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. 2019 Dec 9;9:18622. doi: 10.1038/s41598-019-54738-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Cdc7-ASK phosphorylates Aurora B and increases its kinase activity in vitro. (a) Rat Aurora B (50 ng), Cdc7-ASK (20 ng) and Histone H3 (HH3; 0.5 µg as a substrate) were mixed as indicated in the kinase assays with [γ-³²P] ATP. Half of the reaction was analyzed. HH3 was phosphorylated by Aurora B kinase. Cdc7-ASK increases phosphorylation of both HH3 and Aurora B. (b) Human Aurora B (50 ng), Cdc7-ASK (20 ng), and Plk1 (50 ng) were mixed as indicated in kinase assays. Phosphorylation was detected by western analyses using pT210 (detecting the autophosphorylation of Aurora B; see c). Proteins were also detected by western. (c) Comparison of the amino acid sequences around the T-loop of Aurora B from different species. Thr232 and Thr236 of human Aurora B are conserved. Thr232 is the known autophosphorylation site^22,36. The sequences around Thr232 are similar to those around T210 of Plk. Thus, pT210 antibody detects Aurora B T232 phosphorylation. (d) Titration of human Aurora B-INCENP in the kinase assays with HH3 as a substrate. Phosphorylation was detected by pT210 and HH3 pS10 antibodies. (e) Titration of Cdc7-ASK in the kinase assays with human Aurora B-INCENP complex. Cdc7 increases HH3 S28 phosphorylation, which was detected by HH3 pS28 antibody. (f) Rat Aurora B (wild-type or kinase-dead [K109R]) in a complex with INCENP purified from insect cells was incubated with Cdc7-ASK. Cdc7 increased HH3 phosphorylation detected by pS28 antibody. The pT210 signal in the wild-type Aurora B was not significantly affected by Cdc7 in this experiment. pT210 and HH3 pS28 were undetectable with the kinase-dead Aurora B. In (b,d,e), human Aurora B-INCENP complex from Carna was used, and in (a,b,e,f), Cdc7-ASK complex from Carna was used.