RESUMO
Phosphoinositides are amphipathic lipid molecules derived from phosphatidylinositol that represent low abundance components of biological membranes. Rather than serving as mere structural elements of lipid bilayers, they represent molecular switches for a broad range of biological processes, including cell signaling, membrane dynamics and remodeling, and many other functions. Here, we focus on the molecular mechanisms that turn phosphoinositides into molecular switches and how the dysregulation of these processes can lead to disease.
Assuntos
Doença , Fosfatidilinositóis , Transdução de Sinais , Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , HumanosRESUMO
The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants-R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low-affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called "high"-affinity biological responses arise from activation by isolated receptor dimers, whereas "low"-affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.
Assuntos
Receptores ErbB , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ligantes , Mutação , Fosfolipases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Domínios Proteicos/genética , Células CHO , Animais , Cricetinae , Humanos , Glioblastoma/genéticaRESUMO
Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.
Assuntos
Proteínas de Ligação a DNA , Inibidores Enzimáticos , Chaperonas de Histonas , Alvo Mecanístico do Complexo 1 de Rapamicina , Neoplasias , Complexo Repressor Polycomb 1 , Proteína Fosfatase 2 , Proteínas Proto-Oncogênicas c-akt , Humanos , Inibidores Enzimáticos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Complexo Repressor Polycomb 1/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Chaperonas de Histonas/deficiência , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Transdução de Sinais , Ativação Enzimática , Linhagem Celular TumoralRESUMO
Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.
Assuntos
Ceramidas , Resistência à Insulina , Humanos , Ceramidas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Resistência à Insulina/fisiologia , Células Musculares/metabolismo , Músculo Esquelético/metabolismoRESUMO
The conserved protein kinase mTOR (mechanistic target of rapamycin) responds to diverse environmental cues to control cell metabolism and promote cell growth, proliferation, and survival as part of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTORC2. Our prior work demonstrated that an alkaline intracellular pH (pHi) increases mTORC2 activity and cell survival in complete media in part by activating AMP-activated protein kinase, a kinase best known to sense energetic stress. It is important to note that an alkaline pHi represents an underappreciated hallmark of cancer cells that promotes their oncogenic behaviors. In addition, mechanisms that control mTORC1 and mTORC2 signaling and function remain incompletely defined, particularly in response to stress conditions. Here, we demonstrate that an alkaline pHi increases phosphatidylinositide 3-kinase (PI3K) activity to promote mTORC1 and mTORC2 signaling in the absence of serum growth factors. Alkaline pHi increases mTORC1 activity through PI3K-Akt signaling, which mediates inhibitory phosphorylation of the upstream proteins tuberous sclerosis complex 2 and proline-rich Akt substrate of 40 kDa and dissociates tuberous sclerosis complex from lysosomal membranes, thus enabling Rheb-mediated activation of mTORC1. Thus, alkaline pHi mimics growth factor-PI3K signaling. Functionally, we also demonstrate that an alkaline pHi increases cap-dependent protein synthesis through inhibitory phosphorylation of eIF4E binding protein 1 and suppresses apoptosis in a PI3K- and mTOR-dependent manner. We speculate that an alkaline pHi promotes a low basal level of cell metabolism (e.g., protein synthesis) that enables cancer cells within growing tumors to proliferate and survive despite limiting growth factors and nutrients, in part through elevated PI3K-mTORC1 and/or PI3K-mTORC2 signaling.
RESUMO
Ephrin-B signaling has been implicated in many normal and pathological processes, including neural crest development and tumor metastasis. We showed previously that proteolysis of ephrin-B ligands by the disintegrin metalloprotease ADAM13 is necessary for canonical Wnt signal activation and neural crest induction in Xenopus, but it was unclear if these mechanisms are conserved in mammals. Here, we report that mammalian ADAM9 cleaves ephrin-B1 and ephrin-B2 and can substitute for Xenopus ADAM13 to induce the neural crest. We found that ADAM9 expression is elevated in human colorectal cancer (CRC) tissues and that knockdown (KD) of ADAM9 inhibits the migration and invasion of SW620 and HCT116 CRC cells by reducing the activity of Akt kinase, which is antagonized by ephrin-Bs. Akt is a signaling node that activates multiple downstream pathways, including the Wnt and mTOR pathways, both of which can promote CRC cell migration/invasion. Surprisingly, we also found that KD of ADAM9 downregulates Wnt signaling but has negligible effects on mTOR signaling in SW620 cells; in contrast, mTOR activity is suppressed while Wnt signaling remains unaffected by ADAM9 KD in HCT116 cells. These results suggest that mammalian ADAM9 cleaves ephrin-Bs to derepress Akt and promote CRC migration and invasion; however, the signaling pathways downstream of Akt are differentially regulated by ADAM9 in different CRC cell lines, reflecting the heterogeneity of CRC cells in responding to manipulations of upstream Akt regulators.
