RESUMEN
RNF144A is a DNA damage-induced E3 ubiquitin ligase that targets proteins involved in genome instability for degradation, e.g., DNA-PKcs and BMI1. RNF144A is frequently mutated or epigenetically silenced in cancer, providing the rationale to evaluate RNF144A loss of function in tumorigenesis. Here we report that RNF144A-deficient mice are more prone to the development of bladder tumors upon carcinogen exposure. In addition to DNA-PKcs and BMI1, we identify the immune checkpoint protein PD-L1 as a novel degradation target of RNF144A, since these proteins are expressed at higher levels in Rnf144a KO tumors. RNF144A interacts with PD-L1 in the plasma membrane and intracellular vesicles and promotes poly-ubiquitination and degradation of PD-L1. Therefore, Rnf144a KO stabilizes PD-L1 and leads to a reduction of tumor-infiltrating CD8+ T cell populations in the BBN-induced bladder tumors. The bladder tumors developed in WT and Rnf144a KO mice primarily express CK5 and CK14, markers of basal cancer subtype, as expected in BBN-induced bladder tumors. Intriguingly, the Rnf144a KO tumors also express GATA3, a marker for the luminal subtype, suggesting that RNF144A loss of function promotes features of cellular differentiation. Such differentiation features in Rnf144a KO tumors likely result from a decrease of EGFR expression, consistent with the reported role of RNF144A in maintaining EGFR expression. In summary, for the first time our study demonstrates the in vivo tumor suppressor activity of RNF144A upon carcinogenic insult. Loss of RNF144A promotes the expression of DNA-PKcs, BMI1 and PD-L1, likely contributing to the carcinogen-induced bladder tumorigenesis.
Asunto(s)
Antígeno B7-H1/genética , Carcinogénesis/genética , Proteínas Portadoras/genética , Complejo Represivo Polycomb 1/genética , Proteínas Proto-Oncogénicas/genética , Ubiquitina-Proteína Ligasas/genética , Neoplasias de la Vejiga Urinaria/genética , Animales , Carcinógenos/toxicidad , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Inestabilidad Genómica/genética , Humanos , Ratones , Ratones Noqueados , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitinación , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: CGRRF1 is a growth suppressor and consists of a transmembrane domain and a RING-finger domain. It functions as a RING domain E3 ubiquitin ligase involved in endoplasmic reticulum-associated degradation. The expression of CGRRF1 is decreased in cancer tissues; however, the role of CGRRF1 in breast cancer and the mechanism(s) of its growth suppressor function remain to be elucidated. METHODS: To investigate whether CGRRF1 inhibits the growth of breast cancer, we performed MTT assays and a xenograft experiment. Tumors harvested from mice were further analyzed by reverse phase protein array (RPPA) analysis to identify potential substrate(s) of CGRRF1. Co-immunoprecipitation assay was used to verify the interaction between CGRRF1 and its substrate, followed by in vivo ubiquitination assays. Western blot, subcellular fractionation, and reverse transcription quantitative polymerase chain reaction (qRT-PCR) were performed to understand the mechanism of CGRRF1 action in breast cancer. Publicly available breast cancer datasets were analyzed to examine the association between CGRRF1 and breast cancer. RESULTS: We show that CGRRF1 inhibits the growth of breast cancer in vitro and in vivo, and the RING-finger domain is important for its growth-inhibitory activity. To elucidate the mechanism of CGRRF1, we identified EGFR as a new substrate of CGRRF1. CGRRF1 ubiquitinates EGFR through K48-linked ubiquitination, which leads to proteasome degradation. In addition to regulating the stability of EGFR, knockout of CGRRF1 enhances AKT phosphorylation after EGF stimulation. By analyzing the breast cancer database, we found that patients with low CGRRF1 expression have shorter survival. As compared to normal breast tissues, the mRNA levels of CGRRF1 are lower in breast carcinomas, especially in HER2-positive and basal-like breast cancers. We further noticed that CGRRF1 promoter methylation is increased in breast cancer as compared to that in normal breast tissue, suggesting that CGRRF1 is epigenetically modified in breast cancer. Treatment of 5-azactidine and panobinostat restored CGRRF1 expression, supporting that the promoter of CGRRF1 is epigenetically modified in breast cancer. Since 5-azactidine and panobinostat can increase CGRRF1 expression, they might be potential therapies for breast cancer treatment. CONCLUSION: We demonstrated a tumor-suppressive function of CGRRF1 in breast cancer and identified EGFR as its target.
