RESUMO
The tumor suppressor Merlin/NF2, a key activator of the Hippo pathway in growth control, is regulated by phosphorylation. However, it is uncertain whether additional post-translational modifications regulate Merlin. Here, we show that ubiquitination is required to activate Merlin in the Hippo pathway. Ubiquitinated Merlin is mostly conjugated by one or two ubiquitin molecules. Such modification is promoted by serine 518 dephosphorylation in response to Ca2+ signaling or cell detachment. Merlin ubiquitination is mediated by the E3 ubiquitin ligase, NEDD4L, which requires a scaffold protein, AMOTL1, to approach Merlin. Several NF2-patient-derived Merlin mutations disrupt its binding to AMOTL1 and its regulation by the AMOTL1-NEDD4L apparatus. Lysine (K) 396 is the major ubiquitin conjugation residue. Disruption of Merlin ubiquitination by the K396R mutation or NEDD4L depletion diminishes its binding to Lats1 and inhibits Lats1 activation. These effects are also accompanied by loss of Merlin's anti-mitogenic and tumor suppressive properties. Thus, we propose that dephosphorylation and ubiquitination compose an intramolecular relay to activate Merlin functions in activating the Hippo pathway during growth control.
Assuntos
Neurofibromina 2 , Proteínas Serina-Treonina Quinases , Genes Supressores de Tumor , Via de Sinalização Hippo , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , UbiquitinaçãoRESUMO
YAP1 fusion-positive supratentorial ependymomas predominantly occur in infants, but the molecular mechanisms of oncogenesis are unknown. Here we show YAP1-MAMLD1 fusions are sufficient to drive malignant transformation in mice, and the resulting tumors share histo-molecular characteristics of human ependymomas. Nuclear localization of YAP1-MAMLD1 protein is mediated by MAMLD1 and independent of YAP1-Ser127 phosphorylation. Chromatin immunoprecipitation-sequencing analyses of human YAP1-MAMLD1-positive ependymoma reveal enrichment of NFI and TEAD transcription factor binding site motifs in YAP1-bound regulatory elements, suggesting a role for these transcription factors in YAP1-MAMLD1-driven tumorigenesis. Mutation of the TEAD binding site in the YAP1 fusion or repression of NFI targets prevents tumor induction in mice. Together, these results demonstrate that the YAP1-MAMLD1 fusion functions as an oncogenic driver of ependymoma through recruitment of TEADs and NFIs, indicating a rationale for preclinical studies to block the interaction between YAP1 fusions and NFI and TEAD transcription factors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Encefálicas/metabolismo , Carcinogênese/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ependimoma/metabolismo , Fatores de Transcrição NFI/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/genética , Ependimoma/genética , Ependimoma/patologia , Células HEK293 , Humanos , Camundongos , Fatores de Transcrição NFI/genética , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca2+ entry by enhancing store-operated Ca2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca2+-induced Lats1/2 activation. Ca2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca2+ is a crucial intracellular cue that regulates the Hippo pathway and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.