RESUMEN
Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl--homeostasis, and Ppb1 deletion induces MgCl2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1+. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1+ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl2 in wild-type cells, the arsenite tolerance, but not MgCl2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl- homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.
Asunto(s)
Arsenitos , Calcineurina , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Calcineurina/metabolismo , Calcineurina/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Arsenitos/toxicidad , Arsenitos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Transducción de Señal , Proteínas de Unión al ADN , Proteínas MuscularesRESUMEN
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.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular , Neoplasias Pancreáticas , Humanos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fosforilación , Transducción de Señal , Neoplasias Pancreáticas/tratamiento farmacológico , Apoptosis , Sistema de Señalización de MAP Quinasas , Neoplasias PancreáticasRESUMEN
Protein kinase C (PKC) signaling is a highly conserved signaling module that plays a central role in a myriad of physiological processes, ranging from cell proliferation to cell death, via various signaling pathways, including MAPK signaling. Stress granules (SGs) are non-membranous cytoplasmic foci that aggregate in cells exposed to environmental stresses. Here, we explored the role of SGs in PKC/MAPK signaling activation in fission yeast. High-heat stress (HHS) induced Pmk1 MAPK activation and Pck2 translocation from the cell tips into poly(A)-binding protein (Pabp)-positive SGs. Pck2 dispersal from the cell tips required Pck2 kinase activity, and constitutively active Pck2 exhibited increased translocation to SGs. Importantly, Pmk1 deletion impaired Pck2 recruitment to SGs, indicating that MAPK activation stimulates Pck2 SG translocation. Consistently, HHS-induced SGs delayed Pck2 relocalization at the cell tips, thereby blocking subsequent Pmk1 reactivation after recovery from HHS. HHS partitioned Pck2 into the Pabp-positive SG-containing fraction, which resulted in reduced Pck2 abundance and kinase activity in the soluble fraction. Taken together, these results indicate that MAPK-dependent Pck2 SG recruitment serves as a feedback mechanism to intercept PKC/MAPK activation induced by HHS, which might underlie PKC-related diseases.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Gránulos Citoplasmáticos/metabolismo , Retroalimentación , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Estrés FisiológicoRESUMEN
Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3+ gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3+ and puf4+, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3+ and puf4+ genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Alcoholes Bencílicos/farmacología , Regulación hacia Abajo/genética , Fosfatasa 6 de Especificidad Dual/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptor ErbB-2/metabolismo , Animales , Apoptosis/genética , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Fosfatasa 6 de Especificidad Dual/metabolismo , Ratones , Células 3T3 NIH , OncogenesRESUMEN
FTY720, a sphingosine-1-phosphate (S1P) analog, is used as an immune modulator to treat multiple sclerosis. Accumulating evidence has suggested the mode of action of FTY720 independent of an S1P modulator. In fission yeast, FTY720 induces an increase in intracellular Ca2+ and ROS levels. We have previously identified 49 genes of which deletion causes FTY720 sensitivity. Here, we characterized the FTY720-sensitive mutants in terms of their relevance to the Ca2+ homeostasis and identified the 16 FTY720- and Ca2+ -sensitive mutants (fcs mutants). Most of the FTY720-sensitive mutants showed elevated Ca2+ levels and exhibited Ca2+ dysregulation by FTY720 treatment. One of the functional categories among the genes whose deletion renders cells susceptible to FTY720 and Ca2+ include the Golgi/endosomal membrane trafficking. Notably, FTY720, but not phosphorylated FTY720 incapable of inducing Ca2+ increase, inhibited the secretion of acid phosphatase in the wild-type cells. Importantly, secretory defects of the Golgi/endosomal trafficking mutants, Vps45, or Ryh1 deletion, were further exacerbated by FTY720. Our fcs mutant screen also identified the adenylyl cyclase-associated protein Cap1 and a Rictor homolog Ste20, whose deletion markedly exacerbated FTY720-sensitive secretory impairment. Collectively, our data may suggest a synergistic impact of FTY720 combined with secretion perturbation on proliferation and Ca2+ homeostasis.
