biosynthesis during neural differentiation, in part to induce ankrd1, and to maintain appropriate TH levels. This pathway is disrupted in pathological conditions.