RESUMEN
Occurrence of resistance to olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) approved in ovarian carcinoma, has already been shown in clinical settings. Identifying combination treatments to sensitize tumor cells and/or overcome resistance to olaparib is critical. Polo-like kinase 1 (PLK1), a master regulator of mitosis, is also involved in the DNA damage response promoting homologous recombination (HR)-mediated DNA repair and in the recovery from the G2/M checkpoint. We hypothesized that PLK1 inhibition could sensitize tumor cells to PARP inhibition. Onvansertib, a highly selective PLK1 inhibitor, and olaparib were tested in vitro and in vivo in BRCA1 mutated and wild-type (wt) ovarian cancer models, including patient-derived xenografts (PDXs) resistant to olaparib. The combination of onvansertib and olaparib was additive or synergic in different ovarian cancer cell lines, causing a G2/M block of the cell cycle, DNA damage, and apoptosis, much more pronounced in cells treated with the two drugs as compared to controls and single agents treated cells. The combined treatment was well tolerated in vivo and resulted in tumor growth inhibition and a statistically increased survival in olaparib-resistant-BRCA1 mutated models. The combination was also active, although to a lesser extent, in BRCA1 wt PDXs. Pharmacodynamic analyses showed an increase in mitotic, apoptotic, and DNA damage markers in tumor samples derived from mice treated with the combination versus vehicle. We could demonstrate that in vitro onvansertib inhibited both HR and non-homologous end-joining repair pathways and in vivo induced a decrease in the number of RAD51 foci-positive tumor cells, supporting its ability to induce HR deficiency and favoring the activity of olaparib. Considering that the combination was well tolerated, these data support and foster the clinical evaluation of onvansertib with PARPis in ovarian cancer, particularly in the PARPis-resistant setting.
Asunto(s)
Resistencia a Antineoplásicos , Neoplasias Ováricas , Ftalazinas , Piperazinas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Femenino , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Animales , Línea Celular Tumoral , Ratones , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacosRESUMEN
Abiraterone is a standard treatment for metastatic castrate-resistant prostate cancer (mCRPC) that slows disease progression by abrogating androgen synthesis and antagonizing the androgen receptor (AR). Here we report that inhibitors of the mitotic regulator polo-like kinase-1 (Plk1), including the clinically active third-generation Plk1 inhibitor onvansertib, synergizes with abiraterone in vitro and in vivo to kill a subset of cancer cells from a wide variety of tumor types in an androgen-independent manner. Gene-expression analysis identified an AR-independent synergy-specific gene set signature upregulated upon abiraterone treatment that is dominated by pathways related to mitosis and the mitotic spindle. Abiraterone treatment alone caused defects in mitotic spindle orientation, failure of complete chromosome condensation, and improper cell division independently of its effects on AR signaling. These effects, although mild following abiraterone monotherapy, resulted in profound sensitization to the antimitotic effects of Plk1 inhibition, leading to spindle assembly checkpoint-dependent mitotic cancer cell death and entosis. In a murine patient-derived xenograft model of abiraterone-resistant metastatic castration-resistant prostate cancer (mCRPC), combined onvansertib and abiraterone resulted in enhanced mitotic arrest and dramatic inhibition of tumor cell growth compared with either agent alone. Overall, this work establishes a mechanistic basis for the phase II clinical trial (NCT03414034) testing combined onvansertib and abiraterone in mCRPC patients and indicates this combination may have broad utility for cancer treatment. SIGNIFICANCE: Abiraterone treatment induces mitotic defects that sensitize cancer cells to Plk1 inhibition, revealing an AR-independent mechanism for this synergistic combination that is applicable to a variety of cancer types.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Animales , Ratones , Receptores Androgénicos/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Andrógenos , MitosisRESUMEN
