Protocol to detect OLA1 polyubiquitination by Aurora A in vivo and in vitro

Zhenzhou Fang; Xingming Li; Yuki Yoshino; Natsuko Chiba

doi:10.1016/j.xpro.2024.103008

Protocol to detect OLA1 polyubiquitination by Aurora A in vivo and in vitro

STAR Protoc. 2024 Apr 10;5(2):103008. doi: 10.1016/j.xpro.2024.103008. Online ahead of print.

Authors

Zhenzhou Fang¹, Xingming Li¹, Yuki Yoshino², Natsuko Chiba³

Affiliations

¹ Department of Cancer Biology, Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan; Laboratory of Cancer Biology, Graduate School of Life Sciences, Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan.
² Department of Cancer Biology, Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan; Laboratory of Cancer Biology, Graduate School of Life Sciences, Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan; Department of Cancer Biology, Tohoku University Graduate School of Medicine, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan.
³ Department of Cancer Biology, Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan; Laboratory of Cancer Biology, Graduate School of Life Sciences, Tohoku University, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan; Department of Cancer Biology, Tohoku University Graduate School of Medicine, 4-1 Seiryomachi Aoba-ku, Sendai 980-8575, Japan. Electronic address: natsuko.chiba.c7@tohoku.ac.jp.

Abstract

Aurora A is a critical kinase that functions in centrosome maturation and bipolar spindle assembly. On the other hand, Aurora A has E3 ubiquitin ligase activity and polyubiquitinates Breast cancer gene 1 (BRCA1)-interacting protein Obg-like ATPase 1 (OLA1), targeting it for proteasomal degradation. Here, we present a protocol to detect OLA1 ubiquitination. We describe steps for recovering frozen cells and protein purification. We then detail assays for both in vivo and in vitro ubiquitination of OLA1 by Aurora A. For complete details on the use and execution of this protocol, please refer to Fang et al.¹.

Keywords: Cancer; Molecular Biology; cell Biology.