APCcdh1 Mediates Degradation of the Oncogenic Rho-GEF Ect2 after Mitosis

Caroline Liot,Jacques Bertoglio,Catherine Crouin,Susanne Schmidt,Laetitia Seguin,Aurélie Siret,Jean Kanellopoulos

doi:10.1371/journal.pone.0023676

Abstract

BackgroundBesides regulation of actin cytoskeleton-dependent functions, Rho GTPase pathways are essential to cell cycle progression and cell division. Rho, Rac and Cdc42 regulate G1 to S phase progression and are involved in cytokinesis. RhoA GDP/GTP cycling is required for normal cytokinesis and recent reports have shown that the exchange factor Ect2 and the GTPase activating protein MgcRacGAP regulate RhoA activity during mitosis. We previously showed that the transcription factors E2F1 and CUX1 regulate expression of MgcRacGAP and Ect2 as cells enter S-phase.Methodology/Principal FindingsWe now report that Ect2 is subject to proteasomal degradation after mitosis, following ubiquitination by the APC/C complex and its co-activator Cdh1. A proper nuclear localization of Ect2 is necessary for its degradation. APC-Cdh1 assembles K11-linked poly-ubiquitin chains on Ect2, depending upon a stretch of ∼25 amino acid residues that contain a bi-partite NLS, a conventional D-box and two TEK-like boxes. Site-directed mutagenesis of target sequences generated stabilized Ect2 proteins. Furthermore, such degradation-resistant mutants of Ect2 were found to activate RhoA and subsequent signalling pathways and are able to transform NIH3T3 cells.Conclusions/SignificanceOur results identify Ect2 as a bona fide cell cycle-regulated protein and suggest that its ubiquitination-dependent degradation may play an important role in RhoA regulation at the time of mitosis. Our findings raise the possibility that the overexpression of Ect2 that has been reported in some human tumors might result not only from deregulated transcription, but also from impaired degradation.

Highlights

Rho GTPase pathways control the actin cytoskeleton and are involved in a wide array of cellular functions including cell morphology, cell migration and regulation of gene expression
We previously showed that expression of MgcRacGAP and Ect2 is upregulated when cells enter S-phase and found that the transcription factors E2F1 and CUX1 are responsible for transcriptional induction of these genes [14,15]
Ect2 levels fluctuate during the cell cycle To study expression of Ect2 during the cell cycle, western blot analyses were performed in two different cell types: the interleukin2 (IL2)-dependent human lymphocyte cell line Kit 225 and the HEK 293 epithelial adherent cell line

Summary

Introduction

Rho GTPase pathways control the actin cytoskeleton and are involved in a wide array of cellular functions including cell morphology, cell migration and regulation of gene expression They are essential to cell cycle progression and cell division [1,2]. Failed cytokinesis can occur either as a result of Rho inhibition or from expressing constitutively activated versions of the GTPase, indicating that GDP/GTP cycling is required for normal cytokinesis [3,4,5] Consistent with this hypothesis, recent reports have shed light on the complementary roles of at least two exchange factors, GEF-H1 and Ect, which control the loading of GTP onto Rho and of two GTPase activating proteins (GAPs), p190RhoGAP and MgcRacGAP, which increase the rate of GTP hydrolysis [6,7,8,9]. We previously showed that the transcription factors E2F1 and CUX1 regulate expression of MgcRacGAP and Ect as cells enter S-phase

Methods

Results

Conclusion