RESUMO
ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Drosophila Crumbs (Crb) and its mammalian ortholog CRB3 control epithelial polarity through poorly understood molecular mechanisms. Elucidating these mechanisms is crucial, because the physiology of epithelia largely depends on the polarized architecture of individual epithelial cells. In addition, loss of CRB3 favors tumor cell growth, metastasis and epithelial to mesenchymal transition (EMT). Using Drosophila embryos, we report that Rac1 sustains PI3K signaling, which is required for Rac1 activation. Crb represses this positive-feedback loop. Notably, this property confers to Crb its ability to promote epithelial integrity in vivo, because attenuation of either Rac1 or PI3K activity rescues the crb mutant phenotype. Moreover, inhibition of Rac1 or PI3K results in Crb-dependent apical membrane growth, whereas Rac1 activation restricts membrane localization of Crb and interferes with apical domain formation. This illustrates that Crb and the Rac1-PI3K module are antagonists, and that the fine balance between the activities of these proteins is crucial to maintain epithelial organization and an appropriate apical to basolateral ratio. Together, our results elucidate a mechanism that mediates Crb function and further define the role of PI3K and Rac1 in epithelial morphogenesis, allowing for a better understanding of how distinct membrane domains are regulated in polarized epithelial cells.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Epitélio/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Polaridade Celular/genética , Extensões da Superfície Celular/genética , Células Cultivadas , Drosophila/fisiologia , Proteínas de Drosophila/genética , Embrião não Mamífero , Transição Epitelial-Mesenquimal/genética , Epitélio/patologia , Retroalimentação Fisiológica , Proteínas de Membrana/genética , Morfogênese/genética , Mutação/genética , Transdução de Sinais/fisiologia , Proteínas rac de Ligação ao GTP/genéticaRESUMO
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.
Assuntos
Polaridade Celular , Proteínas de Drosophila/antagonistas & inibidores , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Células Epiteliais/metabolismo , Proteína Quinase C/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Western Blotting , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Células Epiteliais/citologia , Imunofluorescência , Técnicas Imunoenzimáticas , Imunoprecipitação , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1-3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1-NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1-NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.