Figure 4. Binding of PUM2 to Aurora-A protects Aurora-A from APC/C^Cdh1-mediated degradation.

(A) PUM2 blocks the ubiquitination of Aurora-A. HEK293T cells were transfected with FLAG-tagged Aurora-A, together with different amounts of FLAG-tagged PUM2. Myc-tagged ubiquitin was also added to reveal the ubiquitination of Aurora-A. 24 hrs after transfection, the cells were synchronized in the G2/M phase by treatment with nocodazole for 16 hrs. Subsequently, the synchronized cells were released into cell cycle progression in the presence of a proteasome inhibitor (MG132) for 9 hrs. High molecular weight ubiquitinated Aurora-A accumulated in the transfected cells that were treated with MG132, as shown. The relative intensity of protein bands represented the steady-state protein level of Aurora-A on immunoblotting analysis were quantified and normalized to GAPDH. (B) The D-box of Aurora-A mediates its association with PUM2. HEK293T cells were transfected with FLAG-tagged PUM2 and various HA-tagged Aurora-A fragments. The cell lysates were immunoprecipitated with an anti-HA antibody and immunoblotted with an anti-FLAG antibody to detect PUM2.