Asunto(s)
Neoplasias de la Mama/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Neoplasias de la Mama/etiología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Metilación de ADN , Modelos Animales de Enfermedad , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Mutación , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , UbiquitinaciónRESUMEN
RNF144A is a single-pass transmembrane RBR E3 ligase that interacts with and degrades cytoplasmic DNA-PKcs, which is an epidermal growth factor receptor (EGFR)-interacting partner. Interestingly, RNF144A expression is positively correlated with EGFR mRNA and protein levels in several types of cancer. However, the relationship between RNF144A and EGFR is poorly understood. This study reports an unexpected role for RNF144A in the regulation of EGF/EGFR signaling and EGF-dependent cell proliferation. EGFR ligands, but not DNA-damaging agents, induce a DNA-PKcs-independent interaction between RNF144A and EGFR. RNF144A promotes EGFR ubiquitination, maintains EGFR protein, and prolongs EGF/EGFR signaling during EGF stimulation. Moreover, depletion of RNF144A by multiple independent approaches results in a decrease in EGFR expression and EGF/EGFR signaling. RNF144A knockout cells also fail to mount an immediate response to EGF for activation of G1/S progression genes. Consequently, depletion of RNF144A reduces EGF-dependent cell proliferation. These defects may be at least in part due to a role for RNF144A in regulating EGFR transport in the intracellular vesicles during EGF treatment.
Asunto(s)
Proteínas Portadoras/fisiología , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Transducción de Señal , Ubiquitina-Proteína Ligasas/fisiología , Transporte Biológico , Receptores ErbB/metabolismo , Humanos , UbiquitinaciónRESUMEN
RNF144A, an E3 ubiquitin ligase for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), can promote DNA damage-induced cell apoptosis. Here we characterize an important regulation of RNF144A through its transmembrane (TM) domain. The TM domain of RNF144A is highly conserved among species. Deletion of the TM domain abolishes its membrane localization and also significantly reduces its ubiquitin ligase activity. Further evidence shows that the TM domain is required for RNF144A self-association and that the self-association may be partially mediated through a classic GXXXG interaction motif. A mutant RNF144A-G252L/G256L (in the G(252)XXXG(256) motif) preserves membrane localization but is defective in self-association and ubiquitin ligase activity. On the other hand, a membrane localization loss mutant of RNF144A still retains self-association and E3 ligase activity, which can be blocked by additional G252L/G256L mutations. Therefore, our data demonstrate that the TM domain of RNF144A has at least two independent roles, membrane localization and E3 ligase activation, to regulate its physiological function. This regulatory mechanism may be applicable to other RBR (RING1-IBR-RING2) E3 ubiquitin ligases because, first, RNF144B also self-associates. Second, all five TM-containing RBR E3 ligases, including RNF144A and RNF144B, RNF19A/Dorfin, RNF19B, and RNF217, have the RBR-TM(GXXXG) superstructure. Mutations of the GXXXG motifs in RNF144A and RNF217 have also be found in human cancers, including a G252D mutation of RNF144A. Interestingly, RNF144A-G252D still preserves self-association and ubiquitin ligase activity but loses membrane localization and is turned over rapidly. In conclusion, both proper membrane localization and self-association are important for RNF144A function.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Proteínas Portadoras/genética , Línea Celular Tumoral , Membrana Celular/genética , Células HEK293 , Humanos , Mutación Missense , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Several ring between ring fingers (RBR) -domain proteins, such as Parkin and Parc, have been shown to be E3 ligases involved in important biological processes. Here, we identify a poorly characterized RBR protein, Ring Finger protein 144A (RNF144A), as the first, to our knowledge, mammalian E3 ubiquitin ligase for DNA-PKcs. We show that DNA damage induces RNF144A expression in a p53-dependent manner. RNF144A is mainly localized in the cytoplasmic vesicles and plasma membrane and interacts with cytoplasmic DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). DNA-PKcs plays a critical role in the nonhomologous end-joining DNA repair pathway and provides prosurvival signaling during DNA damage. We show that RNF144A induces ubiquitination of DNA-PKcs in vitro and in vivo and promotes its degradation. Depletion of RNF144A leads to an increased level of DNA-PKcs and resistance to DNA damaging agents, which is reversed by a DNA-PK inhibitor. Taken together, our data suggest that RNF144A may be involved in p53-mediated apoptosis through down-regulation of DNA-PKcs when cells suffer from persistent or severe DNA damage insults.