RESUMO
The nucleocytoplasmic transport of proteins is an important mechanism to control cell fate. Pap1 is a fission yeast nucleocytoplasmic shuttling transcription factor of which localization is redox regulated. The nuclear export factor Crm1/exportin negatively regulates Pap1 by exporting it from the nucleus to the cytoplasm. Here, we describe the effect of an anti-cancer compound ACA-28, an improved derivative of 1'-acetoxychavicol acetate (ACA), on the subcellular distribution of Pap1. ACA-28 induced nuclear accumulation of Pap1 more strongly than did ACA. ROS inhibitor N-acetyl-L-cysteine (NAC) partly antagonized the Pap1 nuclear accumulation induced by ACA-28. NAC almost abolished Pap1 nuclear localization upon H 2 O 2 , whereas leptomycin B (LMB)-mediated inhibition of Pap1 nuclear export was resistant to NAC. Collectively, ACA-28-mediated apoptosis in cancer cells may involve ROS-dependent and -independent mechanisms.
RESUMO
The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Neoplasias Pancreáticas , Humanos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação , Transdução de Sinais , Neoplasias Pancreáticas/tratamento farmacológico , Apoptose , Sistema de Sinalização das MAP Quinases , Neoplasias PancreáticasRESUMO
The mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways are dysregulated in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), which has a very poor prognosis due to its lack of efficient therapies. We have previously identified ACAGT-007a (GT-7), an anti-cancer compound that kills ERK-active melanoma cells by inducing ERK-dependent apoptosis. Here, we investigated the apoptosis-inducing effect of GT-7 on three PDAC cell lines and its relevance with the MAPK/ERK and PI3K/AKT signaling pathways. GT-7 induced apoptosis in PDAC cells with different KRAS mutations (MIA-Pa-Ca-2 (KRAS G12C), T3M4 (KRAS Q61H), and PANC-1 (KRAS G12D)), being T3M4 most susceptible, followed by MIA-Pa-Ca-2, and PANC-1 was most resistant to apoptosis induction by GT-7. GT-7 stimulated ERK phosphorylation in the three PDAC cells, but only T3M4 displayed ERK-activation-dependent apoptosis. Furthermore, GT-7 induced a marked down-regulation of AKT phosphorylation after a transient peak in T3M4, whereas PANC-1 displayed the strongest and most sustained AKT activation, followed by MIA-Pa-Ca-2, suggesting that sustained AKT phosphorylation as a determinant for the resistance to GT-7-mediated apoptosis. Consistently, a PI3K inhibitor, Wortmannin, abolished AKT phosphorylation and enhanced GT-7-mediated apoptosis in T3M4 and MIA-Pa-Ca-2, but not in PANC-1, which showed residual AKT phosphorylation. This is the first report that ERK stimulation alone or in combination with AKT signaling inhibition can effectively induce apoptosis in PDAC and provides a rationale for a novel concurrent targeting of the PI3K/AKT and ERK pathways.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMO
The RAF/MEK/ERK signaling pathway regulates diverse cellular processes as exemplified by cell proliferation, differentiation, motility, and survival. Activation of ERK1/2 generally promotes cell proliferation, and its deregulated activity is a hallmark of many cancers. Therefore, components and regulators of the ERK pathway are considered potential therapeutic targets for cancer, and inhibitors of this pathway, including some MEK and BRAF inhibitors, are already being used in the clinic. Notably, ERK1/2 kinases also have pro-apoptotic functions under certain conditions and enhanced ERK1/2 signaling can cause tumor cell death. Although the repertoire of the compounds which mediate ERK activation and apoptosis is expanding, and various anti-cancer compounds induce ERK activation while exerting their anti-proliferative effects, the mechanisms underlying ERK1/2-mediated cell death are still vague. Recent studies highlight the importance of dual-specificity phosphatases (DUSPs) in determining the pro- versus anti-apoptotic function of ERK in cancer. In this review, we will summarize the recent major findings in understanding the role of ERK in apoptosis, focusing on the major compounds mediating ERK-dependent apoptosis. Studies that further define the molecular targets of these compounds relevant to cell death will be essential to harnessing these compounds for developing effective cancer treatments.
Assuntos
Apoptose/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose/fisiologia , Fosfatases de Especificidade Dupla/metabolismo , Humanos , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.
Assuntos
Apoptose/efeitos dos fármacos , Álcoois Benzílicos/farmacologia , Regulação para Baixo/genética , Fosfatase 6 de Especificidade Dupla/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Animais , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fosfatase 6 de Especificidade Dupla/metabolismo , Camundongos , Células NIH 3T3 , OncogenesRESUMO
The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.
