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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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The effector region of Eps8 requires Rac for its actin remodelling activity. Nuclei of quiescent mouse embryo fibroblasts were comicroinjected with the expression vector for GFP-Eps8 (648–821) (all panels) together with one of the indicated (left) dominant negative mutants (ctr, comicroinjection with control vector). In the bottom panels (Wrt), microinjection was with GFP-Eps8 (648–821) alone followed by treatment with wortmannin for 60 min. After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green), whereas F-actin was detected by phalloidin staining (red). All the comicroinjected cells expressed the GFP-Eps8 (648–821) fragment and the individual dominant negative mutant, as determined in parallel experiments, by directly staining the microinjected cells with antibodies specific to the various dominant negative mutants (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bar, 10 μm.
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fig4: The effector region of Eps8 requires Rac for its actin remodelling activity. Nuclei of quiescent mouse embryo fibroblasts were comicroinjected with the expression vector for GFP-Eps8 (648–821) (all panels) together with one of the indicated (left) dominant negative mutants (ctr, comicroinjection with control vector). In the bottom panels (Wrt), microinjection was with GFP-Eps8 (648–821) alone followed by treatment with wortmannin for 60 min. After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green), whereas F-actin was detected by phalloidin staining (red). All the comicroinjected cells expressed the GFP-Eps8 (648–821) fragment and the individual dominant negative mutant, as determined in parallel experiments, by directly staining the microinjected cells with antibodies specific to the various dominant negative mutants (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bar, 10 μm.

Mentions: We then attempted to interfere with the ruffling activity of GFP-Eps8 (648–821) by comicroinjecting the dominant negative mutants or by treating cells microinjected with this latter construct with wortmannin. Ruffling activity was unaffected by coexpression of dominant negative Ras, Cdc42, Rho, or PI3-K, or by treatment with wortmannin (Fig. 4) . However, the expression of a dominant negative Rac abrogated the GFP-Eps8 (648–821)–induced ruffles (Fig. 4). Thus, the ruffling activity of the effector region of Eps8 is apparently exerted at a step upstream of Rac and does not require the functions of Ras and PI3-K.


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)

The effector region of Eps8 requires Rac for its actin remodelling activity. Nuclei of quiescent mouse embryo fibroblasts were comicroinjected with the expression vector for GFP-Eps8 (648–821) (all panels) together with one of the indicated (left) dominant negative mutants (ctr, comicroinjection with control vector). In the bottom panels (Wrt), microinjection was with GFP-Eps8 (648–821) alone followed by treatment with wortmannin for 60 min. After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green), whereas F-actin was detected by phalloidin staining (red). All the comicroinjected cells expressed the GFP-Eps8 (648–821) fragment and the individual dominant negative mutant, as determined in parallel experiments, by directly staining the microinjected cells with antibodies specific to the various dominant negative mutants (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196181&req=5

fig4: The effector region of Eps8 requires Rac for its actin remodelling activity. Nuclei of quiescent mouse embryo fibroblasts were comicroinjected with the expression vector for GFP-Eps8 (648–821) (all panels) together with one of the indicated (left) dominant negative mutants (ctr, comicroinjection with control vector). In the bottom panels (Wrt), microinjection was with GFP-Eps8 (648–821) alone followed by treatment with wortmannin for 60 min. After 3 h, cells were fixed and detection of GFP was performed by epifluorescence (green), whereas F-actin was detected by phalloidin staining (red). All the comicroinjected cells expressed the GFP-Eps8 (648–821) fragment and the individual dominant negative mutant, as determined in parallel experiments, by directly staining the microinjected cells with antibodies specific to the various dominant negative mutants (not shown). More than 100 injected cells were analyzed in each experiment. Similar results were obtained in transfection experiments. Arrows point to ruffles. Bar, 10 μm.
Mentions: We then attempted to interfere with the ruffling activity of GFP-Eps8 (648–821) by comicroinjecting the dominant negative mutants or by treating cells microinjected with this latter construct with wortmannin. Ruffling activity was unaffected by coexpression of dominant negative Ras, Cdc42, Rho, or PI3-K, or by treatment with wortmannin (Fig. 4) . However, the expression of a dominant negative Rac abrogated the GFP-Eps8 (648–821)–induced ruffles (Fig. 4). Thus, the ruffling activity of the effector region of Eps8 is apparently exerted at a step upstream of Rac and does not require the functions of Ras and PI3-K.

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.

Show MeSH