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1.
Cell ; 174(1): 72-87.e32, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29861175

RESUMO

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.


Assuntos
Hipóxia Celular , Relógios Circadianos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Aminoácidos Dicarboxílicos/farmacologia , Animais , Proteínas CLOCK/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Relógios Circadianos/efeitos dos fármacos , Meios de Cultura/química , Fatores de Iniciação em Eucariotos , Concentração de Íons de Hidrogênio , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/metabolismo , Transcriptoma/efeitos dos fármacos , Proteína 2 do Complexo Esclerose Tuberosa/deficiência , Proteína 2 do Complexo Esclerose Tuberosa/genética
2.
J Biol Chem ; 299(4): 104595, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36898579

RESUMO

The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.


Assuntos
Fator de Iniciação 2 em Eucariotos , Proteínas Serina-Treonina Quinases , Humanos , Trifosfato de Adenosina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
3.
Semin Cancer Biol ; 33: 3-15, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25920797

RESUMO

A variety of cell intrinsic or extrinsic stresses evoke perturbations in the folding environment of the endoplasmic reticulum (ER), collectively known as ER stress. Adaptation to stress and re-establishment of ER homeostasis is achieved by activation of an integrated signal transduction pathway called the unfolded protein response (UPR). Both ER stress and UPR activation have been implicated in a variety of human cancers. Although at early stages or physiological conditions of ER stress, the UPR generally promotes survival, when the stress becomes more stringent or prolonged, its role can switch to a pro-cell death one. Here, we discuss historical and recent evidence supporting an involvement of the UPR in malignancy, describe the main mechanisms by which tumor cells overcome ER stress to promote their survival, tumor progression and metastasis and discuss the current state of efforts to develop therapeutic approaches of targeting the UPR.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Resposta a Proteínas não Dobradas , Adaptação Fisiológica , Animais , Apoptose , Autofagia , Linhagem da Célula , Senescência Celular , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Hipóxia , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Neoplasias/terapia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo
4.
J Med Chem ; 67(7): 5259-5271, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38530741

RESUMO

A series of activators of GCN2 (general control nonderepressible 2) kinase have been developed, leading to HC-7366, which has entered the clinic as an antitumor therapy. Optimization resulted in improved permeability compared to that of the original indazole hinge binding scaffold, while maintaining potency at GCN2 and selectivity over PERK (protein kinase RNA-like endoplasmic reticulum kinase). The improved ADME properties of this series led to robust in vivo compound exposure in both rats and mice, allowing HC-7366 to be dosed in xenograft models, demonstrating that activation of the GCN2 pathway by this compound leads to tumor growth inhibition.


Assuntos
Proteínas Serina-Treonina Quinases , eIF-2 Quinase , Humanos , Camundongos , Ratos , Animais , Proteínas Serina-Treonina Quinases/metabolismo , eIF-2 Quinase/metabolismo , Camundongos Endogâmicos C57BL , RNA , Retículo Endoplasmático/metabolismo
5.
Nat Cell Biol ; 21(7): 889-899, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31263264

RESUMO

The c-Myc oncogene drives malignant progression and induces robust anabolic and proliferative programmes leading to intrinsic stress. The mechanisms enabling adaptation to MYC-induced stress are not fully understood. Here we reveal an essential role for activating transcription factor 4 (ATF4) in survival following MYC activation. MYC upregulates ATF4 by activating general control nonderepressible 2 (GCN2) kinase through uncharged transfer RNAs. Subsequently, ATF4 co-occupies promoter regions of over 30 MYC-target genes, primarily those regulating amino acid and protein synthesis, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation. 4E-BP1 relieves MYC-induced proteotoxic stress and is essential to balance protein synthesis. 4E-BP1 activity is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation and inhibition of mTORC1 signalling rescues ATF4-deficient cells from MYC-induced endoplasmic reticulum stress. Acute deletion of ATF4 significantly delays MYC-driven tumour progression and increases survival in mouse models. Our results establish ATF4 as a cellular rheostat of MYC activity, which ensures that enhanced translation rates are compatible with survival and tumour progression.


Assuntos
Fator 4 Ativador da Transcrição/genética , Genes myc/genética , Ativação Transcricional/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Estresse do Retículo Endoplasmático/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Transgênicos , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas/fisiologia , Serina-Treonina Quinases TOR/metabolismo
7.
Cancer Discov ; 9(3): 396-415, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30563872

