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

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.

Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation

“…This augmentation remains unexplained; although it is possible that the over accumulation of p105HEF1 might result in increased phosphorylation. Alternatively, there are examples of proteins in which a single residue is the target of different kinases [41,42]. So we can not exclude that other kinase(s) phosphorylate HEF1 on ser-369 but also on another residue leading to an HEF1 isoform that would be less sensitive to degradation and would be part of the p115 band.…”

Phosphorylation of Ataxin-10 by polo-like kinase 1 is required for cytokinesis

“…Second, we asked whether phosphorylation of Ataxin-10 by the Plk1-kinase domain was essential for Ataxin-10 to interact with Plk1-PBD, since other Plk1 substrates involved in cytokinesis are also found to interact with Plk1 PBD, e.g., Ect2 19 and …”

Polo Kinase and Cytokinesis Initiation in Mammalian Cells: Harnessing the Awesome Power of Chemical Genetics

“…Given that Ect2 is an excellent Plk1 substrate, 30 it is possible that Plk1 phosphorylation triggers a conformational change in Ect2 that induces its central spindle relocalization. Alternatively, Plk1 may phosphorylate Ect2's binding partner CYK-4, creating a docking site for the N-terminal BRCA1 C-terminus (BRCT) domain of Ect2, a potential phosphopeptide-recognition motif.…”

“…Prior studies indicated that such forced exit is accompanied by violent blebbing of the plasma membrane, an actomyosin-and RhoA-dependent process that resembles ectopic furrow formation. 30 Interestingly, whereas control cells blebbed violently, bleb formation was suppressed in Plk1-depleted and BI 2536-treated cells. Thus, cortical contractility that ensues after Cdk1 inactivation appears to require Plk1 activity, consistent with the observed defects in activation of the Ect2-RhoA network and cytokinesis initiation in BI 2536-treated cells.…”