Assuntos
Proteínas ADAM/metabolismo , Neoplasias Colorretais , Efrinas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Humanos , Ligantes , Mamíferos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Via de Sinalização WntRESUMO
Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.
Assuntos
Fator 1 de Ribosilação do ADP , Mapas de Interação de Proteínas , Proteína Companheira de mTOR Insensível à Rapamicina , Serina-Treonina Quinases TOR , Humanos , Fator 1 de Ribosilação do ADP/metabolismo , Insulina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Mapeamento de Interação de Proteínas/métodosRESUMO
Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase-inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase-inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway.
Assuntos
Desenvolvimento Fetal , MAP Quinase Quinase Quinase 4 , Placenta , Placentação , Receptor IGF Tipo 1 , Receptor de Insulina , Adulto , Animais , Proteína de Ligação a CREB/metabolismo , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Desacetilase 6 de Histona/metabolismo , Humanos , MAP Quinase Quinase Quinase 4/genética , MAP Quinase Quinase Quinase 4/metabolismo , Camundongos , Placenta/enzimologia , Gravidez , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
One of the theories related to aging is the increase in oxidative stress. Given this, the objective of the study is to evaluate the cellular mechanisms responsible for the resveratrol antioxidant effect on leukocytes from donors aged between 20 and 80 years old. For this, leukocytes from donors of three age groups (20-39, 40-59 and 60-80) were isolated. Image-iT™LIVE Green Reactive Oxygen Species (ROS) Kit was used. Reactive Nitrogen Species (RNS) analysis was performed by measuring nitric oxide and peroxynitrite. The PKA, Akt/PKB and p38-MAPK were evaluated by chemiluminescence. The statistical analysis between age and treatments were performed by Pearson correlation (*p < 0.05). It was possible to observe the antioxidant effect of resveratrol in all age groups. The correlation results show loss of resveratrol effect in decreasing ROS in leukocytes from older donors. We observed an active antioxidant effect of p38-MAPK in all ages, with resveratrol acting on it. The PKA and Akt/PKB were active in leukocytes from donors aged 20-59. In cells from donors older than 60, these pathways are silenced, and an effect is also not observed in cells treated with resveratrol. Therefore, resveratrol showed antioxidant effect in all age, although it was more pronounced in leukocytes from younger. One of resveratrol's mechanisms is due to the activation of the PKA and Akt/PKB, which were activated in younger donor cells.
Assuntos
Antioxidantes , Proteínas Proto-Oncogênicas c-akt , Antioxidantes/farmacologia , Resveratrol/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Activating transcription factor 3 (ATF3) is a stress-induced transcription factor and a familiar neuronal marker for nerve injury. This factor has been shown to protect neurons from hypoxic insult in vitro by suppressing carboxyl-terminal modulator protein (CTMP) transcription, and indirectly activating the anti-apoptotic Akt/PKB cascade. Despite prior studies in vitro, whether this neuroprotective pathway also exists in the brain in vivo after ischemic insult remains to be determined. In the present study, we showed a rapid and marked induction of ATF3 mRNA throughout ischemia-reperfusion in a middle cerebral artery (MCA) occlusion model. Although the level of CTMP mRNA was quickly induced upon ischemia, its level showed only a mild increase after reperfusion. With the gain-of-function approach, both pre- and post-ischemic administration of Ad-ATF3 ameliorated brain infarct and neurological deficits. Whereas, with the loss-of-function approach, ATF3 knockout (KO) mice showed bigger infarct and worse functional outcome after ischemia. In addition, these congenital defects were rescued upon reintroducing ATF3 to the brain of KO mice. ATF3 overexpression led to a lower level of CTMP and a higher level of p-Akt(473) in the ischemic brain. On the contrary, ATF3 KO resulted in upregulation of CTMP and downregulation of p-Akt(473) instead. Furthermore, post-ischemic CTMP siRNA knockdown led to smaller infarct and better behaviors. CTMP siRNA knockdown increased the level of p-Akt(473), but did not alter the ATF3 level in the ischemic brain, upholding the ATF3âCTMP signal cascade. In summary, our proof-of-principle experiments support the existence of neuroprotective ATF3âCTMP signal cascade regulating the ischemic brain. Furthermore, these results suggest the therapeutic potential for both ATF3 overexpression and CTMP knockdown for stroke treatment.