RESUMO

Resistance to BRAF and MEK inhibitors (BRAFi + MEKi) in BRAF-mutant tumors occurs through heterogeneous mechanisms, including ERK reactivation and autophagy. Little is known about the mechanisms by which ERK reactivation or autophagy is induced by BRAFi + MEKi. Here, we report that in BRAF-mutant melanoma cells, BRAFi + MEKi induced SEC61-dependent endoplasmic reticulum (ER) translocation of the MAPK pathway via GRP78 and KSR2. Inhibition of ER translocation prevented ERK reactivation and autophagy. Following ER translocation, ERK exited the ER and was rephosphorylated by PERK. Reactivated ERK phosphorylated ATF4, which activated cytoprotective autophagy. Upregulation of GRP78 and phosphorylation of ATF4 were detected in tumors of patients resistant to BRAFi + MEKi. ER translocation of the MAPK pathway was demonstrated in therapy-resistant patient-derived xenografts. Expression of a dominant-negative ATF4 mutant conferred sensitivity to BRAFi + MEKi in vivo. This mechanism reconciles two major targeted therapy resistance pathways and identifies druggable targets, whose inhibition would likely enhance the response to BRAFi + MEKi. SIGNIFICANCE: ERK reactivation and autophagy are considered distinct resistance pathways to BRAF + MEK inhibition (BRAFi + MEKi) in BRAF V600E cancers. Here, we report BRAFi + MEKi-induced ER translocation of the MAPK pathway is necessary for ERK reactivation, which drives autophagy. The ER translocation mechanism is a major druggable driver of resistance to targeted therapy.This article is highlighted in the In This Issue feature, p. 305.


Assuntos
Retículo Endoplasmático/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Autofagia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Masculino , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transporte Proteico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Cell Biol ; 20(1): 104-115, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29230015

RESUMO

The unfolded protein response (UPR) is a stress-activated signalling pathway that regulates cell proliferation, metabolism and survival. The circadian clock coordinates metabolism and signal transduction with light/dark cycles. We explore how UPR signalling interfaces with the circadian clock. UPR activation induces a 10 h phase shift in circadian oscillations through induction of miR-211, a PERK-inducible microRNA that transiently suppresses both Bmal1 and Clock, core circadian regulators. Molecular investigation reveals that miR-211 directly regulates Bmal1 and Clock via distinct mechanisms. Suppression of Bmal1 and Clock has the anticipated impact on expression of select circadian genes, but we also find that repression of Bmal1 is essential for UPR-dependent inhibition of protein synthesis and cell adaptation to stresses that disrupt endoplasmic reticulum homeostasis. Our data demonstrate that c-Myc-dependent activation of the UPR inhibits Bmal1 in Burkitt's lymphoma, thereby suppressing both circadian oscillation and ongoing protein synthesis to facilitate tumour progression.


Assuntos
Neoplasias Ósseas/genética , Relógios Circadianos/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Osteossarcoma/genética , eIF-2 Quinase/genética , Fatores de Transcrição ARNTL/antagonistas & inibidores , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas CLOCK/antagonistas & inibidores , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Xenoenxertos , Humanos , Transdução de Sinal Luminoso , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fotoperíodo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/metabolismo
9.
Sci Transl Med ; 10(439)2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720449

RESUMO

Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2-α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Antineoplásicos/uso terapêutico , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/fisiologia
10.
Cell Death Dis ; 9(11): 1108, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382078

RESUMO

Terminal differentiation opposes proliferation in the vast majority of tissue types. As a result, loss of lineage differentiation is a hallmark of aggressive cancers, including soft tissue sarcomas (STS). Consistent with these observations, undifferentiated pleomorphic sarcoma (UPS), an STS subtype devoid of lineage markers, is among the most lethal sarcomas in adults. Though tissue-specific features are lost in these mesenchymal tumors they are most commonly diagnosed in skeletal muscle, and are thought to develop from transformed muscle progenitor cells. We have found that a combination of HDAC (Vorinostat) and BET bromodomain (JQ1) inhibition partially restores differentiation to skeletal muscle UPS cells and tissues, enforcing a myoblast-like identity. Importantly, differentiation is partially contingent upon downregulation of the Hippo pathway transcriptional effector Yes-associated protein 1 (YAP1) and nuclear factor (NF)-κB. Previously, we observed that Vorinostat/JQ1 inactivates YAP1 and restores oscillation of NF-κB in differentiating myoblasts. These effects correlate with reduced tumorigenesis, and enhanced differentiation. However, the mechanisms by which the Hippo/NF-κB axis impact differentiation remained unknown. Here, we report that YAP1 and NF-κB activity suppress circadian clock function, inhibiting differentiation and promoting proliferation. In most tissues, clock activation is antagonized by the unfolded protein response (UPR). However, skeletal muscle differentiation requires both Clock and UPR activity, suggesting the molecular link between them is unique in muscle. In skeletal muscle-derived UPS, we observed that YAP1 suppresses PERK and ATF6-mediated UPR target expression as well as clock genes. These pathways govern metabolic processes, including autophagy, and their disruption shifts metabolism toward cancer cell-associated glycolysis and hyper-proliferation. Treatment with Vorinostat/JQ1 inhibited glycolysis/MTOR signaling, activated the clock, and upregulated the UPR and autophagy via inhibition of YAP1/NF-κB. These findings support the use of epigenetic modulators to treat human UPS. In addition, we identify specific autophagy, UPR, and muscle differentiation-associated genes as potential biomarkers of treatment efficacy and differentiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Musculares/genética , NF-kappa B/genética , Sarcoma/genética , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagia/efeitos dos fármacos , Azepinas/farmacologia , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/genética , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Musculares/tratamento farmacológico , Neoplasias Musculares/metabolismo , Neoplasias Musculares/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Mioblastos/patologia , NF-kappa B/metabolismo , Sarcoma/tratamento farmacológico , Sarcoma/metabolismo , Sarcoma/patologia , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologia , Triazóis/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Vorinostat/farmacologia , Proteínas de Sinalização YAP , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
11.
Cell Rep ; 6(6): 1046-1058, 2014 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-24613355

