RESUMO
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
SLX4, a scaffold for structure-specific DNA repair nucleases, is important for several types of DNA repair. Many repair proteins bind to sites of DNA damage, resulting in subnuclear "foci," but SLX4 forms foci in human cells even without DNA damage. Using several approaches, we show that most, but not all, SLX4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length. The SLX1 Holliday-junction-processing enzyme is recruited to telomeres by SLX4, and SLX4, in turn, is recruited by a motif that binds to the shelterin subunit TRF2 directly. We also show that TRF2-dependent recruitment of SLX4 prevents telomere damage. Furthermore, SLX4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association. These findings reveal that SLX4 plays multiple roles in regulating telomere homeostasis.