Asunto(s)
Calcio/metabolismo , Endosomas/efectos de los fármacos , Clorhidrato de Fingolimod/farmacología , Aparato de Golgi/efectos de los fármacos , Transporte Biológico , Endosomas/metabolismo , Eliminación de Gen , Aparato de Golgi/metabolismo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Compuestos de Bencidrilo/farmacología , Carbonatos/farmacología , Descubrimiento de Drogas , Ésteres/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Compuestos de Bencidrilo/síntesis química , Compuestos de Bencidrilo/química , Carbonatos/síntesis química , Carbonatos/química , Relación Dosis-Respuesta a Droga , Ésteres/síntesis química , Ésteres/química , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/metabolismo , Melanoma/patología , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
We have previously identified the KH-type RNA-binding protein Rnc1 as an important regulator of the posttranscriptional expression of the MAPK phosphatase Pmp1 in fission yeast. Rnc1 localization in response to stress has not been elucidated thus far. Here, we report the dual roles of Rnc1 in assembly of stress granules (SGs), nonmembranous cytoplasmic foci composed of messenger ribonucleoproteins. Rnc1 can localize to poly(A)-binding protein (Pabp)-positive SGs upon various stress stimuli, including heat shock (HS) and arsenite treatment. Furthermore, Rnc1 deletion results in decreased SGs, indicating that Rnc1 is a new component and a regulator of SGs. Notably, Rnc1 translocates to the dot-like structures faster than Pabp, and this stress-induced Rnc1 translocation does not require its RNA-binding ability, as the Rnc1KH1,2,3GD mutant protein with impaired RNA-binding activity forms dots rather more efficiently than the wild-type Rnc1 upon HS. Interestingly, in the absence of stress, Rnc1 overproduction induced massive aggregation of Pabp-positive SGs and eIF2α phosphorylation. In clear contrast, overproduction of the Rnc1KH1,2,3GD mutant failed to induce Pabp aggregation and eIF2α phosphorylation, indicating that Rnc1 overproduction-induced SG assembly requires Rnc1 RNA-binding activity. Collectively, Rnc1 regulates SG assembly, dependently or independently of its RNA-binding activity.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Arsenitos/farmacología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Mutación , Fosforilación , Proteínas de Unión a Poli(A)/metabolismo , Dominios Proteicos , Proteínas de Unión al ARN/genética , Schizosaccharomyces/efectos de los fármacos , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/metabolismo , Células Germinativas/fisiología , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Animales , Animales Modificados Genéticamente , Blastómeros/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina , Femenino , Regulación de la Expresión Génica/genética , Células Germinativas/citología , Células Germinativas/metabolismo , Masculino , ARN/genética , ARN Mensajero/genéticaRESUMEN
Chromatin immunoprecipitation identifies specific interactions between genomic DNA and proteins, advancing our understanding of gene-level and chromosome-level regulation. Based on chromatin immunoprecipitation experiments using validated antibodies, we define the genome-wide distributions of 19 histone modifications, one histone variant, and eight chromatin-associated proteins in Caenorhabditis elegans embryos and L3 larvae. Cluster analysis identified five groups of chromatin marks with shared features: Two groups correlate with gene repression, two with gene activation, and one with the X chromosome. The X chromosome displays numerous unique properties, including enrichment of monomethylated H4K20 and H3K27, which correlate with the different repressive mechanisms that operate in somatic tissues and germ cells, respectively. The data also revealed striking differences in chromatin composition between the autosomes and between chromosome arms and centers. Chromosomes I and III are globally enriched for marks of active genes, consistent with containing more highly expressed genes, compared to chromosomes II, IV, and especially V. Consistent with the absence of cytological heterochromatin and the holocentric nature of C. elegans chromosomes, markers of heterochromatin such as H3K9 methylation are not concentrated at a single region on each chromosome. Instead, H3K9 methylation is enriched on chromosome arms, coincident with zones of elevated meiotic recombination. Active genes in chromosome arms and centers have very similar histone mark distributions, suggesting that active domains in the arms are interspersed with heterochromatin-like structure. These data, which confirm and extend previous studies, allow for in-depth analysis of the organization and deployment of the C. elegans genome during development.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Cromosomas/metabolismo , Histonas/metabolismo , Animales , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Genes Ligados a X/genética , Histonas/genética , Metilación , Metiltransferasas/metabolismo , Membrana Nuclear/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
Various stress conditions, such as heat stress (HS) and oxidative stress, can cause biomolecular condensates represented by stress granules (SGs) via liquid-liquid phase separation. We have previously shown that Hsp90 forms aggregates in response to HS and that Hsp90 aggregates transiently co-localize with SGs as visualized by Pabp. Here, we showed that arsenite, one of the well-described SG-inducing stimuli, induces Hsp90 aggregates distinct from conventional SGs in fission yeast. Arsenite induced Hsp90 granules in a dose-dependent manner, and these granules were significantly diminished by the co-treatment with a ROS scavenger N-acetyl cysteine (NAC), indicating that ROS are required for the formation of Hsp90 granules upon arsenite stress. Notably, Hsp90 granules induced by arsenite do not overlap with conventional SGs as represented by eIF4G or Pabp, while HS-induced Hsp90 granules co-localize with SGs. Nrd1, an RNA-binding protein known as a HS-induced SG component, was recruited into Hsp90 aggregates but not to the conventional SGs upon arsenite stress. The non-phosphorylatable eIF2α mutants significantly delayed the Hsp90 granule formation upon arsenite treatment. Importantly, inhibition of Hsp90 by geldanamycin impaired the Hsp90 granule formation and reduced the arsenite tolerance. Collectively, arsenite stimulates two types of distinct aggregates, namely conventional SGs and a novel type of aggregates containing Hsp90 and Nrd1, wherein Hsp90 plays a role as a center for aggregation, and stress-specific compartmentalization of biomolecular condensates.