Assuntos
Isquemia Encefálica , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Proteínas de Transporte/metabolismo , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Camundongos Knockout , Infarto Encefálico/genética , RNA Interferente Pequeno/genética , Infarto Cerebral , Palmitoil-CoA Hidrolase/metabolismoRESUMO
Cells produce free radicals and antioxidants when exposed to toxic compounds during cellular metabolism. However, free radicals are deleterious to lipids, proteins, and nucleic acids. Antioxidants neutralize and eliminate free radicals from cells, preventing cell damage. Therefore, the study aims to determine whether the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) will ameliorate the maximum dose of acrylamide and alpha (α)-solanine synergistic toxic effects in exposed BEAS-2B cells. These toxic compounds are consumed worldwide by eating potato products. BEAS-2B cells were simultaneously treated with BHA 10 µM and BHT 20 µM and incubated in a 5% CO2 humidified incubator for 24 h, followed by individual or combined treatment with acrylamide (3.5 mM) and α-solanine (44 mM) for 48 h, including the controls. Cell morphology, DNA, RNA, and protein were analyzed. The antioxidants did not prevent acrylamide and α-solanine synergistic effects in exposed BEAS-2B cells. However, cell morphology was altered; polymerase chain reaction (PCR) showed reduced RNA constituents but not DNA. In addition, the toxic compounds synergistically inhibited AKT/PKB expression and its downstream genes. The study showed BHA and BHT are not protective against the synergetic toxic effects of acrylamide and α-solanine in exposed BEAS-2B cells.
Assuntos
Antioxidantes , Solanina , Antioxidantes/farmacologia , Hidroxitolueno Butilado , Hidroxianisol Butilado/farmacologia , Acrilamida/toxicidade , Proteínas , DNA , RNARESUMO
TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of â¼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis-Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[18O4]-ATP. These data revealed that Ser324 (KM â¼6 µM) and Thr649 (KM â¼25 µM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM â¼10 µM), Ser711 (KM â¼79 µM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminopeptidases/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Insulina/farmacologia , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Aminopeptidases/genética , Animais , Proteínas Ativadoras de GTPase/genética , Hipoglicemiantes/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with â¼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1-ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.
