RESUMEN
Post-translational modifications of Notch3 and their functional role with respect to Notch3 overexpression in T-cell leukemia are still poorly understood. We identify here a specific novel property of Notch3 that is acetylated and deacetylated at lysines 1692 and 1731 by p300 and HDAC1, respectively, a balance impaired by HDAC inhibitors (HDACi) that favor hyperacetylation. By using HDACi and a non-acetylatable Notch3 mutant carrying K/R(1692-1731) mutations in the intracellular domain, we show that Notch3 acetylation primes ubiquitination and proteasomal-mediated degradation of the protein. As a consequence, Notch3 protein expression and its transcriptional activity are decreased both in vitro and in vivo in Notch3 transgenic (tg) mice, thus impairing downstream signaling upon target genes. Consistently, Notch3-induced T-cell proliferation is inhibited by HDACi, whereas it is enhanced by the non-acetylatable Notch3-K/R(1692-1731) mutant. Finally, HDACi-induced Notch3 hyperacetylation prevents in vivo growth of T-cell leukemia/lymphoma in Notch3 tg mice. Together, our findings suggest a novel level of Notch signaling control in which Notch3 acetylation/deacetylation process represents a key regulatory switch, thus representing a suitable druggable target for Notch3-sustained T-cell acute lymphoblastic leukemia therapy.
Asunto(s)
Leucemia de Células T/etiología , Receptores Notch/fisiología , Acetilación , Animales , Células HEK293 , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Leucemia de Células T/tratamiento farmacológico , Activación de Linfocitos , Ratones , Complejo de la Endopetidasa Proteasomal/fisiología , Receptor Notch3 , Linfocitos T/inmunología , UbiquitinaciónRESUMEN
Notch3 and pTalpha signaling events are essential for T-cell leukemogenesis and characterize murine and human T-cell acute lymphoblastic leukemia. Genetic ablation of pTalpha expression in Notch3 transgenic mice abrogates tumor development, indicating that pTalpha signaling is crucial to the Notch3-mediated leukemogenesis. Here we report a novel direct interaction between Notch3 and pTalpha. This interaction leads to the recruitment and persistence of the E3 ligase protein c-Cbl to the lipid rafts in Notch3-IC transgenic thymocytes. Conversely, deletion of pTalpha in Notch3 transgenic mice leads to cytoplasmic retention of c-Cbl that targets Notch3 protein to the proteasomal-degradative pathway. It appears that protein kinase C theta (PKCtheta), by regulating tyrosine and serine phosphorylation of Cbl, is able to control its function. We report here that the increased Notch3-IC degradation correlates with higher levels of c-Cbl tyrosine phosphorylation in Notch3-IC/pTalpha(-/-) double-mutant thymocytes, which also display a decreased PKCtheta activity. Our data indicate that pTalpha/pre-T-cell receptor is able to regulate the different subcellular localization of c-Cbl and, by regulating PKCtheta activity, is also able to influence its ubiquitin ligase activity upon Notch3 protein.