RESUMEN
The extracellular signal-regulated kinase (ERK) MAPK pathway is dysregulated in various human cancers and is considered an attractive therapeutic target for cancer. Therefore, several inhibitors of this pathway are being developed, and some are already used in the clinic. We have previously identified an anticancer compound, ACA-28, with a unique property to preferentially induce ERK-dependent apoptosis in melanoma cells. To comprehensively understand the biological cellular impact induced by ACA-28, we performed a global gene expression analysis of human melanoma SK-MEL-28 cells exposed to ACA-28 using a DNA microarray. The transcriptome analysis identified nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcription factor that combats oxidative stress, as the most upregulated genetic pathway after ACA-28 treatment. Consistently, ACA-28 showed properties to increase the levels of reactive oxygen species (ROS) as well as Nrf2 protein, which is normally repressed by proteasomal degradation and activated in response to oxidative stresses. Furthermore, the ROS scavenger N-acetyl cysteine significantly attenuated the anticancer activity of ACA-28. Thus, ACA-28 activates Nrf2 signaling and exerts anticancer activity partly via its ROS-stimulating property. Interestingly, human A549 cancer cells with constitutively high levels of Nrf2 protein showed resistance to ACA-28, as compared with SK-MEL-28. Transient overexpression of Nrf2 also increased the resistance of cells to ACA-28, while knockdown of Nrf2 exerted the opposite effect. Thus, upregulation of Nrf2 signaling protects cancer cells from ACA-28-mediated cell death. Notably, the Nrf2 inhibitor ML385 substantially enhanced the cell death-inducing property of ACA-28 in pancreatic cancer cells, T3M4 and PANC-1. Our data suggest that Nrf2 plays a key role in determining cancer cell susceptibility to ACA-28 and provides a novel strategy for cancer therapy to combine the Nrf2 inhibitor and ACA-28.
Asunto(s)
Melanoma , Neoplasias Pancreáticas , Humanos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Melanoma/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Neoplasias Pancreáticas/tratamiento farmacológicoRESUMEN
Methylation of histone H3K36 in higher eukaryotes is mediated by multiple methyltransferases. Set2-related H3K36 methyltransferases are targeted to genes by association with RNA Polymerase II and are involved in preventing aberrant transcription initiation within the body of genes. The targeting and roles of the NSD family of mammalian H3K36 methyltransferases, known to be involved in human developmental disorders and oncogenesis, are not known. We used genome-wide chromatin immunoprecipitation (ChIP) to investigate the targeting and roles of the Caenorhabditis elegans NSD homolog MES-4, which is maternally provided to progeny and is required for the survival of nascent germ cells. ChIP analysis in early C. elegans embryos revealed that, consistent with immunostaining results, MES-4 binding sites are concentrated on the autosomes and the leftmost approximately 2% (300 kb) of the X chromosome. MES-4 overlies the coding regions of approximately 5,000 genes, with a modest elevation in the 5' regions of gene bodies. Although MES-4 is generally found over Pol II-bound genes, analysis of gene sets with different temporal-spatial patterns of expression revealed that Pol II association with genes is neither necessary nor sufficient to recruit MES-4. In early embryos, MES-4 associates with genes that were previously expressed in the maternal germ line, an interaction that does not require continued association of Pol II with those loci. Conversely, Pol II association with genes newly expressed in embryos does not lead to recruitment of MES-4 to those genes. These and other findings suggest that MES-4, and perhaps the related mammalian NSD proteins, provide an epigenetic function for H3K36 methylation that is novel and likely to be unrelated to ongoing transcription. We propose that MES-4 transmits the memory of gene expression in the parental germ line to offspring and that this memory role is critical for the PGCs to execute a proper germline program.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Embrión no Mamífero/enzimología , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/enzimología , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Sitios de Unión , Biocatálisis , Caenorhabditis elegans/embriología , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/genética , Embrión no Mamífero/citología , Femenino , Genes de Helminto/genética , Células Germinativas/citología , Histona Metiltransferasas , Histonas/metabolismo , Lisina/metabolismo , Metilación , Sistemas de Lectura Abierta/genética , Unión Proteica , Interferencia de ARN , ARN Polimerasa II/metabolismo , Cromosoma X/metabolismoRESUMEN
Autophagy promotes or inhibits cell death depending on the environment and cell type. Our previous findings suggested that Atg1 is genetically involved in the regulation of Pmk1 MAPK in fission yeast. Here, we showed that Δatg1 displays lower levels of Pmk1 MAPK phosphorylation than did the wild-type (WT) cells upon treatment with a 1,3-ß-D-glucan synthase inhibitor micafungin or CaCl2, both of which activate Pmk1. Moreover, the overproduction of Atg1, but not that of the kinase inactivating Atg1D193A activates Pmk1 without any extracellular stimuli, suggesting that Atg1 may promote Pmk1 MAPK signaling activation. Notably, the overproduction of Atg1 induces a toxic effect on the growth of WT cells and the deletion of Pmk1 failed to suppress the cell death induced by Atg1, indicating that the Atg1-mediated cell death requires additional mechanism(s) other than Pmk1 activation. Moreover, atg1 gene deletion induces tolerance to micafungin and CaCl2, whereas pmk1 deletion induces severe sensitivities to these compounds. The Δatg1Δpmk1 double mutants display intermediate sensitivities to these compounds, showing that atg1 deletion partly suppressed growth inhibition induced by Δpmk1. Thus, Atg1 may act to promote cell death upon micafungin and CaCl2 stimuli regardless of Pmk1 MAPK activity. Since micafungin and CaCl2 are intracellular calcium inducers, our data reveal a novel role of the autophagy regulator Atg1 to induce cell death upon calcium overload independent of its role in Pmk1 MAPK activation.
