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An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.

Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, Di Fiore PP - J. Cell Biol. (2001)

Bottom Line: Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus.This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization.Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.

ABSTRACT
Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

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A dominant negative Arf6 mutant inhibits ruffling induced by GFP-Eps8 (648–821). (A) Nuclei of quiescent mouse embryo fibroblasts were microinjected with the expression vector encoding a dominant negative Arf6 (Arf6T27N). After 3 h cells were stimulated with PDGF (10 ng/ml) for 10 min, and then fixed and stained with FITC-conjugated phalloidin (green) to detect F-actin and with the anti-myc antibody to detect Arf6T27N (red). Arf6T27N inhibited PDGF-induced ruffling in 90 ± 4% (mean ± SE) of the injected cells, as compared with cells injected with an empty vector. (B) Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors for GFP-Eps8 (648–821) (all panels) together with either a control vector (ctr) or a vector encoding myc-Arf6T27N (Arf6T27N). After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green). Phalloidin (red) staining was also performed. All the comicroinjected cells expressed both the GFP-Eps8 (648–821) fragment and the Arf6T27N mutant, as determined in parallel experiments by directly staining the microinjected cells with anti-myc antibodies to detect Arf6T27N (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bars, 10 μm.
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fig5: A dominant negative Arf6 mutant inhibits ruffling induced by GFP-Eps8 (648–821). (A) Nuclei of quiescent mouse embryo fibroblasts were microinjected with the expression vector encoding a dominant negative Arf6 (Arf6T27N). After 3 h cells were stimulated with PDGF (10 ng/ml) for 10 min, and then fixed and stained with FITC-conjugated phalloidin (green) to detect F-actin and with the anti-myc antibody to detect Arf6T27N (red). Arf6T27N inhibited PDGF-induced ruffling in 90 ± 4% (mean ± SE) of the injected cells, as compared with cells injected with an empty vector. (B) Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors for GFP-Eps8 (648–821) (all panels) together with either a control vector (ctr) or a vector encoding myc-Arf6T27N (Arf6T27N). After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green). Phalloidin (red) staining was also performed. All the comicroinjected cells expressed both the GFP-Eps8 (648–821) fragment and the Arf6T27N mutant, as determined in parallel experiments by directly staining the microinjected cells with anti-myc antibodies to detect Arf6T27N (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bars, 10 μm.

Mentions: The dependence on Rac activity of the effect of GFP-Eps8 (648–821) was further supported by experiments performed with a dominant negative Arf6 mutant. Arf6 is a protein belonging to the Arf subfamily of small GTPases (Donaldson and Radhakrishna, 2001). A dominant negative Arf6 blocks Rac-dependent actin reorganization, likely by preventing the translocation of GTP-bound Rac from an endosomal compartment to the plasma membrane (Radhakrishna et al., 1999; Zhang et al., 1999; Fig. 5 A). Microinjection, in mouse fibroblasts, of a dominant negative mutant Arf6 (Arf6T27N) capable of abrogating Rac-dependent PDGF-induced ruffles inhibited GFP-Eps8 (648–821)–dependent actin cytoskeleton rearrangement (Fig. 5 B), compatible with the requirement for proper localization of Rac.


An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.

Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, Di Fiore PP - J. Cell Biol. (2001)

A dominant negative Arf6 mutant inhibits ruffling induced by GFP-Eps8 (648–821). (A) Nuclei of quiescent mouse embryo fibroblasts were microinjected with the expression vector encoding a dominant negative Arf6 (Arf6T27N). After 3 h cells were stimulated with PDGF (10 ng/ml) for 10 min, and then fixed and stained with FITC-conjugated phalloidin (green) to detect F-actin and with the anti-myc antibody to detect Arf6T27N (red). Arf6T27N inhibited PDGF-induced ruffling in 90 ± 4% (mean ± SE) of the injected cells, as compared with cells injected with an empty vector. (B) Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors for GFP-Eps8 (648–821) (all panels) together with either a control vector (ctr) or a vector encoding myc-Arf6T27N (Arf6T27N). After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green). Phalloidin (red) staining was also performed. All the comicroinjected cells expressed both the GFP-Eps8 (648–821) fragment and the Arf6T27N mutant, as determined in parallel experiments by directly staining the microinjected cells with anti-myc antibodies to detect Arf6T27N (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bars, 10 μm.
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fig5: A dominant negative Arf6 mutant inhibits ruffling induced by GFP-Eps8 (648–821). (A) Nuclei of quiescent mouse embryo fibroblasts were microinjected with the expression vector encoding a dominant negative Arf6 (Arf6T27N). After 3 h cells were stimulated with PDGF (10 ng/ml) for 10 min, and then fixed and stained with FITC-conjugated phalloidin (green) to detect F-actin and with the anti-myc antibody to detect Arf6T27N (red). Arf6T27N inhibited PDGF-induced ruffling in 90 ± 4% (mean ± SE) of the injected cells, as compared with cells injected with an empty vector. (B) Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors for GFP-Eps8 (648–821) (all panels) together with either a control vector (ctr) or a vector encoding myc-Arf6T27N (Arf6T27N). After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green). Phalloidin (red) staining was also performed. All the comicroinjected cells expressed both the GFP-Eps8 (648–821) fragment and the Arf6T27N mutant, as determined in parallel experiments by directly staining the microinjected cells with anti-myc antibodies to detect Arf6T27N (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bars, 10 μm.
Mentions: The dependence on Rac activity of the effect of GFP-Eps8 (648–821) was further supported by experiments performed with a dominant negative Arf6 mutant. Arf6 is a protein belonging to the Arf subfamily of small GTPases (Donaldson and Radhakrishna, 2001). A dominant negative Arf6 blocks Rac-dependent actin reorganization, likely by preventing the translocation of GTP-bound Rac from an endosomal compartment to the plasma membrane (Radhakrishna et al., 1999; Zhang et al., 1999; Fig. 5 A). Microinjection, in mouse fibroblasts, of a dominant negative mutant Arf6 (Arf6T27N) capable of abrogating Rac-dependent PDGF-induced ruffles inhibited GFP-Eps8 (648–821)–dependent actin cytoskeleton rearrangement (Fig. 5 B), compatible with the requirement for proper localization of Rac.

Bottom Line: Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus.This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization.Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.

ABSTRACT
Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

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