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Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA

Oncogene volume 25, pages 827–837 (2006)Cite this article

Abstract

The epithelial cell transforming gene 2 (ECT2) protooncogene encodes a Rho exchange factor, and regulates cytokinesis. ECT2 is phosphorylated in G2/M phases, but its role in the biological function is not known. Here we show that two mitotic kinases, Cdk1 and polo-like kinase 1 (Plk1), phosphorylate ECT2 in vitro. We identified an in vitro Cdk1 phosphorylation site (T412) in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding was inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, did not exhibit strong association with Plk1 PBD compared with wild-type (WT) ECT2. Moreover, ECT2 T412A, but not phosphomimic T412D, displayed a diminished accumulation of GTP-bound RhoA compared with WT ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of WT ECT2 or the T412D mutant caused cortical hyperactivity in U2OS cells during cell division, this activity was not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert.

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Acknowledgements

We thank Dr Michael Gottesman for support, Dr Keiju Kamijo for assistance with microscopic analysis, and Dr Hiroki Inoue and George Leiman for critical reading of the manuscript. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

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  1. T Tatsumoto

    Present address: Fukuoka Teishin Hospital, 2-6-11 Yakuin, Chuo-ku, Fukuoka, 810-8798, Japan

Authors and Affiliations

  1. Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD, USA

    F Niiya, T Tatsumoto & T Miki

  2. Laboratory of Metabolism, National Cancer Institute, Bethesda, MD, USA

    K S Lee

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Correspondence to T Miki.

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Niiya, F., Tatsumoto, T., Lee, K. et al. Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA. Oncogene 25, 827–837 (2006). https://doi.org/10.1038/sj.onc.1209124

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