Assuntos
Antineoplásicos/farmacologia , Compostos Benzidrílicos/farmacologia , Carbonatos/farmacologia , Descoberta de Drogas , Ésteres/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Melanoma/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Compostos Benzidrílicos/síntese química , Compostos Benzidrílicos/química , Carbonatos/síntese química , Carbonatos/química , Relação Dose-Resposta a Droga , Ésteres/síntese química , Ésteres/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/metabolismo , Melanoma/patologia , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Mitogen-activated protein kinase (MAPK) is a highly conserved serine/threonine kinase that regulates multiple cellular processes such as cell proliferation, differentiation, apoptosis, and inflammation. Rnc1 has been identified as a regulator of Pmk1 MAPK signaling, a homologue of extracellular signal-regulated kinase (ERK)-1 MAPK in mammals. Rnc1 encodes a K-homology (KH)-type RNA-binding protein (RBP). Previously, it was reported that Rnc1 acts as a negative regulator of Pmk1 MAPK signaling through the mRNA stabilization of Pmp1, the MAPK phosphatase for Pmk1 in our laboratory. We analyzed the spatial regulation of Rnc1 and discovered that Rnc1 is exported from the nucleus by the mRNA-export system. The nuclear export of Rnc1 is important for exerting its function to stabilize Pmp1 mRNA. Therefore, the spatial regulation of Rnc1 affects MAPK signaling activity. We also reported that Nrd1, an RRM-type RBP, plays a critical role in cytokinesis by binding to and stabilizing myosin mRNA. Notably, Rnc1 and Nrd1 localize to stress granules (SGs) in response to various environmental stresses. Moreover, SG formation is inhibited in the Nrd1 or Rnc1 deletion cells, whereas the overproduction of Nrd1 or Rnc1, as well as that of mammalian RBP TIA-1, induces granule formation. These data show that Nrd1 and Rnc1 regulate SG formation as a novel SG component. Alterations of SG formation are linked to neurodegenerative diseases and resistance to anti-cancer drugs, thus conferring remarkable clinical importance to SGs. This review discusses the spatial regulation of RBPs or SG formation as novel targets for drug discovery.
Assuntos
Descoberta de Drogas , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Terapia de Alvo Molecular , Proteínas de Ligação a RNA/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Humanos , Metaloendopeptidases , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombeRESUMO
Fingolimod hydrochloride (FTY720) is a first-in-class of sphingosine-1-phosphate (S1P) receptor modulator approved to treat multiple sclerosis by its phosphorylated form (FTY720-P). Recently, a novel role of FTY720 as a potential anticancer drug has emerged. One of the anticancer mechanisms of FTY720 involves the induction of reactive oxygen species (ROS) and subsequent apoptosis, which is largely independent of its property as an S1P modulator. ROS have been considered as a double-edged sword in tumor initiation/progression. Intriguingly, prooxidant therapies have attracted much attention due to its efficacy in cancer treatment. These strategies include diverse chemotherapeutic agents and molecular targeted drugs such as sulfasalazine which inhibits the CD44v-xCT (cystine transporter) axis. In this review, we introduce our recent discoveries using a chemical genomics approach to uncover a signaling network relevant to FTY720-mediated ROS signaling and apoptosis, thereby proposing new potential targets for combination therapy as a means to enhance the antitumor efficacy of FTY720 as a ROS generator. We extend our knowledge by summarizing various measures targeting the vulnerability of cancer cells' defense mechanisms against oxidative stress. Future directions that may lead to the best use of FTY720 and ROS-targeted strategies as a promising cancer treatment are also discussed.
Assuntos
Apoptose/efeitos dos fármacos , Organofosfatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Organofosfatos/uso terapêutico , Esfingosina/farmacologia , Esfingosina/uso terapêuticoRESUMO
Fingolimod hydrochloride (FTY720), a sphingosine-1-phosphate (S1P) analogue, is an approved immune modulator for the treatment of multiple sclerosis (MS). Notably, in addition to its well-known mode of action as an S1P modulator, accumulating evidence suggests that FTY720 induces apoptosis in various cancer cells via reactive oxygen species (ROS) generation. Although the involvement of multiple signaling molecules, such as JNK (Jun N-terminal kinase), Akt (alpha serine/threonine-protein kinase) and Sphk has been reported, the exact mechanisms how FTY720 induces cell growth inhibition and the functional relationship between FTY720 and these signaling pathways remain elusive. Our previous reports using the fission yeast Schizosaccharomyces pombe as a model system to elucidate FTY720-mediated signaling pathways revealed that FTY720 induces an increase in intracellular Ca2+ concentrations and ROS generation, which resulted in the activation of the transcriptional responses downstream of Ca2+/calcineurin signaling and stress-activated MAPK signaling, respectively. Here, we performed a genome-wide screening for genes whose deletion induces FTY720-sensitive growth in S. pombe and identified 49 genes. These gene products are related to the biological processes involved in metabolic processes, transport, transcription, translation, chromatin organization, cytoskeleton organization and intracellular signal transduction. Notably, most of the FTY720-sensitive deletion cells exhibited NAC-remedial FTY720 sensitivities and dysregulated ROS homeostasis. Our results revealed a novel gene network involving ROS homeostasis and the possible mechanisms of the FTY720 toxicity.
