RESUMEN
The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is known to target the degradation of cyclin B1, which is crucial for mitotic progression in animal cells. In this study, we show that the ubiquitin ligase CRL2ZYG-11 redundantly targets the degradation of cyclin B1 in Caenorhabditis elegans and human cells. In C. elegans, both CRL2ZYG-11 and APC/C are required for proper progression through meiotic divisions. In human cells, inactivation of CRL2ZYG11A/B has minimal effects on mitotic progression when APC/C is active. However, when APC/C is inactivated or cyclin B1 is overexpressed, CRL2ZYG11A/B-mediated degradation of cyclin B1 is required for normal progression through metaphase. Mitotic cells arrested by the spindle assembly checkpoint, which inactivates APC/C, often exit mitosis in a process termed "mitotic slippage," which generates tetraploid cells and limits the effectiveness of antimitotic chemotherapy drugs. We show that ZYG11A/B subunit knockdown, or broad cullin-RING ubiquitin ligase inactivation with the small molecule MLN4924, inhibits mitotic slippage in human cells, suggesting the potential for antimitotic combination therapy.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Mitosis , Proteolisis , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Caenorhabditis elegans/efectos de los fármacos , Línea Celular Tumoral , Células HEK293 , Humanos , Mitosis/efectos de los fármacos , Nocodazol/farmacología , Unión Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Imagen de Lapso de TiempoRESUMEN
The cullin CUL-2 is a crucial component of a subclass of multisubunit cullin-RING ubiquitin-ligases. The specificity of CUL-2-based complexes is provided by variable substrate-recognition subunits that bind to specific substrates. In Caenorhabditis elegans, CUL-2 regulates several key processes in cell division and embryonic development, including meiotic progression, anterior-posterior polarity and mitotic chromatin condensation. However, the substrate recognition subunits that work in these CUL-2-dependent processes were unknown. Here, we present evidence that ZYG-11 is the substrate-recognition subunit for a CUL-2-based complex that regulates these functions. We show that ZYG-11 interacts with CUL-2 in vivo and binds to the complex adaptor protein Elongin C using a nematode variant of the VHL-box motif. We show that the ZYG11 gene family encompasses two main branches in metazoa, and provide evidence that members of the extended ZYG11 family in nematodes and humans are conserved components of CUL2-based ubiquitin-ligases.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas Cullin/genética , Evolución Molecular , Complejos Multiproteicos/genética , Filogenia , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Análisis por Conglomerados , Proteínas Cullin/metabolismo , Elonguina , Humanos , Datos de Secuencia Molecular , Especificidad por Sustrato , Factores de Transcripción/metabolismoRESUMEN
The faithful segregation of chromosomes during meiosis is vital for sexual reproduction. Currently, little is known about the molecular mechanisms regulating the initiation and completion of meiotic anaphase. We show that inactivation of CUL-2, a member of the cullin family of ubiquitin ligases, delays or abolishes meiotic anaphase II with no effect on anaphase I, indicating differential regulation during the two meiotic stages. In cul-2 mutants, the cohesin REC-8 is removed from chromosomes normally during meiosis II and sister chromatids separate, suggesting that the failure to complete anaphase results from a defect in chromosome movement rather than from a failure to sever chromosome attachments. CUL-2 is required for the degradation of cyclin B1 in meiosis and inactivation of cyclin B1 partially rescued the meiotic delay in cul-2 mutants. In cul-2 mutants, the failure to degrade cyclin B1 precedes the metaphase II arrest. CUL-2 is also required for at least two aspects of embryonic polarity. The extended meiosis II in cul-2 mutants induces polarity reversals that include reversed orientation of polarity proteins, P granules, pronuclei migration and asymmetric cell division. Independently of its role in meiotic progression, CUL-2 is required to limit the initiation/spread of the polarity protein PAR-2 in regions distant from microtubule organizing centers. Finally, we show that inactivation of the leucine-rich repeat protein ZYG-11 produces meiotic and polarity reversal defects similar to those observed in cul-2 mutants, suggesting that the two proteins function in the same pathways.