Figure 3. Aurora B phosphorylates YY1 at serine 184 in vitro.

(A) Radioactive in vitro kinase assay using purified Aurora kinase isoforms and GST-YY1 as substrate. The kinase reactions include GST-YY1 only (no kinase), Aurora A, Aurora B and Aurora C only (no substrate) and GST-YY1 with each Aurora isoform. The reactions were performed at 30°C for 30 minutes. The reaction mixture was separated on a 10% SDS-PAGE gel and stained with Coomassie blue to visualize the protein bands and exposed to a phosphorimager screen. (B) Cold in vitro kinase assay reactions using purified Aurora A, Aurora B, Aurora C, Plk1 and PAK1 kinases and purified non-tagged YY1 as substrate. The reactions were performed at 30°C for 30 minutes followed by Western blot. The blot was probed with anti-pS184 antibody, then stripped and reprobed with anti-YY1 antibody. (C) Radioactive in vitro kinase assay using purified Aurora B kinase and GST-YY1 as substrate. The kinase reactions include GST-YY1 only (no kinase), Aurora B only (no substrate) and GSTY-YY1, GST-YY1 S180A, GST-YY1 S184A or GST-YY1 S180,184A with Aurora B. The reactions were performed as described for panel A. (D) Cold in vitro kinase assay reactions using purified Aurora B and GST-YY1 as substrate. The kinase reactions include GST-YY1 only (no kinase), Aurora B only (no substrate) and GST-YY1, GST-YY1 S180A, GST-YY1 S184A or GST-YY1 S180,184A with Aurora B. The reactions were performed at 30°C for 30 minutes followed by Western blot. The blot was probed with anti-pS184 antibody, then anti-YY1 antibody. (E) Cold in vitro kinase assay reactions using purified Aurora B and GST-YY1 as substrate. The kinase reactions were performed at 30°C for 30 minutes. After the reaction, GST-YY1 was washed with lysis buffer and resuspended in phosphatase buffer, and incubated with protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) or λ-phosphatase at 30°C for 30 minutes, followed by Western blot. The blot was probed with anti-pS184 antibody, then stripped and reprobed with anti-YY1.