RESUMO
A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N,N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 × 100 × 30 µm) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.
Assuntos
Resinas Acrílicas/química , Eletroforese em Microchip , Eletroforese em Gel de Poliacrilamida , Peptídeos/análise , Fosforilação , PiridinasRESUMO
The extracellular signal-regulated kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as melanoma. Melanoma remains incurable despite the use of conventional chemotherapy; consequently, development of new therapeutic agents for melanoma is highly desirable. Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. Notably, the ACA-28-induced apoptosis in melanoma and HER2-transformed cells was abrogated when ERK activation was blocked with a specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells, as compared in NHEM. ACA-28 might serve as a promising seed compound for melanoma treatment.
Assuntos
Antineoplásicos/farmacologia , Álcoois Benzílicos/farmacologia , Melanoma/tratamento farmacológico , Células 3T3 , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Álcoois Benzílicos/química , Butadienos/farmacologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
PKN, a conserved family member related to PKC, was the first protein kinase identified as a target of the small GTPase Rho. PKN is involved in various functions including cytoskeletal arrangement and cell adhesion. Furthermore, the enrichment of PKN3 mRNA in some cancer cell lines as well as its requirement in malignant prostate cell growth suggested its involvement in oncogenesis. Despite intensive research efforts, physiological as well as pathological roles of PKN3 in vivo remain elusive. Here, we generated mice with a targeted deletion of PKN3. The PKN3 knockout (KO) mice are viable and develop normally. However, the absence of PKN3 had an impact on angiogenesis as evidenced by marked suppressions of micro-vessel sprouting in ex vivo aortic ring assay and in vivo corneal pocket assay. Furthermore, the PKN3 KO mice exhibited an impaired lung metastasis of melanoma cells when administered from the tail vein. Importantly, PKN3 knock-down by small interfering RNA (siRNA) induced a glycosylation defect of cell-surface glycoproteins, including ICAM-1, integrin ß1 and integrin α5 in HUVECs. Our data provide the first in vivo genetic demonstration that PKN3 plays critical roles in angiogenesis and tumor metastasis, and that defective maturation of cell surface glycoproteins might underlie these phenotypes.
Assuntos
Carcinoma Pulmonar de Lewis/genética , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Proteína Quinase C/genética , Animais , Aorta/metabolismo , Aorta/patologia , Bioensaio , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Córnea/irrigação sanguínea , Córnea/metabolismo , Córnea/patologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/deficiência , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[(14)C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.
Assuntos
Adenosina Difosfato Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Amido/metabolismo , Genes de Plantas , Proteínas Facilitadoras de Transporte de Glucose/genética , Mutação , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/metabolismo , Zea mays/enzimologia , Zea mays/genéticaRESUMO
Rapamycin and its derivatives have now emerged as an attractive therapeutic strategy with both immunosuppressant and antitumor properties. In addition, rapamycin has been proposed as a calorie restriction mimetic to extend the life span of various organisms. The fission yeast Schizosaccharomyces pombe (S. pombe) serves as a valuable genetic model system to study the mechanism(s) of drug action as well as to determine genetic contexts associated with drug sensitivity or resistance. Here, we identified genes that when deleted modulate the rapamycin-sensitive strains in S. pombe. We carried out a chemical genomics screen for rapamycin-sensitive mutants using the genome-deletion library which covers 95.3% of all nonessential fission yeast genes and confirmed 59 genes to be rapamycin sensitive. Gene Ontology (GO) enrichment analysis showed that strains sensitive to rapamycin are highly enriched in processes regulating tRNA modification and mitochondria as well as other ontologies, including cellular metabolic process, chromatin organization, cell cycle, signaling, translation, transport and other cellular processes. Analysis also showed that components of the Elongator complex are overrepresented in the sensitive strains. Here, the data obtained will provide valuable information for speculation on the actions of rapamycin as well as on TORC signaling, thereby presenting a strategy to enhance sensitivity to rapamycin.
