Fig. 4: AURKA-mediated phosphorylation of SMC2 T574 leads to mitotic catastrophe.

A HeLa cells were transfected with AURKA siRNA-1, AURKA siRNA-2, or control siRNA. These cells were treated with BMH-21 or DMSO, and were synchronized at M phase by nocodazole and harvested by shaking off. Immunoprecipitation was performed with anti-pan-phospho-serine/threonine antibody. The immunoprecipitates were immunoblotted with the indicated antibodies. B HeLa cells were transfected with the indicated doses of Flag-AURKA plasmid or Flag-vector. These cells were treated with BMH-21 or DMSO, and were synchronized at M phase and harvested as described in (A). Immunoprecipitation was performed with anti-pan-phospho-serine/threonine antibody. The immunoprecipitates were immunoblotted with the indicated antibodies. C HeLa cells were treated with 0 μM, 5 μM, 10 μM, and 15 μM TCS7010. These cells were treated with BMH-21 or DMSO, and were synchronized at M phase and harvested as described in (A). Immunoprecipitation was performed with anti-pan-phospho-serine/threonine antibody. The immunoprecipitates were immunoblotted with the indicated antibodies. D In vitro phosphorylation experiment was performed using purified His-AURKA and GST-SMC2. Proteins were resolved by SDS-PAGE and probed with anti-pan-phospho-lysine antibody. Coomassie blue staining showed the purified proteins. E Sequence alignment of the AURKA phosphorylation consensus within SMC2 and substrates of AURKA. Phosphorylated threonine residues are highlighted in red, arginine at the n − 3 position is highlighted in green, and hydrophobic residues at the n + 1 position are highlighted in blue. F In vitro phosphorylation experiment was performed using purified His-AURKA and GST-SMC2 T574A. Proteins were resolved by SDS-PAGE and probed with the indicated antibodies. Coomassie blue staining showed the purified proteins. G HeLa cells were transfected with the indicated siRNAs and the indicated plasmids (left). A quantitative comparison of multinucleated cells in HeLa cells in the indicated circumstances is shown (n > 500) (right). **p < 0.01. ***p < 0.001. ****p < 0.0001. n.s. denotes no significance.