RESUMEN
We had previously reported that RBEL1A, a novel Ras-like GTPase, was overexpressed in multiple human malignancies and that its depletion suppressed cell growth. However, the underlying molecular mechanism remained to be elucidated. Here we report that depletion of endogenous RBEL1A results in p53 accumulation due to increased p53 half-life whereas increased expression of RBEL1A reduces p53 levels under unstressed and genotoxic stress conditions. RBEL1A directly interacts with p53 and MDM2, and strongly enhances MDM2-dependent p53 ubiquitylation and degradation. We also found that RBEL1A modulation of p53 ubiquitylation by MDM2 does not depend on its GTPase activity. We have also defined the p53 oligomeric domain and RBEL1A GTPase domain to be the crucial regions for p53-RBEL1A interactions. Importantly, we have found that RBEL1A strongly interferes with p53 transactivation function; thus our results indicate that RBEL1A appears to function as a novel p53 negative regulator that facilitates MDM2-dependent p53 ubiquitylation and degradation.
Asunto(s)
Daño del ADN , Proteolisis , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación , Proteínas ras/metabolismo , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteínas ras/genéticaRESUMEN
Loss of epithelial organization is a hallmark of carcinomas, but whether polarity regulates tumor growth and metastasis is poorly understood. To address this issue, we depleted the Par3 polarity gene by RNAi in combination with oncogenic Notch or Ras(61L) expression in the murine mammary gland. Par3 silencing dramatically reduced tumor latency in both models and produced invasive and metastatic tumors that retained epithelial marker expression. Par3 depletion was associated with induction of MMP9, destruction of the extracellular matrix, and invasion, all mediated by atypical PKC-dependant JAK/Stat3 activation. Importantly, Par3 expression is significantly reduced in human breast cancers, which correlates with active aPKC and Stat3. These data identify Par3 as a regulator of signaling pathways relevant to invasive breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Polaridad Celular , Proliferación Celular , Transformación Celular Neoplásica , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Metástasis de la Neoplasia , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Interferencia de ARN , Células Tumorales CultivadasRESUMEN
In this article, we report the characterization of a novel DNA damage-regulated gene, named DNA damage-regulated overexpressed in cancer 45 (DOC45). Our results indicate that DNA damage-inducing agents, including doxorubicin (adriamycin), etoposide, and ionizing and UV radiation, strongly downregulate DOC45 expression, whereas endoplasmic reticulum stress-inducing agents do not. Our results also indicate that DOC45 is overexpressed in several human malignancies, including cancers of the colon, rectum, ovary, lung, stomach, and uterus. DOC45 harbors conserved nucleotide triphosphate-binding motifs and is capable of ATP hydrolysis, findings that highlight its function as a novel ATPase. Although predominantly cytoplasmic, DOC45 exhibits a characteristic nucleocytoplasmic distribution and, on inhibition of nuclear export, predominantly accumulates in the nucleoli. These results suggest that DOC45 may shuttle between nucleus and cytoplasm to carry out its function. Our results also indicate that DOC45 expression is enhanced during oncogenic Ras-mediated transformation and that its expression is linked to phosphoinositide 3-kinase signaling pathway. Furthermore, short hairpin RNA-mediated knockdown of DOC45 in human colon cancer cells inhibits their proliferation and enhances cellular sensitivity to doxorubicin-induced cell death, suggesting that DOC45 plays an important role in cell proliferation and survival. Collectively, our results indicate that DOC45 is a novel ATPase that is linked to cellular stress response and tumorigenesis, and may also serve as a valuable tumor marker.
