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

Background: Besides 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 Findings: We 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/Significance: Our 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

Inside-Out Regulation of ICAM-1 Dynamics in TNF-α-Activated Endothelium

Background: During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions. Principal Findings: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions. Conclusions: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesio

Gene Expression Profiles of the NCI-60 Human Tumor Cell Lines Define Molecular Interaction Networks Governing Cell Migration Processes

Although there is extensive information on gene expression and molecular interactions in various cell types, integrating those data in a functionally coherent manner remains challenging. This study explores the premise that genes whose expression at the mRNA level is correlated over diverse cell lines are likely to function together in a network of molecular interactions. We previously derived expression-correlated gene clusters from the database of the NCI-60 human tumor cell lines and associated each cluster with function categories of the Gene Ontology (GO) database. From a cluster rich in genes associated with GO categories related to cell migration, we extracted 15 genes that were highly cross-correlated; prominent among them were RRAS, AXL, ADAM9, FN14, and integrin-beta1. We then used those 15 genes as bait to identify other correlated genes in the NCI-60 database. A survey of current literature disclosed, not only that many of the expression-correlated genes engaged in molecular interactions related to migration, invasion, and metastasis, but that highly cross-correlated subsets of those genes engaged in specific cell migration processes. We assembled this information in molecular interaction maps (MIMs) that depict networks governing 3 cell migration processes: degradation of extracellular matrix, production of transient focal complexes at the leading edge of the cell, and retraction of the rear part of the cell. Also depicted are interactions controlling the release and effects of calcium ions, which may regulate migration in a spaciotemporal manner in the cell. The MIMs and associated text comprise a detailed and integrated summary of what is currently known or surmised about the role of the expression cross-correlated genes in molecular networks governing those processes

Random mutagenesis of Peptide aptamers as an optimization strategy for inhibitor screening.

International audienceAccumulating work over the past decade has shown that peptide aptamer screening represents a valid strategy for inhibitor identification that can be applied to a variety of different targets. Because of the screening method in cells and the highly combinatorial libraries available, this approach yields rapidly highly specific candidate inhibitors. Once a hit peptide has been identified, its interaction strength and affinity towards its target protein can be optimized even more, in order to increase its inhibition efficiency when subsequently applied in vivo. A condition to a successful optimization is that gain of inhibition strength should not result in loss of specificity.Here we present a simple method for peptide aptamer optimization, which can be achieved by PCR-based random mutagenesis combined with a selection screen in yeast using a strong selective drug. The rationale of this approach, which has proven valid and efficient, is that stronger interaction in yeast will also lead to stronger inhibition. Our optimization method is effective, without loss of specificity, which is of a great importance for the discovery of inhibitors that target specific protein-protein interactions

A novel function for Cyclin A2: Control of cell invasion via RhoA signaling

International audienceCyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2-depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration

Regulation of Postsynaptic Function by the Dementia-Related ESCRT-III Subunit CHMP2B

International audienc