RESUMEN
Aurora B is critically involved in ensuring proper cytokinesis and maintaining genomic stability. The tumor suppressor RASSF1A regulates cell cycle progression by regulating mitotic progression, G(1)-S transition, and microtubule stability. We previously reported that both Aurora A and Aurora B phosphorylate RASSF1A, and showed that phosphorylation of RASSF1A by Aurora A blocks the inhibitory function of RASSF1A toward anaphase-promoting complex-Cdc20. However, the role of Aurora B-mediated RASSF1A phosphorylation remains unknown. Here, we show that phosphorylation of RASSF1A on Ser203 by Aurora B during late mitosis has a critical role in regulating cytokinesis. Notably, RASSF1A interacts with Syntaxin16, a member of the t-SNARE family, at the midzone and midbody during late mitosis. Aurora B is required for this interaction and for the subsequent recruitment of Syntaxin16 to the midzone and midbody, a prerequisite for the successful completion of cytokinesis. Furthermore, Aurora B depletion results in a failure of Syntaxin16 to properly localize to the midzone and midbody, a mislocalization that was prevented by overexpression of the phosphomimetic RASSF1A (S203D) mutant. Finally, either depletion of Syntaxin16 or expression of the nonphosphorylatable RASSF1A (S203A) mutant results in cytokinesis defects. Our findings implicate Aurora B-mediated phosphorylation of RASSF1A in the regulation of cytokinesis.
Asunto(s)
Citocinesis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Aurora Quinasa B , Aurora Quinasas , Células HeLa , Humanos , Fosforilación , Transporte de Proteínas/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
The Aurora (Ipl) kinase family plays important roles in the regulation of mitosis and tumorigenesis. The tumor suppressor RASSF1A controls mitotic progression by regulating anaphase-promoting complex (APC)-Cdc20 activity and microtubule stability, but the mechanism by which this action is regulated has not been previously established. Here, we show that Aurora A and B associate with and phosphorylate RASSF1A on serine 203 in vivo at different times and in different subcellular compartments during mitosis. Notably, both depletion of Aurora A by RNA interference and expression of a nonphosphorylatable RASSF1A (S203A) mutant gene led to a marked delay in prometaphase progression. This is likely because of the failure of RASSF1A to dissociate from Cdc20, constitutive inhibition of APC-Cdc20, and accumulation of mitotic cyclins. In contrast, the delay in prometaphase progression caused by Aurora A depletion was largely normalized by phosphomimetic RASSF1A (S203D). Finally, RASSF1A phosphorylation on serine 203 was up-regulated in Aurora A-overexpressing human tumors. These findings indicate that Aurora A plays a critical role in RASSF1A-APC-Cdc20 regulatory mechanisms that control normal prometaphase progression and that are involved in tumorigenesis. [Cancer Res 2009;69(6):2314-23.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Prometafase/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Aurora Quinasas , Proteínas Cdc20 , Proteínas de Ciclo Celular/antagonistas & inhibidores , División Celular/fisiología , Fase G2/fisiología , Células HeLa , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Mitosis/fisiología , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Using gene chip analyses, we have identified novel neuronal activity-dependent genes. Application of 25 mM KCl to mature (14-day culture) rat cortical neurons resulted in more than 1.5-fold induction of 19 genes and reduction of 42 genes among 1200 neural genes. Changes in the overall gene expression profiles appeared to be related to the reduction of excitability and induction of cellular survival signals. Among the genes identified, three transcriptional modulators [encoding Cbp/p300-interacting transactivator with ED-rich tail 2 (CITED2), CCAAT/enhancer binding protein beta (C/EBPbeta) and neuronal orphan receptor-1, (NOR1)] were newly identified as activity-dependent transcription factors, and two of these (CITED2 and NOR1) were found to be influenced by electroconvulsive shock (ECS). NOR1 was induced in specific brain regions by behavioral activation, such as exposure to a novel environment. Because the brain regions that exhibited the induction of these newly identified neuronal activity-dependent transcriptional modulators were distinct from those showing the induction of previously identified activity-dependent genes such as c-fos, these genes might be useful markers for mapping neuronal activity in vivo.