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/fisiología , Dominio Catalítico/fisiología , Daño del ADN/fisiología , Proteína Quinasa Activada por ADN/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Western Blotting , Proteínas Portadoras/metabolismo , Ensayo de Unidades Formadoras de Colonias , Cartilla de ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Inmunoprecipitación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that overexpressed NCOAT(GK), an O-GlcNAcase-inactive spliced variant of the O-GlcNAcase gene, specifically in skeletal muscle using the muscle creatine kinase promoter. Expression of the chimeric enhanced green fluorescent protein-NCOAT(GK) transgene caused an increase of cellular O-GlcNAc levels, along with the accumulation and activation of proapoptotic factors in muscles of bitransgenic mice. The consequence of overexpressing the transgene for a 2-wk period was muscle atrophy and, in some cases, resulted in the death of male mice. Muscle atrophy is a common complication of many diseases, some of which correlate markedly with high cellular O-GlcNAc levels, such as diabetes. Our study provides direct evidence linking muscle atrophy and the disruption of O-GlcNAcase activity.
Asunto(s)
Empalme Alternativo/genética , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Atrofia Muscular/enzimología , Atrofia Muscular/patología , beta-N-Acetilhexosaminidasas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Transgénicos , Fibras Musculares Esqueléticas/metabolismo , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transgenes/genética , Regulación hacia Arriba/genética , beta-N-Acetilhexosaminidasas/química , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
The FOXO4 transcription factor plays an important role in cell survival in response to oxidative stress. The regulation of FOXO4 is orchestrated by post-translational modifications including phosphorylation, acetylation, and ubiquitination. Here, we demonstrate that O-GlcNAcylation also contributes to the FOXO4-dependent oxidative stress response. We show that hydrogen peroxide treatment of HEK293 cells increases FOXO4 association with OGT, the enzyme that adds O-GlcNAc to proteins, causing FOXO4 O-GlcNAcylation and enhanced transcriptional activity under acute oxidative stress. O-GlcNAcylation is known to be protective for cells under stress conditions, including oxidative stress. Our data provide a mechanism of FOXO4 anti-oxidative protection through O-GlcNAcylation.
Asunto(s)
Acetilglucosamina/metabolismo , Regulación de la Expresión Génica , Estrés Oxidativo/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Acilación , Proteínas de Ciclo Celular , Línea Celular , Factores de Transcripción Forkhead , Humanos , N-Acetilglucosaminiltransferasas/metabolismo , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Diminished proteolytic functionality in the lens may cause cataracts. We have reported that O-GlcNAc is an endogenous inhibitor of the proteasome. We hypothesize that in the lens there is a cause-and-effect relationship between proteasome inhibition by O-GlcNAc, and cataract formation. To demonstrate this, we established novel transgenic mouse models to over-express a dominant-negative form of O-GlcNAcase, GK-NCOAT, in the lens. Expression of GK-NCOAT suppresses removal of O-GlcNAc from proteins, resulting in increased levels of O-GlcNAc in the lenses of our transgenic mice, along with decreased proteasome function. We observed that transgenic mice developed markedly larger cataracts than controls and lens fiber cell denucleation was inhibited. Our study suggests that increased O-GlcNAc in the lens could lead to cataract formation and attenuation of lens fiber cell denucleation by inhibition of proteasome function. These findings may explain why cataract formation is a common complication of diabetes since O-GlcNAc is derived from glucose.
