Identification of the first Rho–GEF inhibitor, TRIPα, which targets the RhoA-specific GEF domain of Trio

AbstractThe Rho–guanine nucleotide exchange factors (Rho–GEFs) remodel the actin cytoskeleton via their Rho–GTPase targets and affect numerous physiological processes such as transformation and cell motility. They are therefore attractive targets to design specific inhibitors that may have therapeutic applications. Trio contains two Rho–GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively. Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity. One aptamer, TRIAPα (TRio Inhibitory APtamer), specifically blocks GEFD2-exchange activity on RhoA in vitro. The corresponding peptide sequence, TRIPα, inhibits TrioGEFD2-mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells. Thus TRIPα is the first Rho–GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho–GEFs

Localization of Staf50, a Member of the Ring Finger Family, to 11p15 by Fluorescencein SituHybridization

International audienc

Localization of the Ribonuclease L Inhibitor Gene (RNS4I), a New Member of the Interferon-Regulated 2–5A Pathway, to 4q31 by Fluorescencein SituHybridization

International audienc

Identification of the first Rho-GEF inhibitor, TRIP alpha, which targets the RhoA-specific GEF domain of Trio

The Rho-guanine nucleotide exchange factors (Rho-GEFs) remodel the actin cytoskeleton via their Rho-GTPase targets and affect numerous physiological processes such as transformation and cell motility. They are therefore attractive targets to design specific inhibitors that may have therapeutic applications. Trio contains two Rho-GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively. Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity. One aptamer, TRIAPalpha (TRio Inhibitory APtamer), specifically blocks GEFD2-exchange activity on RhoA in vitro. The corresponding peptide sequence, TRIPalpha, inhibits TrioGEFD2-mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells. Thus TRIPalpha is the first Rho-GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho-GEFs. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

The Human Rho-GEF Trio and Its Target GTPase RhoG Are Involved in the NGF Pathway, Leading to Neurite Outgrowth

International audienceRho-GTPases control a wide range of physiological processes by regulating actin cytoskeleton dynamics. Numerous studies on neuronal cell lines have established that Rac, Cdc42, and RhoG activate neurite extension, while RhoA mediates neurite retraction. Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by accelerating GDP/GTP exchange. Trio displays two Rho-GEF domains, GEFD1, activating the Rac pathway via RhoG, and GEFD2, acting on RhoA, and contains numerous signaling motifs whose contribution to Trio function has not yet been investigated. Genetic analyses in Drosophila and in Caenorhabditis elegans indicate that Trio is involved in axon guidance and cell motility via a GEFD1-dependent process, suggesting that the activity of its Rho-GEFs is strictly regulated. Here, we show that human Trio induces neurite outgrowth in PC12 cells in a GEFD1-dependent manner. Interestingly, the spectrin repeats and the SH3-1 domain of Trio are essential for GEFD1-mediated neurite outgrowth, revealing an unexpected role for these motifs in Trio function. Moreover, we demonstrate that Trio-induced neurite outgrowth is mediated by the GEFD1-dependent activation of RhoG, previously shown to be part of the NGF (nerve growth factor) pathway. The expression of different Trio mutants interferes with NGF-induced neurite outgrowth, suggesting that Trio may be an upstream regulator of RhoG in this pathway. In addition, we show that Trio protein accumulates under NGF stimulation. Thus, Trio is the first identified Rho-GEF involved in the NGF-differentiation signaling

Trio : Un facteur d’échange des GTPases Rho aux multiples facettes impliqué dans le guidage axonal

Les facteurs d’échanges nucléotidiques (GEF) des GTPases Rho remodèlent le cytosquelette d’actine en activant leurs cibles, les GTPases Rho, et sont ainsi impliqués dans de nombreux processus physiologiques. Trio est le premier membre dune famille de protéines multifonctionnelles possédant deux domaines fonctionnels d’échange dont la spécificité est différente, le premier activant Rac et le second RhoA. Trio possède aussi de nombreux domaines d’interaction protéine-protéine, et semble donc se situer à un carrefour de différentes voies de signalisation. La liaison de Trio avec des protéines qui lient l’actine, suggère en outre que l’activation des GTPases par Trio est contrôlée par sa localisation spécifique près du cytosquelette. De plus, l’identification d’orthologues de Trio chez les invertébrés a permis de proposer une fonction essentielle de Trio dans le guidage axonal