RESUMEN
Accurate segregation of chromosomes is critical to ensure that each daughter cell receives the full genetic complement. Maintenance of cohesion between sister chromatids, especially at centromeres, is required to segregate chromosomes precisely during mitosis and meiosis. The Drosophila protein MEI-S332, the founding member of a conserved protein family, is essential in meiosis for maintaining cohesion at centromeres until sister chromatids separate at the metaphase II/anaphase II transition. MEI-S332 localizes onto centromeres in prometaphase of mitosis or meiosis I, remaining until sister chromatids segregate. We elucidated a mechanism for controlling release of MEI-S332 from centromeres via phosphorylation by POLO kinase. We demonstrate that POLO antagonizes MEI-S332 cohesive function and that full POLO activity is needed to remove MEI-S332 from centromeres, yet this delocalization is not required for sister chromatid separation. POLO phosphorylates MEI-S332 in vitro, POLO and MEI-S332 bind each other, and mutation of POLO binding sites prevents MEI-S332 dissociation from centromeres.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Sitios de Unión , Western Blotting/métodos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Células Cultivadas , Ciclina B/metabolismo , Drosophila , Proteínas de Drosophila/genética , Ensayo de Cambio de Movilidad Electroforética/métodos , Embrión no Mamífero , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Inmunoprecipitación/métodos , Indoles/metabolismo , Larva , Masculino , Meiosis/fisiología , Microscopía Confocal/métodos , Mitosis/fisiología , Modelos Biológicos , Mutagénesis/fisiología , Fosforilación , Espermatocitos/metabolismo , Testículo/metabolismo , Xenopus/metabolismoRESUMEN
Tumors utilize hyperactivation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway to cope with deleterious environmental conditions. Activation of the PI3K/AKT pathway has been shown to increase protein expression of the alpha subunit of the hypoxia-inducible factor (HIF) 1, a key regulator of oxygen homeostasis. Elevated levels of HIF-1 alpha induce expression of genes with critical roles in angiogenesis, erythropoiesis, and glucose metabolism, processes that are essential for tumor expansion. Here we examine the involvement of FOXO4 (also known as AFX), a member of the forkhead transcription factor superfamily that is negatively regulated by the PI3K/AKT pathway, in the regulation of HIF-1 alpha protein expression. Nuclear expression of FOXO4 results in the suppression of various responses to hypoxia, including decreased vascular endothelial growth factor, glucose transporter 1, and erythropoietin expression. Interestingly, FOXO4 down-regulates the HIF-1 alpha protein levels, consistent with the lack of hypoxia responsiveness. Previous results have revealed a role for prolyl hydroxylation and resultant von Hippel-Lindau protein (pVHL) interactions in the ubiquitin-proteasome-mediated degradation of HIF-1 alpha. However, neither inhibition of prolyl hydroxylases nor mutation of HIF-1 alpha-hydroxylated prolines involved with pVHL-mediated binding inhibits the observed FOXO4-mediated down-regulation of HIF-1 alpha. These results suggest a novel alternate mechanism for hypoxic regulation that is dependent upon the level of activation of FOXO4-mediated transcription.
Asunto(s)
Regulación hacia Abajo , Ligasas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Western Blotting , Proteínas de Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , ADN/metabolismo , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Células HeLa , Humanos , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Luciferasas/metabolismo , Complejos Multienzimáticos/metabolismo , Oxígeno/metabolismo , Plásmidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Estructura Terciaria de Proteína , Transcripción Genética , Células Tumorales Cultivadas , Ubiquitina/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
The activation of the AKT/protein kinase B kinases by mutation of the PTEN lipid phosphatase results in enhanced survival of a diversity of tumors. This resistance to apoptosis is partly accomplished by the inhibition of genetic programs induced by a subfamily of forkhead transcription factors including AFX. Here we describe an AFX-regulated pathway that appears to account for at least part of this apoptotic regulatory system. Cells induced to synthesize an active form of AFX die by activating the apoptotic death pathway. An analysis of genes regulated by AFX demonstrated that BCL-6, a transcriptional repressor, is up-regulated approximately 4-7-fold. An examination of the BCL-6 promoter demonstrated that AFX bound to specific target sites that could activate transcription. BCL-X(L), an anti-apoptotic protein, contains potential BCL-6 target sites in its promoter. An analysis of endogenous BCL-X(L) levels in AFX-expressing cells revealed enhanced down-regulation of the transcript ( approximately 1.3-1.7-fold) and protein, and BCL-6 directly binds to and suppresses the BCL-X(L) promoter. Finally, macrophages isolated from BCL-6-/- mice show enhanced survival in vitro. These results suggest that AFX regulates apoptosis in part by suppressing the levels of anti-apoptotic BCL-XL through the transcriptional repressor BCL-6.