Asunto(s)
Adenosina Trifosfatasas/genética , Neoplasias/genética , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Clonación Molecular , Citoplasma/genética , Citoplasma/metabolismo , Daño del ADN/genética , Proteínas de Unión al GTP , Regulación Neoplásica de la Expresión Génica , Células HT29 , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Regulación hacia ArribaRESUMEN
Pirh2 is a newly identified E3 ubiquitin ligase known to inhibit tumor suppressor p53 function via ubiquitination and proteasomal degradation. We have identified two novel Pirh2 splice variants that encode different Pirh2 isoforms and named these Pirh2B and Pirh2C. Accordingly, the full-length protein is now classified as isoform Pirh2A. The central region of Pirh2 harbors a RING finger domain that is critical for its ubiquitin ligase function. The Pirh2B isoform lacks amino acids 171-179, whereas Pirh2C is missing C-terminal amino acids 180-261, which for each isoform results in a RING domain deletion and the abrogation of ubiquitin ligase activity. Our findings further indicate that the Pirh2B isoform but not the Pirh2C isoform is capable of binding to Pirh2A, suggesting that the C-terminal region absent in Pirh2C is critical for Pirh2-Pirh2 interactions. Similar to Pirh2A, both Pirh2B and Pirh2C interact with p53; however, interactions between p53 and Pirh2B appear stronger than those between p53 and Pirh2C. Interestingly, although both Pirh2B and Pirh2C are not able to promote in vitro p53 ubiquitination, both are capable of negatively regulating p53 protein stability and promoting the intracellular ubiquitination of p53. Furthermore, like Pirh2A, both isoforms are able to inhibit p53 transcriptional activity. We have also for the first time demonstrated that Pirh2A as well as the novel isoforms also interact directly with MDM2 within a region encompassing MDM2 acidic and zinc finger domains. It is therefore possible that Pirh2A and the novel Pirh2 isoforms identified in this study may also modulate p53 function by engaging MDM2.
Asunto(s)
Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular Tumoral , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Dominios RING Finger , Homología de Secuencia de AminoácidoRESUMEN
Recently, we reported the identification of a novel gene named RBEL1 (Rab-like protein 1) and characterized its two encoded isoforms, RBEL1A and RBEL1B, that function as novel GTPases of Ras superfamily. Here we report the identification of two additional splice variants of RBEL1 that we have named RBEL1C and -D. All four RBEL1 isoforms (A, B, C, and D) have identical N termini harboring the Rab-like GTPase domains but contain variable C termini. Although all isoforms can be detected in both cytoplasm and nucleus, RBEL1A is predominantly cytoplasmic, whereas RBEL1B is mostly nuclear. RBEL1C and -D, by contrast, are evenly distributed between the cytoplasm and nucleus. Furthermore, all four RBEL1 proteins are also capable of associating with cellular membrane. The RBEL1 proteins also exhibit a unique nucleotide-binding potential and, whereas the larger A and B isoforms are mainly GTP-bound, the smaller C and D variants bind to both GTP and GDP. Furthermore, a regulatory region at amino acid position 236-302 immediately adjacent to the GTP-binding domain is important for GTP-binding potential of RBEL1A, because deletion of this region converts RBEL1A from predominantly GTP-bound to GDP-bound. RBEL1 knockdown via RNA interference results in marked cell growth suppression, which is associated with morphological and biochemical features of apoptosis as well as inhibition of extracellular signal-regulated kinase phosphorylation. Taken together, our results indicate that RBEL1 proteins are linked to cell growth and survival and possess unique biochemical, cellular, and functional characteristics and, therefore, appear to form a novel subfamily of GTPases within the Ras superfamily.
Asunto(s)
Empalme Alternativo/fisiología , Apoptosis/fisiología , División Celular/fisiología , Proteínas ras/genética , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama , Núcleo Celular/enzimología , Citoplasma/enzimología , Femenino , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Isomerismo , Sistema de Señalización de MAP Quinasas/fisiología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN Interferente Pequeño , Proteínas ras/químicaRESUMEN
Rab family proteins are generally known as regulators of protein transport and trafficking. A number of Rab proteins have been implicated in cancer development and/or progression. Here we report the identification of a novel Rab-like protein, which we have named RBEL1 (Rab-like protein 1) for its higher similarity to the Rab subfamily members. We have characterized two isoforms of RBEL1 including the predominant RBEL1A and the less abundant RBEL1B that results from alternative splicing. Both isoforms harbor conserved N-terminal guanine trinucleotide phosphate (GTP) binding domains and, accordingly, are capable of binding to GTP. Both isoforms contain variable C termini and exhibit differential subcellular localization patterns. Unlike known Rabs that are mostly cytosolic, RBEL1B predominantly resides in the nucleus, whereas RBEL1A is localized primarily to the cytosol. Interestingly, a point mutation affecting RBEL1B GTP binding also alters the ability of mutant protein to accumulate in the nucleus, suggesting GTP binding potential to be important for RBEL1B nuclear localization. Our results also indicate that RBEL1A is overexpressed in about 67% of primary breast tumors. Thus, RBEL1A and RBEL1B are novel Rab-like proteins that localize in the nucleus and cytosol and may play an important role in breast tumorigenesis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas ras/biosíntesis , Proteínas ras/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citosol/metabolismo , Guanosina Trifosfato/química , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Mutación Puntual , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rab/metabolismo , Proteínas ras/genéticaRESUMEN
Oncogenic Ras proteins transform cells via multiple downstream signaling cascades that are important for cell proliferation and survival. Gadd153, also known as CHOP, is a growth inhibitory and proapoptotic protein and its expression is upregulated by many agents that induce apoptosis. Here, we report our novel findings that oncogenic Ras downregulates Gadd153 expression at both protein and mRNA levels and that such downregulation occurs, at least in part, via decreases in GADD153 mRNA stability. Gadd153 downregulation is specific to oncogenic Ras since another oncogenic family member R-Ras2/TC21 does not downregulate Gadd153. We further demonstrate that the expression of exogenous Gadd153 interferes with Ras-induced oncogenic transformation, which suggests that downregulation of Gadd153 appears to be an important mechanism by which oncogenic Ras promotes cellular transformation. Thus, oncogenic Ras-mediated cellular transformation also involves downmodulation of important molecules such as Gadd153 that negatively regulate cell growth and survival.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Transformación Celular Neoplásica , Regulación hacia Abajo , Proteína Oncogénica p21(ras)/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Regulación hacia Abajo/efectos de los fármacos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína Oncogénica p21(ras)/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factor de Transcripción CHOP , Factores de Transcripción/genéticaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis, bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2, respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors, and transduced cells were cultured in a semisolid medium permissive for the development of erythroid, myeloid, and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro, in contrast to Tax2-transduced cells, which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected, suggesting that Tax1 inhibited the maturation of relatively early, uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis, lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells, an activity that is not displayed by Tax2, and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo, the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2.
Asunto(s)
Antígenos CD34/análisis , Productos del Gen tax/fisiología , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Apoptosis , Linaje de la Célula , Células HeLa , Virus Linfotrópico T Tipo 2 Humano/patogenicidad , Humanos , Células Jurkat , Lentivirus/genética , Secuencias Repetidas Terminales , Transducción GenéticaRESUMEN
Thapsigargin (TG), by inducing perturbations in cellular Ca(2+) homeostasis, can induce apoptosis, but the molecular mechanisms remain to be fully elucidated. We have recently reported that TG-induced apoptosis appears to involve the DR5-dependent apoptotic pathway that cross talks with the mitochondrial pathway via TG-induced Bid cleavage. In this study, we have utilized Bax-proficient and -deficient HCT116 human colon cancer cells to investigate the effect of Bax deficiency on TG-induced apoptosis and TG regulation of the DR5 and mitochondrial pathways. Our results indicate that Bax-deficient cells are less sensitive to undergo apoptosis following TG treatment. Our results further demonstrate that TG-induced apoptosis is coupled with DR5 upregulation and caspases 8 and 3 activation, as well as Bid cleavage in both Bax-proficient and -deficient cells, although caspase 3 activation was reduced in Bax-deficient cells. TG also promoted the release of cytochrome c into cytosol and caspase 9 activation in Bax-proficient cells but not in Bax-deficient cells. These findings suggest that although Bax is not absolutely required for death receptor (DR)-dependent signals, it appears to be a key molecule in TG-regulated mitochondrial events. Bax-deficient cells were relatively more resistant to Apo2L/TRAIL than the Bax-proficient counterparts. However, the combination of Apo2L/TRAIL and TG was more effective in mediating apoptosis in both Bax-proficient and -deficient cells and that was coupled with activation of caspases 8 and 3. Although both agents in combination also induced cytochrome c release into cytosol and caspase 9 activation in Bax-proficient cells, these events were abrogated in Bax-deficient cells. Our results thus suggest that the combination of Apo2L/TRAIL and TG appears to bypass the Bax deficiency-induced defects in the mitochondrial (intrinsic) pathway by engaging the DR5-dependent apoptotic signals (extrinsic pathway).