RESUMO

The ability to interconvert terminally differentiated cells could serve as a powerful tool for cell-based treatment of degenerative diseases, including diabetes mellitus. To determine which, if any, adult tissues are competent to activate an islet ß cell program, we performed an in vivo screen by expressing three ß cell "reprogramming factors" in a wide spectrum of tissues. We report that transient intestinal expression of these factors-Pdx1, MafA, and Ngn3 (PMN)-promotes rapid conversion of intestinal crypt cells into endocrine cells, which coalesce into "neoislets" below the crypt base. Neoislet cells express insulin and show ultrastructural features of ß cells. Importantly, intestinal neoislets are glucose-responsive and able to ameliorate hyperglycemia in diabetic mice. Moreover, PMN expression in human intestinal "organoids" stimulates the conversion of intestinal epithelial cells into ß-like cells. Our results thus demonstrate that the intestine is an accessible and abundant source of functional insulin-producing cells.


Assuntos
Células Secretoras de Insulina/citologia , Insulina/biossíntese , Intestinos/citologia , Ilhotas Pancreáticas/citologia , Animais , Diferenciação Celular/fisiologia , Humanos , Células Secretoras de Insulina/metabolismo , Mucosa Intestinal/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Transgênicos
12.
Autophagy ; 9(4): 612-4, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23328692

RESUMO

Stress in the tumor microenvironment in the form of hypoxia and low glucose/amino acid levels activates the evolutionarily conserved cellular adaptation program called the unfolded protein response (UPR) promoting cell survival in such conditions. Our recent studies showed that cell autonomous stress such as activation of the proto-oncogene MYC/c-Myc, can also trigger the UPR and induce endoplasmic reticulum (ER) stress-mediated autophagy. Amelioration of ER stress or autophagy enhances cancer cell death in vitro and attenuates tumor growth in vivo. Here we will discuss the role of the UPR and autophagy in MYC-induced transformation. Our findings demonstrate that the EIF2AK3/PERK-EIF2S1/eIF2α-ATF4 arm of the UPR promotes tumorigenesis by activating autophagy and enhancing tumor formation. Therefore, the UPR is an attractive target in MYC-driven cancers.


Assuntos
Autofagia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Humanos , Camundongos , Modelos Biológicos , Proto-Oncogene Mas , Transdução de Sinais , Resposta a Proteínas não Dobradas
13.
J Clin Invest ; 122(12): 4621-34, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23143306

RESUMO

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.


Assuntos
Autofagia , Linfoma de Burkitt/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Apoptose , Linfoma de Burkitt/patologia , Sinalização do Cálcio , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Análise por Conglomerados , Estresse do Retículo Endoplasmático , Técnicas de Inativação de Genes , Heterozigoto , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcriptoma , Resposta a Proteínas não Dobradas , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
14.
Mol Biol Cell ; 21(24): 4373-86, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20980617

RESUMO

Deletion of the paralogs ZDS1 and ZDS2 in the budding yeast Saccharomyces cerevisiae causes a mis-regulation of polarized cell growth. Here we show a function for these genes as regulators of the Swe1p (Wee1p) kinase-dependent G2/M checkpoint. We identified a conserved domain in the C-terminus of Zds2p consisting of amino acids 813-912 (hereafter referred to as ZH4 for Zds homology 4) that is required for regulation of Swe1p-dependent polarized bud growth. ZH4 is shown by protein affinity assays to be necessary and sufficient for interaction with Cdc55p, a regulatory subunit of protein phosphatase 2A (PP2A). We hypothesized that the Zds proteins are in a pathway that negatively regulates the Swe1p-dependent G2/M checkpoint via Cdc55p. Supporting this model, deletion of CDC55 rescues the aberrant bud morphology of a zds1Δzds2Δ strain. We also show that expression of ZDS1 or ZDS2 from a strong galactose-inducible promoter can induce mitosis even when the Swe1p-dependent G2/M checkpoint is activated by mis-organization of the actin cytoskeleton. This negative regulation requires the CDC55 gene. Together these data indicate that the Cdc55p/Zds2p module has a function in the regulation of the Swe1p-dependent G2/M checkpoint.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Sequência Conservada , Fase G2 , Deleção de Genes , Mitose , Mutação de Sentido Incorreto , Fosforilação , Mapeamento de Interação de Proteínas , Proteína Fosfatase 2/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Temperatura , Fatores de Tempo
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