Aurora-A interacts with AP-2α and down regulates its transcription activity

Abstract

Aurora-A is a serine/threonine protein kinase and plays an important role in the control of mitotic progression. Dysregulated expression of Aurora-A impairs centrosome separation and maturation, which lead to disrupted cell cycle progression and tumorigenesis. However, the molecular mechanism by which Aurora-A causes cell malignant transformation remains to be further defined. In this report, using transcription factors array and mRNA expression profiling array, we found that overexpression of Aurora-A suppressed transcription activity of AP-2α, a tumor suppressor that is often downregulated in variety of tumors, and inhibited expression of AP-2α-regulated downstream genes. These array-based observations were further confirmed by microwell colorimetric TF assay and luciferase reporter assay. Downregulated transcription activity of AP-2α by Aurora-A was found to be associated with reduced AP-2α protein stability, which appeared to be mediated by Aurora-A enhanced ubiquitin-dependent proteasomal degradation of AP-2α protein. Interestingly, Aurora-A-mediated AP-2α degradation was likely dependent Aurora-A kinase activity since inhibition of Aurora-A kinase activity was able to rescue Aurora-A-induced degradation of AP-2α. Moreover, we defined a physical interaction between Aurora-A and AP-2α, and such interaction might bridge the suppressive effect of Aurora-A on AP-2α protein stability. These findings provide new insights into molecular mechanism by which Aurora-A acts as an oncogenic molecule in tumor occurrence and malignant development.

Figure 1. Transcription activity of AP-2α is downregulated by Aurora-A.

(A) The summary of the transcription factors with significant changes in their activities by transcription factors array when Aurora-A was overexpressed. (B) Fluorescence images of the transcription factors arrays hybridized with nuclear extracts from KYSE150/GFP-Aurora-A and KYSE150/GFP cells. The analysis of arrays was replicated twice, exchanging the Cy3- and Cy5- label. Magnified panels show the spots representing the activity of AP-2 transcription factor that was down-regulated upon Aurora-A overexpression. (C) The summary of the AP-2α-regulated genes with significant down-expression when Aurora-A was overexpressed by mRNA expression profiling array. The black bars represent the AP-2α-regulated genes, and the gray bars represent the genes which were not the AP-2α-regulated genes. (D) The nucleoprotein extraction of KYSE150/GFP-Aurora-A and KYSE150/GFP cells were incubated with AP-2α binding DNA double-stranded oligonucleotidic probe on multi-well plates, then anti-AP-2α antibodies and the peroxidase-conjugated anti-mouse IgG were subsequently added. At last, the samples could be read for the colorimetric detection. (E) KYSE150/GFP-Aurora-A and KYSE150/GFP cells were cotransfected with (AP-2)₃-TK-LUC expression vector and a pRL-SV40 expression vector. Transcription activities were expressed as luciferase values after normalization. All experiments were performed three times and described as mean ± SD.

Figure 2. Regulation of AP-2α gene expression by Aurora-A.

(A) AP-2α protein levels in KYSE150/GFP-Aurora-A and KYSE150/GFP cells were analyzed using immunoblotting assay. The actin levels were measured as loading control. (B) AP-2α protein levels in EC9706/pGCsi???EC9706/pGCsi-Aurora-A cells were examined by Western Blotting assay. (C) The HCT116 p53^+/+ and HCT116 p53^-/- cells were transiently transfected with pEGFP-Aurora-A and control plasmids. Cells were lysed after 48h of incubation and evaluated for the expression of AP-2α by Western Blotting assay.

Figure 3. Overexpression of Aurora-A enhances the degradation of AP-2α.

(A) KYSE150/GFP-Aurora-A and KYSE150/GFP cells were treated with CHX respectively, and then harvested at the indicated time points. AP-2α protein level at each time point was determined by Western Blot. (B) The amounts of AP-2α were calculated by densitometry and normalized to corresponding actin levels. The column diagram represents the amount of normalized AP-2α at each time point comparing with the original levels (0 h). (C) Aurora-A enhances the proteasome-dependent degradation of AP-2α. KYSE150/GFP-Aurora-A and KYSE150/GFP cells were treated with CHX alone or combination with MG-132. AP-2α protein level was determined by Western Blot.

Figure 4. Aurora-A-mediated AP-2α degradation depends on Aurora-A kinase activity.

(A) KYSE150/GFP-Aurora-A cells were exposed to 1 µM of Aurora-A kinase inhibitor and cellular proteins were collected 2 hours later. Western Blot was performed with antibodies to pThr288 on Aurora-A or total Aurora-A. DMSO was used as a negative control. (B) KYSE150/GFP-Aurora-A cells were exposed to Aurora-A kinase inhibitor or DMSO for 2 hours prior to treatment with CHX. Cells were harvested at the indicated time points and analyzed by Western Blot. (C) The amounts of AP-2α were calculated by densitometry and normalized to corresponding actin levels. The column diagram represents the amount of normalized AP-2α at each time point comparing with the original levels (0 h).

Figure 5. Physical association of Aurora-A with AP-2α.

(A‵B) Protein extracts from KYSE150/GFP-Aurora-A cells were prepared and immunoprecipitated with anti-Aurora-A, anti-AP-2α, mouse or rabbit IgG. The immunocomplexes were analyzed by SDS-PAGE and immunoblotted with antibodies against Aurora-A and AP-2α respectively. The visualized bands were shown for Aurora-A and AP-2α. (C) Aurora-A-GST fusion protein was bound to glutathione-sepharose beads, and incubated with protein extracts. The pull-down complexes were analyzed by SDS-PAGE and immunoblotted with antibodies against AP-2α. The visualized band of 48 kDa was AP-2α.