RESUMEN
The stability and membrane localization of the transforming growth factor-ß (TGF-ß) type I receptor (TßRI) determines the levels of TGF-ß signalling. TßRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-ß signalling. USP4 was found to directly interact with TßRI and act as a deubiquitylating enzyme, thereby controlling TßRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-ß-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TßRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-ß and AKT signalling pathways.
Asunto(s)
Neoplasias de la Mama/enzimología , Membrana Celular/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Movimiento Celular , Estabilidad de Enzimas , Transición Epitelial-Mesenquimal , Femenino , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Noqueados , Mutación , Invasividad Neoplásica , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Proteínas Proto-Oncogénicas , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina Tiolesterasa/genética , Proteasas Ubiquitina-Específicas , Ubiquitinación , Pez Cebra/embriologíaRESUMEN
TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.