Assuntos
Comunicação Autócrina/genética , Neuregulina-1/genética , Neurofibromina 2/genética , Receptor ErbB-3/genética , Receptor IGF Tipo 1/genética , Serina-Treonina Quinases TOR/genética , Anticorpos Monoclonais Humanizados/farmacologia , Benzamidas/farmacologia , Benzoxazóis/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Lapatinib/farmacologia , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patologia , Meningioma/genética , Meningioma/metabolismo , Meningioma/patologia , Morfolinas/farmacologia , Neuregulina-1/antagonistas & inibidores , Neuregulina-1/metabolismo , Neurofibromina 2/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptor EphA2/genética , Receptor EphA2/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Triazinas/farmacologiaRESUMO
The phosphatidylinositol 3-kinase-Akt signaling pathway plays an essential role in regulating cell proliferation and apoptosis. Akt kinase is at the center of this signaling pathway and interacts with a variety of proteins. Akt is overexpressed in almost 80% of tumors. However, inhibiting Akt has serious clinical side effects so is not a suitable treatment for cancer. During recent years, Akt scaffold proteins have received increasing attention for their ability to regulate Akt signaling and have emerged as potential targets for cancer therapy. In this paper, we categorize Akt kinase scaffold proteins into four groups based on their cellular location: membrane-bound activator and inhibitor, cytoplasm, and endosome. We describe how these scaffolds interact with Akt kinase, how they affect Akt activity, and how they regulate the specificity of Akt signaling. We also discuss the clinical application of Akt scaffold proteins as targets for cancer therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Terapia de Alvo Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Sestrins represent a family of stress-inducible proteins that prevent the progression of many age- and obesity-associated disorders. Endogenous Sestrins maintain insulin-dependent AKT Ser/Thr kinase (AKT) activation during high-fat diet-induced obesity, and overexpressed Sestrins activate AKT in various cell types, including liver and skeletal muscle cells. Although Sestrin-mediated AKT activation improves metabolic parameters, the mechanistic details underlying such improvement remain elusive. Here, we investigated how Sestrin2, the Sestrin homolog highly expressed in liver, induces strong AKT activation. We found that two known targets of Sestrin2, mTOR complex (mTORC) 1 and AMP-activated protein kinase, are not required for Sestrin2-induced AKT activation. Rather, phosphoinositol 3-kinase and mTORC2, kinases upstream of AKT, were essential for Sestrin2-induced AKT activation. Among these kinases, mTORC2 catalytic activity was strongly up-regulated upon Sestrin2 overexpression in an in vitro kinase assay, indicating that mTORC2 may represent the major link between Sestrin2 and AKT. As reported previously, Sestrin2 interacted with mTORC2; however, we found here that this interaction occurs indirectly through GATOR2, a pentameric protein complex that directly interacts with Sestrin2. Deleting or silencing WDR24 (WD repeat domain 24), the GATOR2 component essential for the Sestrin2-GATOR2 interaction, or WDR59, the GATOR2 component essential for the GATOR2-mTORC2 interaction, completely ablated Sestrin2-induced AKT activation. We also noted that Sestrin2 also directly binds to the pleckstrin homology domain of AKT and induces AKT translocation to the plasma membrane. These results uncover a signaling mechanism whereby Sestrin2 activates AKT through GATOR2 and mTORC2.
Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Obesidade/genética , Peroxidases/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica/genética , Células Hep G2 , Humanos , Insulina/genética , Resistência à Insulina/genética , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação/genética , Ligação Proteica/genética , Proteínas/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by the regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.
Assuntos
Lisossomos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidoresRESUMO
Muscle atrophy is regulated by the balance between protein degradation and synthesis. FOXO1, a transcription factor, helps to determine this balance by activating pro-atrophic gene transcription when present in muscle fiber nuclei. Foxo1 nuclear efflux is promoted by AKT-mediated Foxo1 phosphorylation, eliminating FOXO1's atrophy-promoting effect. AKT activation can be promoted by insulin-like growth factor 1 (IGF1) or insulin via a pathway including IGF1 or insulin, phosphatidylinositol 3-kinase, and AKT. We used confocal fluorescence time-lapse imaging of FOXO1-GFP in adult isolated living muscle fibers maintained in culture to explore the effects of IGF1 and insulin on FOXO1-GFP nuclear efflux with and without pharmacological inhibitors. We observed that although AKT inhibitor blocks the IGF1- or insulin-induced effect on FOXO1 nuclear efflux, phosphatidylinositol 3-kinase inhibitors, which we show to be effective in these fibers, do not. We also found that inhibition of the protein kinase ACK1 or ATM contributes to the suppression of FOXO1 nuclear efflux after IGF1. These results indicate a novel pathway that has been unexplored in the IGF1- or insulin-induced regulation of FOXO1 and present information useful both for therapeutic interventions for muscle atrophy and for further investigative areas into insulin insensitivity and type 2 diabetes.
Assuntos
Núcleo Celular/metabolismo , Proteína Forkhead Box O1/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Núcleo Celular/efeitos dos fármacos , Feminino , Proteína Forkhead Box O1/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Imagem Óptica , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Adesão Celular , Linhagem Celular , Movimento Celular , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citosol/metabolismo , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transdução de SinaisRESUMO
Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Endotélio Vascular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Núcleo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Mitocôndrias/patologia , Biogênese de Organelas , Consumo de Oxigênio , Fatores de Transcrição/genéticaRESUMO
The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.