RESUMO
Survivin operates in a complex with aurora B kinase and is phosphorylated by it on threonine 117 in vitro. Here we ask whether phosphorylation of survivin by aurora B kinase regulates its function during mitosis in vivo. Using a phospho-specific antibody we first establish that survivin is phosphorylated at T117 during mitosis and is present at the midbody during cytokinesis. Next we use two independent RNAi complementation approaches to investigate threonine 117 mutants in survivin depleted cells. Our data suggest that while non-phosphorylatable survivin, survivin(T117A), can substitute for the wild type protein, a phosphomimic, survivin(T117E) cannot restore viability, nor can it complement chromosome congression and spindle checkpoint defects that arise due to depletion of endogenous survivin. Fluorescence imaging and fluorescence recovery after photobleaching analysis suggest that the phosphomimic has reduced affinity for centromeres compared with the non-phosphorylatable form. We conclude that survivin is phosphorylated at T117 during mitosis, and once phosphorylated, dephosphorylation is crucial for chromosome congression and progression into anaphase.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Linhagem Celular Tumoral , Centrômero/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Immunoblotting , Proteínas Inibidoras de Apoptose , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Proteínas de Neoplasias/genética , Fosforilação , Interferência de RNA , SurvivinaRESUMO
The Aurora kinases have been implicated in tumorigenesis and are important regulators of diverse cell cycle events, ranging from the entry into mitosis, centrosome function, mitotic spindle formation, chromosome biorientation and segregation, and cytokinesis. The recent identification of novel binding partners and key downstream effectors, together with new small-molecule inhibitors that display efficacy against tumours, heralds an upsurge of interest in these critical kinases. This review details new developments in the field and analyses the potential of Aurora kinases as anticancer targets.
Assuntos
Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica , Neoplasias/prevenção & controle , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/uso terapêutico , Animais , Aurora Quinases , Ciclo Celular , Centrossomo/ultraestrutura , Cromossomos/ultraestrutura , Humanos , MitoseRESUMO
The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.
Assuntos
Proteínas de Ciclo Celular , Cromossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Autorradiografia , Cromossomos/fisiologia , Eletroforese em Gel de Poliacrilamida , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Carioferinas/metabolismo , Proteínas Luminescentes , Microesferas , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência , Células Tumorais Cultivadas , Xenopus , Proteínas de XenopusRESUMO
Chromosome orientation and alignment within the mitotic spindle requires the Aurora B protein kinase and the mitotic centromere-associated kinesin (MCAK). Here, we report the regulation of MCAK by Aurora B. Aurora B inhibited MCAK's microtubule depolymerizing activity in vitro, and phospho-mimic (S/E) mutants of MCAK inhibited depolymerization in vivo. Expression of either MCAK (S/E) or MCAK (S/A) mutants increased the frequency of syntelic microtubule-kinetochore attachments and mono-oriented chromosomes. MCAK phosphorylation also regulates MCAK localization: the MCAK (S/E) mutant frequently localized to the inner centromere while the (S/A) mutant concentrated at kinetochores. We also detected two different binding sites for MCAK using FRAP analysis of the different MCAK mutants. Moreover, disruption of Aurora B function by expression of a kinase-dead mutant or RNAi prevented centromeric targeting of MCAK. These results link Aurora B activity to MCAK function, with Aurora B regulating MCAK's activity and its localization at the centromere and kinetochore.