RESUMEN
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
In this study, we report the characterization of a novel genotoxic and nongenotoxic stress-regulated gene that we had previously named as SKNY. Our results indicate that SKNY encodes the recently identified neutral sphingomyelinase-3 (nSMase3; hereafter SKNY is referred to as nSMase3). Examination of nSMase3 subcellular distribution reveals nSMase3 to localize to the endoplasmic reticulum (ER), and deletion of a COOH-terminal region containing its putative transmembrane domain and ER targeting signal partly alters its compartmentalization to the ER. Treatment with genotoxic Adriamycin and nongenotoxic tumor necrosis factor-alpha up-regulates endogenous nSMase3 expression, albeit with different kinetics. Tumor necrosis factor-alpha up-regulates nSMase3 expression within 2 h that lasts beyond 24 h and declines to control levels by 36 h. Adriamycin up-regulation of nSMase3 is transient, occurs within 30 min, and declines to control levels by 120 min. Prolonged treatment with Adriamycin by 24 h and beyond, however, causes a down-regulation in nSMase3 expression. Activation of wild-type p53 also down-regulates nSMase3 expression, suggesting that DNA damage-mediated nSMase3 down-regulation seems to occur partly through the tumor suppressor p53. Overexpression of exogenous nSMase3 sensitizes cells to Adriamycin-induced cell killing, a finding consistent with the proposed proapoptotic role of nSMase enzymes and nSMase-generated ceramide. We further investigated nSMase3 expression in various human malignancies and found its expression to be deregulated in several types of primary tumors when compared with their matching normal tissues. Collectively, our results have identified nSMase3 to be an important molecule that is linked to tumorigenesis and cellular stress response.
Asunto(s)
Daño del ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Esfingomielina Fosfodiesterasa/fisiología , Secuencia de Aminoácidos , Apoptosis , Línea Celular Tumoral , Doxorrubicina/farmacología , Retículo Endoplásmico/metabolismo , Humanos , Cinética , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The function of p53 as a tumor suppressor remains undisputed. p53 has a central role in cellular stress responses as well as affecting cancer development and progression. The word "central", however, is becoming increasingly more of an understatement as the list of p53-regulated pathways and processes is ever expanding. Although much focus continues to center on p53-mediated signaling cascades that control cell growth arrest and/or apoptosis, recent work has begun to define a role for p53 in the regulation of metabolic pathways typically thought of as essential for maintaining life. With the first potential link between p53 and glycolysis reported nearly ten years ago, the topic has gained a renewed interest. Recent studies now demonstrate the ability of p53 to regulate the expression of several novel genes including PGM (phosphoglycerate mutase), TIGAR (TP53-induced glycolysis and apoptosis regulator) and, SCO2 (synthesis of cytochrome c oxidase 2), each intimately linked to the processes of glycolysis and oxidative phosphorylation. With this discovery, yet another novel means by which p53 carries out its tumor suppressor function is brought into light.
Asunto(s)
Genes p53 , Proteína p53 Supresora de Tumor/metabolismo , Metabolismo Energético , Glucólisis , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Consumo de Oxígeno , Fosfoglicerato Mutasa/genética , Fosfoglicerato Mutasa/metabolismoRESUMEN
TRB3 has recently been identified as a potential pro-apoptotic protein that may modulate the Akt/PKB-dependent signaling pathway. Here we report that TRB3 expression is strongly upregulated by endoplasmic reticulum (ER) stress-inducing agents that (1) promote ER Ca2+ pool depletion or (2) disrupt protein trafficking. Genotoxic stress (DNA damage)-inducing agents, by contrast, downregulate TRB3 expression and appear to do so through both p53-dependent and -independent mechanisms. To the best of our knowledge, TRB3 is the first gene that is upregulated by ER stress and downregulated following genotoxic stress. Collectively, these findings highlight the importance of stress-specific signaling cascades as well as point out the seemingly divergent roles that TRB3 may play in the cellular stress response.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Daño del ADN , Retículo Endoplásmico/fisiología , Estrés Oxidativo/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HT29 , Humanos , Masculino , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Tapsigargina/farmacologíaRESUMEN
Cyclooxygenase-2 (COX-2) has been implicated in a variety of human malignancies and, accordingly, COX-2 selective inhibitors are being investigated as important chemopreventive and therapeutic agents. How COX-2 overexpression results in tumorigenesis and how COX-2 selective agents mediate their chemopreventive effects are issues that remain poorly understood. Here we report that the tumor suppressor p53 upregulates COX-2 expression and that COX-2 can in turn inhibit p53-dependent transcription. Additionally, a COX-2-selective inhibitor potentiates p53-induced apoptosis, which also supports the notion that COX-2 activity appears to interfere with p53 function. Expression of exogenous COX-2 in p53 wild-type cells does not affect the cytoplasmic or nuclear levels of p53, suggesting that COX-2 may not affect p53 turnover or subcellular localization. We further demonstrate that endogenous COX-2 interacts with p53 and that COX-2 and p53 interactions are a physiologically relevant event. Thus, p53 upregulates COX-2 and COX-2 in turn appears to negatively affect p53 activity via mechanisms that could involve physical interactions between COX-2 and p53. Based on our results, we propose that p53-dependent upregulation and activation of COX-2 appear to be yet another novel mechanism by which p53 could abate its own growth-inhibitory and apoptotic effects.
Asunto(s)
Apoptosis/genética , Genes p53 , Prostaglandina-Endoperóxido Sintasas/metabolismo , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclooxigenasa 2 , Femenino , Humanos , Masculino , Proteínas de la Membrana , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
For years, the growth inhibitory effects of the tumor suppressor p53 were thought to be antagonized predominantly by the ubiquitin ligase, MDM2. It has long been established that MDM2 physically associates with p53 and targets this tumor suppressor for proteasomal degradation. In light of recent findings, it now appears that MDM2 may not be the only ubiquitin ligase that negatively controls p53 function. Two recently discovered proteins, Pirh2 and COP1, are also believed to facilitate p53 degradation via the ubiquitin-proteasome pathway. Both proteins are upregulated by p53 as well as genotoxic stress and each has been found to directly promote p53 ubiquitination and degradation. Future studies in this field will now face the challenge of elucidating the physiological significance of three molecules all apparently able to independently facilitate p53 degradation and abrogate its function.