RESUMEN
Transcriptional induction of cell-cycle regulatory proteins ensures proper timing of subsequent cell-cycle events. Here we show that the Forkhead transcription factor FoxM1 regulates expression of many G2-specific genes and is essential for chromosome stability. Loss of FoxM1 leads to pleiotropic cell-cycle defects, including a delay in G2, chromosome mis-segregation and frequent failure of cytokinesis. We show that transcriptional activation of cyclin B by FoxM1 is essential for timely mitotic entry, whereas CENP-F, another direct target of FoxM1 identified here, is essential for precise functioning of the mitotic spindle checkpoint. Thus, our data uncover a transcriptional cluster regulated by FoxM1 that is essential for proper mitotic progression.
Asunto(s)
Inestabilidad Cromosómica , Mitosis , Factores de Transcripción/fisiología , Animales , Ciclo Celular , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Ciclina B/metabolismo , Ciclina B1 , Proteínas de Unión al ADN/fisiología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Humanos , Ratones , Proteínas de MicrofilamentosRESUMEN
Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.
Asunto(s)
Mitosis/fisiología , Proteínas Quinasas/fisiología , Procesamiento Proteico-Postraduccional , Huso Acromático/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Ciclo Celular , Línea Celular Tumoral , Humanos , Mitosis/genética , Mutación/genética , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , ARN Interferente Pequeño/genética , Serina/metabolismo , Huso Acromático/genética , Treonina/metabolismo , Quinasa Tipo Polo 1RESUMEN
The chromosomal passenger complex (CPC) coordinates chromosomal and cytoskeletal events of mitosis. The enzymatic core of this complex (Aurora-B) is guided through the mitotic cell by its companion chromosomal passenger proteins, inner centromere protein (INCENP), Survivin and Borealin/Dasra-B, thereby allowing it to act at the right place at the right time. Here, we addressed the individual contributions of INCENP, Survivin and Borealin to the proper functioning of this complex. We show that INCENP has an important role in stabilizing the complex, and that Borealin acts to promote binding of Survivin to INCENP. Importantly, when Survivin is directly fused to INCENP, this hybrid can restore CPC function at the centromeres and midbody, even in the absence of Borealin and the centromere-targeting domain of INCENP. Thus, Survivin is an important mediator of centromere and midbody docking of Aurora-B during mitosis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Proteínas Cromosómicas no Histona/genética , Inhibidores de Cisteína Proteinasa/genética , Inhibidores de Cisteína Proteinasa/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Sustancias Macromoleculares , Proteínas Asociadas a Microtúbulos/genética , Mitosis , Proteínas de Neoplasias/genética , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SurvivinRESUMEN
Genetic evidence is mounting that survivin plays a crucial role in mitosis, but its exact role in human cell division remains elusive. We show that mammalian cells lacking survivin are unable to align their chromosomes, fail to recruit Aurora B to kinetochores and become polyploid at a very high frequency. Survivin-depleted cells enter mitosis with normal kinetics, but are delayed in prometaphase in a BubR1/Mad2-dependent fashion. Nonetheless, these cells exit mitosis prior to completion of chromosome congression and without sister chromatid segregation, indicating that the spindle assembly checkpoint is not fully functional. Indeed, in survivin-depleted cells, BubR1 and Mad2 are prematurely displaced from kinetochores, yet no tension is generated at kinetochores. Importantly, these cells fail to respond to drugs that prevent tension, but do arrest in mitosis after depolymerization of the mitotic spindle. This demonstrates that survivin is not required for initial checkpoint activation, or for sustained checkpoint activation by loss of microtubules. However, stable association of BubR1 to kinetochores and sustained checkpoint signalling in response to lack of tension crucially depend on survivin.