Asunto(s)
Acetilglucosamina/metabolismo , Catarata/patología , Diferenciación Celular , Cristalino/citología , Cristalino/patología , gamma-Cristalinas/metabolismo , Animales , Histona Acetiltransferasas/genética , Hialuronoglucosaminidasa/genética , Ratones , Ratones TransgénicosRESUMEN
This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress beta-catenin and cyclin D1 by up-regulating the E3 ligase SCF(beta-TrCP), we hypothesized that beta-transducin repeat-containing protein (beta-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with beta-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed beta-TrCP. Although ectopic expression of beta-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous beta-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box ((727)DSGAGS(732)) that mediates beta-TrCP recognition and encompasses a glycogen synthase kinase 3beta (GSK3beta) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3beta-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate beta-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.
Asunto(s)
Glucosa/deficiencia , Factor de Transcripción Sp1/metabolismo , Tiazolidinedionas/farmacología , Proteínas con Repetición de beta-Transducina/biosíntesis , Línea Celular Tumoral , Femenino , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Humanos , MAP Quinasa Quinasa 1/fisiología , Masculino , Fosforilación , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Regulación hacia ArribaRESUMEN
OBJECTIVES: The relationship of plasma adiponectin levels with various anthropometric and metabolic factors has been surveyed extensively in adults. However, how plasma adiponectin levels are related to various anthropometric indices and cardiovascular risk factors in adolescents is not as vigorously studied. In this study, we investigated this among healthy nondiabetic adolescents. RESEARCH METHODS AND PROCEDURES: Two hundred thirty nondiabetic subjects (125 boys and 105 girls, approximately 10 to 19 years old) were included. The plasma adiponectin, fasting plasma glucose, insulin, lipids and anthropometric indices including body height, weight, waist circumference, and hip circumference were examined. Body fat mass (FM) and percentage were obtained from DXA scan. The homeostasis model assessment was applied to estimate the degree of insulin resistance. RESULTS: The plasma adiponectin levels were significantly higher in girls (30.79 +/- 14.48 micro g/mL) than boys (22.87 +/- 11.41 micro g/mL). The plasma adiponectin levels were negatively related to BMI, FM, FM percentage, waist circumference, waist-to-hip ratio, insulin resistance, plasma insulin, triglycerides, and uric acid levels, but positively with high-density lipoprotein cholesterol (HDL-C) with the adjustment for age and gender. Using different multivariate linear regression models, only age and HDL-C were consistently related to the plasma adiponectin levels after adjustment for the other variables. DISCUSSION: The relationship between plasma adiponectin and various anthropometric indices and metabolic factors, especially HDL-C, previously reported in adults was present in the healthy nondiabetic adolescents. Whether variation of plasma adiponectin levels in healthy nondiabetic adolescents may influence their future coronary artery disease risk warrants further investigation.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Proteínas/análisis , Adiponectina , Adolescente , Adulto , Glucemia/análisis , Composición Corporal , Constitución Corporal , Estatura , Índice de Masa Corporal , Peso Corporal , Niño , HDL-Colesterol/sangre , Ayuno , Femenino , Humanos , Insulina/sangre , Modelos Lineales , Lípidos/sangre , Masculino , Valores de Referencia , Caracteres SexualesRESUMEN
Adiponectin is an adipose-derived plasma protein. Recently the plasma adiponectin levels have been linked to most variables of metabolic syndrome (MS) and risk factors of coronary artery disease (CAD). However, its relation with blood pressure is yet unclear. Here we report the relationship between the plasma adiponectin levels and blood pressures in 68 female adolescents (age 16.1 +/- 1.8 yr). We found that the plasma adiponectin levels correlated significantly with both the systolic blood pressure (SBP) (gamma = -0.47, P = 0.000) and diastolic blood pressure (gamma = -0.30, P = 0.014). In linear regression models with adjustment for age and the other anthropometric or metabolic factors, only the SBP, but not the diastolic blood pressure, was independently related to the plasma adiponectin levels. The mean plasma adiponectin levels between the subjects in the lowest quartile of SBP (SBP < or = 100 mm Hg) and those in the highest quartile (SBP > or = 118 mm Hg) were significantly different (P = 0.035). In conclusion, the SBP is inversely related to the plasma adiponectin independent of the other variables of the MS and other risk factors of CAD in healthy adolescent females. This further strengthens the potential roles of adiponectin in the pathophysiology of MS and CAD.