Stage IA gastric adenocarcinoma, characterized by foci of intramucosal signet ring cells (SRC), is found in nearly all asymptomatic patients with germline pathogenic CDH1 variants and hereditary diffuse gastric cancer syndrome (HDGC). The molecular steps involved in initiating malignant transformation and promoting SRC dormancy in HDGC are unknown. Here, whole-exome bulk RNA sequencing (RNA-seq) of SRCs and adjacent non-SRC epithelium (NEP) was performed on laser-capture microdissected (LCM) regions of interest found in risk-reducing total gastrectomy specimens from patients with HDGC (Clinicaltrials.gov ID: NCT03030404). In total, 20 patients (6 male, 14 female) with confirmed HDGC were identified. Analysis of differentially expressed genes (DEG) demonstrated upregulation of certain individual EMT and proliferation genes. However, no oncogenic pathways were found to be upregulated in SRCs. Rather, SRC regions had significant enrichment in pathways involved in T-cell signaling. CIBERSORTx predicted significant increases in the presence of regulatory T cells (Treg) specific to SRC regions. IHC confirmed an increase in FOXP3+ cells in SRC foci, as well as elevations in CD4+ T cells and HLA-DR staining. In summary, the tumor immune microenvironment is microscopically inseparable from stage IA gastric SRCs using a granular isolation technique. An elevation in CD4+ T cells within SRC regions correlates with clinically observed SRC dormancy, while Treg upregulation represents a potential immune escape mechanism. IMPLICATIONS: Characterization of the tumor-immune microenvironment in HDGC underscores the potential for the immune system to shape the transcriptional profile of the earliest tumors, which suggests immune-directed therapy as a potential cancer interception strategy in diffuse-type gastric cancer.
Asunto(s)
Adenocarcinoma , Carcinoma de Células en Anillo de Sello , Neoplasias Gástricas , Humanos , Masculino , Femenino , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Predisposición Genética a la Enfermedad , Gastrectomía , Mutación de Línea Germinal , Carcinogénesis/genética , Carcinoma de Células en Anillo de Sello/genética , Carcinoma de Células en Anillo de Sello/patología , Carcinoma de Células en Anillo de Sello/cirugía , Cadherinas/genética , Microambiente Tumoral , Antígenos CDRESUMEN
5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.
RESUMEN
Elevated external solute stimulates a conserved MAPK cascade that elicits responses that maintain osmotic balance. The yeast high-osmolarity glycerol (HOG) pathway activates Hog1 MAPK (mammalian ortholog p38alpha/SAPKalpha), which enters the nucleus and induces expression of >50 genes, implying that transcriptional up-regulation is necessary to cope with hyperosmotic stress. Contrary to this expectation, we show here that cells lacking the karyopherin required for Hog1 nuclear import or in which Hog1 is anchored at the plasma membrane (or both) can withstand long-term hyperosmotic challenge by ionic and nonionic solutes without exhibiting the normal change in transcriptional program (comparable with hog1Delta cells), as judged by mRNA hybridization and microarray analysis. For such cells to survive hyperosmotic stress, systematic genetic analysis ruled out the need for any Hog1-dependent transcription factor, the Hog1-activated MAPKAP kinases, or ion, glycerol, and water channels. By contrast, enzymes needed for glycerol production were essential for viability. Thus, control of intracellular glycerol formation by Hog1 is critical for maintenance of osmotic balance but not transcriptional induction of any gene.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/fisiología , Presión Osmótica , Proteínas de Saccharomyces cerevisiae/fisiología , Transducción de Señal , Glicerol/metabolismo , Sistema de Señalización de MAP Quinasas , Redes y Vías Metabólicas , Saccharomyces cerevisiae/fisiología , Transcripción GenéticaRESUMEN
Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Feromonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión al GTP cdc42/genética , Dominio Catalítico/genética , Regulación Fúngica de la Expresión Génica/genética , Genes del Tipo Sexual de los Hongos/genética , Fosfoproteínas Fosfatasas/genética , Fosforilación/genética , Saccharomyces cerevisiae/genética , Transducción de Señal/genéticaRESUMEN