Assuntos
Antifúngicos/metabolismo , Farmacorresistência Fúngica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Sirolimo/metabolismo , Ciclo Celular , Cromatina/genética , Genoma Fúngico , Genômica/métodos , Mitocôndrias/genética , Mutação , Naftiridinas/metabolismo , Biossíntese de Proteínas , Inibidores de Proteínas Quinases/metabolismo , RNA de Transferência/genética , Schizosaccharomyces/citologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKBα using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKBα protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1â mg PKBα/20 larvae was recorded. Kinase assays showed that the recombinant PKBα possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKBα with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
Assuntos
Bombyx/genética , Bombyx/metabolismo , Larva/metabolismo , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Humanos , Larva/genética , Fosforilação , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/genética , Transdução de SinaisRESUMO
Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1(+), which encodes a multi-KH type RNA-binding protein, and pab1(+), which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1(+) and pab1(+) genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2α, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2α phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Proteína I de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The interaction between Rnc1, an RNA interactive protein, and a Pmp1 mRNA was investigated by affinity capillary electrophoresis (ACE). Prior to the ACE experiments, the column performances of three capillaries (an untreated fused silica capillary, a polybrene-polyacrylic acid (PB-PAA) double layer coating capillary, and a carboxylated capillary with a covalent modification) were studied with model proteins including ribonuclease B (RNase B) and bovine serum albumin (BSA). Using an untreated fused silica and a PB-PAA double layer coating capillaries, both of the protein peaks were broad and tailing. However, using a carboxylated capillary, the protein peaks were sharp and symmetric, and migration times were repeatable (RSD<0.4%). Further, the proteins in human serum also gave sharp peaks and its repeatability was kept at a high level by pre-treatment of a capillary inner wall with 1M sodium chloride solution before each run. An Rnc1 protein was analyzed by ACE with background electrolytes containing various concentrations of Pmp1 sense mRNA using a carboxylated capillary. Increase in the concentration of the mRNA was found to delay the migration time of the protein. But the migration time of the protein was kept constant with increasing Pmp1 anti-sense mRNA instead of Pmp1 sense mRNA. A straight line (r=0.987) was obtained by plotting 1/(migration time shift) versus 1/(Pmp1 sense mRNA concentration) and the association constant of Rnc1 protein with Pmp1 sense mRNA could be estimated to be 4.15x10(6)M(-1). These results suggest that the association constants of proteins with mRNAs as ligands were easily determined by the proposed method.
Assuntos
Ácidos Carboxílicos/metabolismo , Desoxirribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteolipídeos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , tRNA Metiltransferases/metabolismo , Animais , Ácidos Carboxílicos/química , Bovinos , Desoxirribonucleases/genética , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Humanos , Proteínas de Membrana/genética , Ligação Proteica/genética , Proteolipídeos/genética , ATPases Translocadoras de Prótons/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , tRNA Metiltransferases/genéticaRESUMO
Myosin II is an essential component of the actomyosin contractile ring and plays a crucial role in cytokinesis by generating the forces necessary for contraction of the actomyosin ring. Cdc4 is an essential myosin II light chain in fission yeast and is required for cytokinesis. In various eukaryotes, the phosphorylation of myosin is well documented as a primary means of activating myosin II, but little is known about the regulatory mechanisms of Cdc4. Here, we isolated Nrd1, an RNA-binding protein with RNA-recognition motifs, as a multicopy suppressor of cdc4 mutants. Notably, we demonstrated that Nrd1 binds and stabilizes Cdc4 mRNA, thereby suppressing the cytokinesis defects of the cdc4 mutants. Importantly, Pmk1 mitogen-activated protein kinase (MAPK) directly phosphorylates Nrd1, thereby negatively regulating the binding activity of Nrd1 to Cdc4 mRNA. Consistently, the inactivation of Pmk1 MAPK signaling, as well as Nrd1 overexpression, stabilized the Cdc4 mRNA level, thereby suppressing the cytokinesis defects associated with the cdc4 mutants. In addition, we demonstrated the cell cycle-dependent regulation of Pmk1/Nrd1 signaling. Together, our results indicate that Nrd1 plays a role in the regulation of Cdc4 mRNA stability; moreover, our study is the first to demonstrate the posttranscriptional regulation of myosin expression by MAPK signaling.