RESUMEN
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.
RESUMEN
Apart from the highly conserved role in the cellular degradation process, autophagy also appears to play a key role in cellular proliferation. Here, we describe the genetic interaction of autophagy-related genes and Pmk1 MAPK signaling in fission yeast. atg1 deletion cells (Δ atg1 ) exhibit the vic (viable in the presence of immunosuppressant and Cl - ) phenotype, indicative of Pmk1 signaling inhibition. Moreover, the Δ atg1 Δ pmk1 double mutant resembles the single Δ pmk1 mutant, suggesting that Atg1 functions in the Pmk1 pathway. In addition, the growth defect induced by overexpression of Pck2, an upstream activator of Pmk1 MAPK was alleviated by the deletion of atg1 + . Finally, the deletion of autophagy-related genes recapitulates Pmk1 MAPK signaling inhibition. Our data suggest a novel role for autophagy in MAPK signaling regulation.
RESUMEN
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.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Apoptosis , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMEN
Calcineurin (CN) is a conserved Ca2+-calmodulin activated protein phosphatase, which plays important roles in immune regulation, cardiac hypertrophy, and apoptosis in humans. In pathogenic fungi, CN is essential for stress survival, sexual development, and virulence. The immunosuppressant tacrolimus (FK506) is a specific inhibitor of CN in humans and fungi including nonpathogenic fission yeast. Although calcineurin inhibition by FK506 or CN deletion in fission yeast does not induce growth defects, treatment with some anti-fungal drugs such as micafungin and valproic acid, induced synthetic lethality with calcineurin inhibition. Here, we searched for the compounds that induce synthetic growth defects with CN inhibition in fission yeast. We found that ellagic acid (EA) preferentially induced growth inhibition in CN deletion cells. Consistently, co-treatment with EA and FK506 induced severe growth inhibition in the wild-type cells, whereas neither of the single treatment with each compound did so. Moreover, deletion of the calcineurin-regulated transcription factor Prz1 also induced a marked EA sensitivity. Intriguingly, EA also enhanced the growth inhibitory effect of other anti-fungal drugs, including micafungin and miconazole. Thus, our data suggesting the synergistic growth inhibitory effect of the calcineurin inhibitor FK506 and EA may be useful to understand the mechanism to overcome the antifungal resistance.
Asunto(s)
Ácido Elágico , Schizosaccharomyces , Tacrolimus , Calcineurina/metabolismo , Inhibidores de la Calcineurina/farmacología , Ácido Elágico/farmacología , Humanos , Schizosaccharomyces/efectos de los fármacos , Tacrolimus/farmacologíaRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Apoptosis/fisiología , Fosfatasas de Especificidad Dual/metabolismo , Humanos , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The molecular chaperone Hsp90 is highly conserved from bacteria to mammals. In fission yeast, Hsp90 is essential in many cellular processes and its expression is known to be increased by heat stress (HS). Here, we describe the distinct spatiotemporal distribution of Hsp90 under high-heat stress (HHS: 45ËC) and mild-heat stress (MHS: 37ËC). Hsp90 is largely distributed in the cytoplasm under non-stressed conditions (27ËC). Under HHS, Hsp90 forms several cytoplasmic granules within 5 minutes, then the granules disappear within 60 minutes. Under MHS, Hsp90 forms fewer granules than under HHS within 5 minutes and strikingly the granules persist and grow in size. In addition, nuclear enrichment of Hsp90 was observed after 60 minutes under both HS conditions. Our data suggest that assembly/disassembly of Hsp90 granules is differentially regulated by temperatures.