Chronic inflammation increases the risk for colorectal cancer through a variety of mechanisms involving the tumor microenvironment. MAPK-activated protein kinase 2 (MK2), a major effector of the p38 MAPK stress and DNA damage response signaling pathway, and a critical regulator of pro-inflammatory cytokine production, has been identified as a key contributor to colon tumorigenesis under conditions of chronic inflammation. We have previously described how genetic inactivation of MK2 in an inflammatory model of colon cancer results in delayed tumor progression, decreased tumor angiogenesis, and impaired macrophage differentiation into a pro-tumorigenic M2-like state. The molecular mechanism responsible for the impaired angiogenesis and tumor progression, however, has remained contentious and poorly defined. Here, using RNA expression analysis, assays of angiogenesis factors, genetic models, in vivo macrophage depletion and reconstitution of macrophage MK2 function using adoptive cell transfer, we demonstrate that MK2 activity in macrophages is necessary and sufficient for tumor angiogenesis during inflammation-induced cancer progression. We identify a critical and previously unappreciated role for MK2-dependent regulation of the well-known pro-angiogenesis factor CXCL-12/SDF-1 secreted by tumor associated-macrophages, in addition to MK2-dependent regulation of Serpin-E1/PAI-1 by several cell types within the tumor microenvironment.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Neoplasias Asociadas a Colitis/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neovascularización Patológica , Proteínas Serina-Treonina Quinasas/metabolismo , Macrófagos Asociados a Tumores/enzimología , Traslado Adoptivo , Proteínas Angiogénicas/genética , Animales , Células Cultivadas , Neoplasias Asociadas a Colitis/genética , Neoplasias Asociadas a Colitis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Transcripción Genética , Microambiente Tumoral , Macrófagos Asociados a Tumores/trasplanteRESUMEN
In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Reparación del ADN/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Células HCT116 , Humanos , Immunoblotting , Ratones , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Nanomedicina/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Although elevated levels of reactive oxygen species (ROS) have been observed in cancer cells and cancer cells aberrantly proliferate, it is not known whether the level of reactive oxygen species and the accumulation of oxidative damage to macromolecules vary across the cell cycle. Here, we measure the prevalence of reactive oxygen species and of biomolecule oxidation across the cell cycle in freely cycling cancer cells. We report that reactive oxygen species vary during the cell cycle and peak in mitosis, resulting in mitotic accumulation of oxidized protein cysteine residues. Prolonged mitotic arrest further increased the levels of ROS and the abundance of oxidatively damaged biomolecules, including cysteine-sulfenic-acid-containing proteins and 8-oxoguanine. These finding suggest that mitotic arrest agents may enhance the effects of ROS-dependent anticancer therapies.
Asunto(s)
Ciclo Celular/genética , Especies Reactivas de Oxígeno/metabolismo , Humanos , Mitosis , Estrés OxidativoRESUMEN
There is an unmet need for new antimitotic drug combinations that target cancer-specific vulnerabilities. Based on our finding of elevated biomolecule oxidation in mitotically arrested cancer cells, we combined Plk1 inhibitors with TH588, an MTH1 inhibitor that prevents detoxification of oxidized nucleotide triphosphates. This combination showed robust synergistic killing of cancer, but not normal, cells that, surprisingly, was MTH1-independent. To dissect the underlying synergistic mechanism, we developed VISAGE, a strategy integrating experimental synergy quantification with computational-pathway-based gene expression analysis. VISAGE predicted, and we experimentally confirmed, that this synergistic combination treatment targeted the mitotic spindle. Specifically, TH588 binding to ß-tubulin impaired microtubule assembly, which when combined with Plk1 blockade, synergistically disrupted mitotic chromosome positioning to the spindle midzone. These findings identify a cancer-specific mitotic vulnerability that is targetable using Plk1 inhibitors with microtubule-destabilizing agents and highlight the general utility of the VISAGE approach to elucidate molecular mechanisms of drug synergy.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores de Crecimiento/uso terapéutico , Neoplasias/tratamiento farmacológico , Pirimidinas/uso terapéutico , Huso Acromático/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Biología Computacional , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Humanos , Terapia Molecular Dirigida , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Huso Acromático/fisiología , Tubulina (Proteína)/metabolismo , Quinasa Tipo Polo 1RESUMEN
Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability.
