Function And Regulation Of Human Sgo1

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Proper alignment of chromosomes and their segregation in mitosis also relies on cohesion between sister chromatids. Vertebrate Sgo1 localizes to kinetochores and is required to prevent premature sister centromere separation in mitosis. Sgo1 is degraded by the anaphase-promoting complex, allowing the separation of sister centromeres in anaphase. It was then speculated that Sgo accounts for the differential cohesion observed between the arms and the centromeres of mitotic sister chromatids. Sgo interacts strongly with microtubules in vitro and that it regulates kinetochore microtubule stability in vivo, consistent with a direct microtubule interaction. Human Sgo1 binds to protein phosphatase 2A (PP2A), the Sgo1-PP2A interaction is required for proper chromosome segregation. However, little is known on the molecular basis of Sgo1 degradation and its temporal control during mitosis.Here we show that human Sgo1 (hSgo1) is phosphorylated by NEK2A kinase and such phosphorylation regulates hSgo1 function. Using a combination of computational prediction and mass spectrometric analysis, we have mapped the Nek2A-mediated phosphorylation sites. We have also proved that NEK2A can phosphorylate hSgo1 at S14/507 sites by combination of biochemical assays in vitro and mass spectrometric analysis. We have demonstrated that NEK2A-mediated protein phosphorylation of hSgo1 at Ser^14,507 is not required for the localization of hSgo1 at the kinetochore. However, expression of non-phosphorylatable mutant GFP-Sgo1^S14/507A-mutant results in a significant increase in attachment errors and perturbed chromosome congression. We propose that Nek2A-mediated phosphorylation of hSgo1 is critical for mitotic progression and chromosome segregation and provides a link between mitotic clock and sister centromere cohesion and microtubule interactions at kinetochores. Moreover, we demonstrated that such phosphorylation downregulates phosphatase activity of PP2A, thereby facilitating the onset of anaphase in mitosis. In this research, we have explored some molecular basis of hSgo1 degradation and its temporal control during mitosis, we show that APC/C targets human hSgo1 for degradation through a destruction box motif (D-box) in its C-terminus. Mutation of the D-box causes human cells to transiently arrest in metaphase more than 1 hour and chromosomes were not separated until the D-box-mutant entirely disappeared. These results establish a role for D-box in hSgo1 degradation and faithful chromosome segregation. Besides, the D-box mutation of hSgo1 resulted in significant increases in cells bearing lagging chromosomes, but after arrest for some time, chromosomes still started to become scattered and cytokinesis was initiated in spite of the existence of lagging chromosomes. Because there is an overlap between the D-box domain and phosphorylationconsensus motifs of Aurora B kinases, we examined whether mutation of D-box altered localization of Aurora B to the kinetochore. As expected, mutation of the D-box affects the localization of Aurora B and CENP-E. According to these results, we suggest that mutation in the D-box leads to defects of chromosome alignment and segregation through its effect on the localization of Aurora B and CENP-E. On the other hand, hSgo1 may sensor the tension across the sister kinetochore through the D-box in its C-terminus.Finally, we have studied some biochemical character of hSgo1. We indicate that both N- and C- terminus are essential for kinetochore localization of hSgo1. There is an interaction between N- and C- terminus, and this interaction also exist in the hSgo1 localized at kinetochore by the results of FRET (Fluorescence Resonance Energy Transfer) assay in vivo. According to these results, we propose a model to explain the mechanism of the hSgo1 function for protecting cohesin. hSgo1 can be a dimer through the interaction between its N- and C- terminaus, which forms a ring around cohesin ring to protect the cohesin complex. hSgo1 will become inactive by cleavage, degradation, remove and so on, allowing the separation of sister centromeres in anaphase.