RESUMEN
SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mama/patología , Proteína Quinasa CDC2/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Secuencia de Aminoácidos , Mama/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Fosforilación , Pronóstico , Regiones Promotoras Genéticas , Proteolisis , Proteínas Ligasas SKP Cullina F-box/análisis , Proteínas Ligasas SKP Cullina F-box/genética , Transducción de Señal , Análisis de Supervivencia , Activación Transcripcional , UbiquitinaciónRESUMEN
Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G(1)-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G(1) and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCF(Fbw7) (also known as SCF(Cdc4)), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7ß is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCF(Fbw7α) in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCF(Fbw7γ). Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCF(Fbw7γ) prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Ciclina E/metabolismo , Proteínas F-Box/metabolismo , Proteínas Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Ciclina E/genética , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Peptidilprolil Isomerasa de Interacción con NIMA , Nucleoplasminas/metabolismo , Proteínas Oncogénicas/genética , Isomerasa de Peptidilprolil/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Fase S , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The F-box protein FBW7/hCDC4 is a tumor suppressor that acts as the substrate recognition component of an SCF ubiquitin ligase that targets numerous oncoproteins for proteasomal degradation. In this study, we investigated whether FBW7 is regulated by microRNAs, using a screen combining bioinformatic analysis, luciferase reporters and microRNA libraries. The ubiquitous miR-27a was identified as a major suppressor of FBW7 and in line with this, miR-27a prohibited ubiquitylation and turnover of the key FBW7 substrate cyclin E. Notably, we found that miR-27a only suppresses FBW7 during specific cell cycle phases, relieving its negative impact at the G1 to S-phase transition, prior to cyclin E protein degradation. We also demonstrate that attenuation of FBW7 by miR-27a overexpression leads to improper cell cycle progression and DNA replication stress, consistent with dysregulation of cyclin E expression. Finally, in the context of human cancer, miR-27a was discovered to be generally overexpressed in pediatric B-ALL and its expression to be inversely correlated with that of FBW7 in hyperdiploid cases of B-ALL. These data provide evidence for microRNA-mediated regulation of FBW7, and highlight the role of miR-27a as a novel factor fine-tuning the periodic events regulating cell cycle progression.