Asunto(s)
Neoplasias Encefálicas/patología , Glioma/patología , Intrones , Proteína-Arginina N-Metiltransferasas/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Isoquinolinas/farmacología , Ratones , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Pirimidinas/farmacología , Empalme del ARNRESUMEN
Yeast prions, based on self-seeded highly ordered fibrous aggregates (amyloids), serve as a model for human amyloid diseases. Propagation of yeast prions depends on the balance between chaperones of the Hsp100 and Hsp70 families. The yeast prion [PSI(+)] can be eliminated by an excess of the chaperone Hsp104. This effect is reversed by an excess of the chaperone Hsp70-Ssa. Here we show that the actions of Hsp104 and Ssa on [PSI(+)] are modulated by the small glutamine-rich tetratricopeptide cochaperone Sgt2. Sgt2 is conserved from yeast to humans, has previously been implicated in the guided entry of tail-anchored proteins (GET) trafficking pathway, and is known to interact with Hsps, cytosolic Get proteins, and tail-anchored proteins. We demonstrate that Sgt2 increases the ability of excess Ssa to counteract [PSI(+)] curing by excess Hsp104. Deletion of SGT2 also restores trafficking of a tail-anchored protein in cells with a disrupted GET pathway. One region of Sgt2 interacts both with the prion domain of Sup35 and with tail-anchored proteins. Sgt2 levels are increased in response to the presence of a prion when major Hsps are not induced. Our data implicate Sgt2 as an amyloid "sensor" and a regulator of chaperone targeting to different types of aggregation-prone proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Chaperonas Moleculares/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Portadoras/genética , Eliminación de Gen , Genes Fúngicos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Modelos Biológicos , Chaperonas Moleculares/genética , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eukaryotic cells use multiple mitogen-activated protein kinase (MAPK) cascades to evoke appropriate responses to external stimuli. In Saccharomyces cerevisiae, the MAPK Fus3 is activated by pheromone-binding heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptors to promote mating, whereas the MAPK Hog1 is activated by hyperosmotic stress to elicit the high-osmolarity glycerol (HOG) response. Although these MAPK pathways share several upstream components, exposure to either pheromone or osmolyte alone triggers only the appropriate response. We used fluorescence localization- and transcription-specific reporters to assess activation of these pathways in individual cells on the minute and hour time scale, respectively. Dual activation of these two MAPK pathways occurred over a broad range of stimulant concentrations and temporal regimes in wild-type cells subjected to costimulation. Thus, signaling specificity is achieved through an "insulation" mechanism, not a "cross-inhibition" mechanism. Furthermore, we showed that there was a critical period during which Hog1 activity had to occur for proper insulation of the HOG pathway.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/genética , Presión Osmótica/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Cellular responses to many external stimuli are mediated by mitogen-activated protein kinases (MAPKs). We investigated whether dynamic intracellular movement contributes to the spatial and temporal characteristics of the responses elicited by a prototypic MAPK, Fus3, in the mating pheromone response pathway in budding yeast (Saccharomyces cerevisiae). Confining Fus3 in the nucleus, via fusion to a histone H2B, reduced MAPK activation and diminished all responses (pheromone-induced gene expression, cell cycle arrest, projection formation, and mating). Elimination of MAPK phosphatases restored more robust outputs for all responses, indicating that nuclear sequestration impedes full MAPK activation but does not abrogate its functional competence. Restricting Fus3 to the plasma membrane, via fusion to a lipid-modified CCaaX motif, led to MAPK hyperactivation yet severely impaired all response outputs. Fus3-CCaaX also caused aberrant cell morphology and a proliferation defect. Unlike similar phenotypes induced by pathway hyperactivation via upstream components, these deleterious effects were independent of the downstream transcription factor Ste12. Thus, appropriate cellular responses require free subcellular MAPK transit to disseminate MAPK activity optimally because preventing dynamic MAPK movement either markedly impaired signal-dependent activation and/or resulted in improper biological outputs.