Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint

Abstract

The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

Keywords: Aurora B; Skp2; cell mitosis; genomic stability; spindle checkpoint; tumorigenesis; ubiquitination.

Figure 1.

Skp2 is required for activation of Aurora B in cell mitosis. (A) HeLa luciferase knockdown control cells transfected with p27 or not, and Skp2 knockdown cells were synchronized by nocodazole for 18 hrs before harvested. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 3 hrs) and following immunoblotted with indicated antibodies. Since the ectopic expression of p27 was too strong to see the endogenous p27 clearly, we rerun the last 9 lanes and incubated with p27 and GAPDH antibody again. (C) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs before harvested. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 2, 3 hrs) and following immunoblotted with indicated antibodies. (B, D) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs and then were fixed for IF analysis with indicated antibodies. (E) HeLa cells with luciferase control (mock), Skp2 knockdown restored with vector control, Xp-Skp2 or Skp2-LRR plasmids were synchronized by nocodazole for 18 hrs. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 3 hrs), followed by western blot analysis with indicated antibodies.

Figure 2.

Skp2 and Aurora B interact with each other. (A) 293T cells were transfected with Xp-Skp2 and/or HA-Aurora B, and cells were harvested for coimmmunoprecipitation assay with HA antibody, followed by western blot analysis. (B) 293T cells were transfected with Flag-Skp2 and/or HA-Aurora B, and cells were harvested for coimmmunoprecipitation assay with Flag antibody, followed by western blot analysis. (C) 293T cells with luciferase control or Skp2 knockdown were harvested and coimmmunoprecipitated with Skp2 antibody, followed by western blot analysis. (D) HeLa cells were transfected with Xp-Skp2, and then were synchronized by nocodazole for 18 hrs, followed by IF analysis with Xpress antibody and phospho-Aurora B antibody.

Figure 3.

Skp2 triggers Aurora B ubiquitination. (A) 293T cells were transfected with the indicated plasmids and harvested for in vivo ubiquitination assay (see the Materials and Methods for details). (B) 293T cells with luciferase control or Skp2 knockdown were transfected with the indicated plasmids and harvested for in vivo ubiquitination assay. (C) 293T cells with luciferase control or Skp2 knockdown were harvested and coimmmunoprecipitated with Aurora B antibody, followed by western blot analysis with ubiquitin antibody. (D) 293T cells with Cul-1 or luciferase control knockdown were transfected with indicated plasmids and harvested for in vivo ubiquitination assay. (E) 293T cells were transfected with HA-Aurora B, Xp-Skp2, along with His-Ub WT, K48R, or K63R mutant and harvested for in vivo ubiquitination assay.

Figure 4.

For figure legend, see page 3882.Figure 4. (See previous page) Skp2 is essential for cell mitosis progression and spindle checkpoint. (A) Wide Type (WT) and Skp2^−/− MEFs were harvested for cell cycle analysis. The left panel shows the representative flow cytometry profile and the right panel displays the statistical results. Error bars represent the standard deviation. (B) Skp2 level was shown for the luciferase control and Skp2 knockdown clone#1 and #2 HeLa cells, which were used for the following FASCS assay (C, D) and Live cell time-lapse imaging assay (E). (C) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs and then all the cells were harvested for FASCS assay. The mitotic index was measured based on Histone H3S10p staining. The left panel shows the representative flow cytometry profile and the right panel displays the statistical results. Error bars represent the standard deviation. (D) Skp2 knockdown clone#1 HeLa cells were transfected with vector control or HA-Aurora B plasmids for 30 hrs, synchronized by nocodazole for 18 hrs and then all the cells were harvested for FASCS assay. The mitotic index was measured based on Histone H3S10p staining. The left panel shows the representative flow cytometry profile, the middle panel displays the statistical results, and the right panel shows the Aurora B expression level of the cells used in the assay. Error bars represent the standard deviation. (E) Luciferase control or Skp2 knocking-down HeLa cells expressing H2B-GFP were imaged at every 5 minutes, and Skp2 knockdown clone#1 HeLa cells expressing H2B-GFP transfected with pmCherry-C1-Aurora B was also included. The left panel shows the representative fluorescence video microscopy series from the onset of mitosis to monitor chromosome dynamics and the right panel displays the statistical results. Cell numbers were 100 (Control shRNA), 94(Skp2 shRNA#1), 100(Skp2 shRNA#2), and 72(Skp2 shRNA#1+ Aurora B). Error bars represent the standard deviation.