RESUMEN
Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an "ancient" RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Células T Asesinas Naturales/metabolismo , ARN Helicasas/metabolismo , Espermatogénesis/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Activación de Linfocitos/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismoRESUMEN
The Wnt, TGF-ß, and Notch signaling pathways are essential for the regulation of cellular polarity, differentiation, proliferation, and migration. Differential activation and mutual crosstalk of these pathways during animal development are crucial instructive forces in the initiation of the body axis and the development of organs and tissues. Due to the ability to initiate cell proliferation, these pathways are vulnerable to somatic mutations selectively producing cells, which ultimately slip through cellular and organismal checkpoints and develop into cancer. The architecture of the Wnt, TGF-ß, and Notch signaling pathways is simple. The transmembrane receptor, activated by the extracellular stimulus, induces nuclear translocation of the transcription factor, which subsequently changes the expression of target genes. Nevertheless, these pathways are regulated by a myriad of factors involved in various feedback mechanisms or crosstalk. The most prominent group of regulators is the ubiquitin-proteasome system (UPS). To open the door to UPS-based therapeutic manipulations, a thorough understanding of these regulations at a molecular level and rigorous confirmation in vivo are required. In this quest, mouse models are exceptional and, thanks to the progress in genetic engineering, also an accessible tool. Here, we reviewed the current understanding of how the UPS regulates the Wnt, TGF-ß, and Notch pathways and we summarized the knowledge gained from related mouse models.
Asunto(s)
Ratones/embriología , Ratones/genética , Ubiquitina-Proteína Ligasas/fisiología , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Homeostasis/genética , Ligasas/metabolismo , Receptores Notch/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismoRESUMEN
BACKGROUND INFORMATION: Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin ß and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined. RESULTS: Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import. SIGNIFICANCE: Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair.
Asunto(s)
Arginina/metabolismo , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Lisina/metabolismo , Señales de Localización Nuclear , Osteosarcoma/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Transporte Activo de Núcleo Celular , Arginina/química , Arginina/genética , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Núcleo Celular/genética , Células HEK293 , Humanos , Carioferinas/genética , Lisina/química , Lisina/genética , Osteosarcoma/genética , Osteosarcoma/patología , Fosforilación , Unión Proteica , Células Tumorales Cultivadas , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.