1. ¹Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD, USA
2. ²Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick Inc., National Cancer Institute-Frederick, Frederick, MD, USA
3. ³Laboratory of Metabolism, National Cancer Institute, Bethesda, MD, USA

Correspondence: Dr T Miki, Molecular Tumor Biology Section, Laboratory of Cell Biology, National Cancer Institute, Building 37, Room 2144, 37 Convent Dr MSC 4256, Bethesda, MD 20892-4256, USA. E-mail: toru@helix.nih.gov

⁴Current address: Department of Urology, Yamaguchi Red Cross Hospital, 53-1, Hachimanbaba, Yamaguchi 753-8519, Japan.

Received 12 May 2005; Revised 1 August 2005; Accepted 1 August 2005; Published online 19 September 2005.

Abstract

The Rho activator ECT2 functions as a key regulator in cytokinesis. ECT2 is phosphorylated during G2/M phase, but the physiological significance of this event is not well known. In this study, we show that phosphorylation of ECT2 at threonine-341 (T341) affects the autoregulatory mechanism of ECT2. In G2/M phase, ECT2 was phosphorylated at T341 most likely by Cyclin B/Cyclin-dependent kinase 1 (Cdk1), and then dephosphorylated before cytokinesis. Depletion of ECT2 by RNA interference (RNAi) efficiently induced multinucleate cells. Expression of the phospho-deficient mutant of ECT2 at T341 suppressed the multinucleation induced by RNAi to ECT2, indicating that ECT2 is biologically active even when it is not phosphorylated at T341. However, the phospho-mimic mutation at T341 weakly stimulates the catalytic activity of ECT2 as detected by serum response element reporter gene assays. As T341 is located at the hinge region of the N-terminal regulatory domain and C-terminal catalytic domain, phosphorylation of T341 may help accessing downstream signaling molecules to further activate ECT2. We found that the phospho-mimic mutation T341D increases binding with itself or the N-terminal half of ECT2. These results suggest a conformational change of ECT2 upon phosphorylation at T341. Therefore, ECT2 activity might be regulated by the phosphorylation status of T341. We propose that T341 phosphorylation by Cyclin B/Cdk1 could be a trigger for further activation of ECT2.