Figure 6

AZD1152 reduces Aurora B protein level by increasing poly-ubiquitination and degradation via the proteasome. (A) Western blots were performed for Aurora B, phospho-Histone H3 and actin levels. HER18 cells were treated with increasing duration of AZD1152 20 nM (left panel) and with increasing concentration of AZD1152 for 48 hours (right panel) as labeled. (B) HER18 cells were treated with or without 20 nM AZD1152 for 48 hours and then in the presence of cycloheximide for up to four hours. Immunoblots of Aurora B and actin are shown. (C) The integrated optical densities of the protein bands in B. The amount of Aurora B protein relative to 0 h of cycloheximide treatment was calculated for each group and corrected for gel loading differences based on actin. The relative rates of Aurora B turnover were estimated by the slope of the linear regression line for the control and AZD1152-HQPA-treated cells. (D) MDA-MB-231 cells show increased Aurora B protein levels when treated with the proteasome inhibitor MG132 and AZD1152-HQPA versus AZD1152-HQPA treatment alone. (E) AZD1152-HQPA causes increased poly-ubiquitinated Aurora B versus control. HER18 cells were transfected with the plasmids (Flag-Aurora B and HA-ubiquitin) and treated with the drugs (AZD1152-HQPA and the proteasome inhibitor MG132) as labeled above the top panel. Top panel: anti-HA immunoblot of anti-Flag-immunoprecipitates. Bottom panels: Anti-Flag immunoblot of whole cell lysates. (F) AZD1152-HQPA may disturb mitosis in at least two pathways: 1) through inhibition of Aurora B kinase activity, and 2) through increasing poly-ubiquitination and proteasomal degradation of Aurora B protein leading to decreased Aurora B function.