RESUMO
Aurora B kinase plays essential roles in mitosis. Its protein levels increase before the onset of mitosis and sharply decrease during mitosis exit. The latter decrease is due to a balance between the actions of the E3 ubiquitin ligase anaphase-promoting complex or cyclosome (activated by the Cdh1 adapter), and the deubiquitinating enzyme USP35. Aurora B also executes important functions in interphase. Abnormal modulation of Aurora B in interphase leads to cell cycle defects often linked to aberrant chromosomal condensation and segregation. Very little is however known about how Aurora B levels are regulated in interphase. Here we found that USP13-associates with and stabilizes Aurora B in cells, especially before their entry into mitosis. In order for USP13 to exert its stabilizing effect on Aurora B, their association is promoted by the Aurora B-mediated phosphorylation of USP13 at Serine 114. We also present evidence that USP13 instigates Aurora B deubiquitination and/or protect it from degradation in a non-catalytic manner. In addition, we report that genetic or chemical modulation of the cellular levels/activity of USP13 affects unperturbed cell-cycle progression. Overall our study unveils the molecular and cellular connections of the USP13-Aurora B axis, which potentially participates in the rewiring of the cell cycle happening in cancer cells.
Assuntos
Aurora Quinase B/metabolismo , Ciclo Celular/genética , Endopeptidases/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Progressão da Doença , Endopeptidases/metabolismo , Estabilidade Enzimática , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fosforilação , Ligação Proteica , Serina/metabolismo , Proteases Específicas de UbiquitinaRESUMO
The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclo Celular/fisiologia , Ubiquitinação/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Ciclo Celular/genética , Humanos , Fosforilação/genética , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Ubiquitina/metabolismo , Ubiquitinação/genéticaRESUMO
SUMOylation is a widely used protein posttranslational mechanism capable of regulating substrates localization, stability, and/or activity. Identification and characterization of bona fide SUMO substrates is a laborious task but its discovery can shed light to exquisite and crucial regulatory signaling events occurring within the cell. Experiments performed in the SUMOylation field often demand a good understanding of the putative substrate's function and necessitate a solid knowledge regarding both in vitro and in vivo approaches. This contribution offers a simplified view into some of the most common experiments performed in biochemical and cell biological research of the SUMO pathway in mammalian systems. It also summarizes and updates well established protocols and tricks in order to improve the likelihood to obtain reliable and reproducible results.
Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/fisiologia , Animais , Humanos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação/genéticaRESUMO
Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt.