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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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Identification of the minimal effector region of Eps8 capable of inducing actin polymerization into ruffles. (A) Schematic of the various Eps8 fragments, all engineered as GFP fusion proteins (amino acid boundaries are given on the left of the constructs. The SH3 domain is indicated by a solid box), and their biological characterization. Mouse embryo fibroblasts, transfected with the indicated GFP-tagged expression vectors, were serum starved and treated with 10 ng/ml of PDGF for 10 min (+PDGF) or mock treated (−PDGF). Cells were then fixed for subsequent detection of GFP by epifluorescence and F-actin by rhodamine-conjugated phalloidin. (B) Mouse embryo fibroblasts, transfected with the indicated expression vectors, were serum-starved and fixed for subsequent detection of GFP by epifluorescence (green), and F-actin by rhodamine-conjugated phalloidin (red). Actin polymerization into ruffles induced by the biologically active Eps8 fragments was detected in >80% of the transfected cells. The effect of selected constructs is shown. Arrows point to ruffles. Bar, 10 μm.
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fig2: Identification of the minimal effector region of Eps8 capable of inducing actin polymerization into ruffles. (A) Schematic of the various Eps8 fragments, all engineered as GFP fusion proteins (amino acid boundaries are given on the left of the constructs. The SH3 domain is indicated by a solid box), and their biological characterization. Mouse embryo fibroblasts, transfected with the indicated GFP-tagged expression vectors, were serum starved and treated with 10 ng/ml of PDGF for 10 min (+PDGF) or mock treated (−PDGF). Cells were then fixed for subsequent detection of GFP by epifluorescence and F-actin by rhodamine-conjugated phalloidin. (B) Mouse embryo fibroblasts, transfected with the indicated expression vectors, were serum-starved and fixed for subsequent detection of GFP by epifluorescence (green), and F-actin by rhodamine-conjugated phalloidin (red). Actin polymerization into ruffles induced by the biologically active Eps8 fragments was detected in >80% of the transfected cells. The effect of selected constructs is shown. Arrows point to ruffles. Bar, 10 μm.

Mentions: To identify the region of Eps8 that mediates its localization, we used deletion mutants of Eps8 fused to the green fluorescence protein (GFP). A GFP full length Eps8 (amino acids 1–821) displayed a sub-cellular localization indistinguishable from that of the endogenous protein (Fig. 1 B). In contrast, a NH2-terminal fragment (amino acids 1–535) showed a diffuse cytoplasmic and nuclear staining unaltered by treatment with growth factors, suggesting that the COOH terminus contains residues important for Eps8 localization (Fig. 1 B). Accordingly, a COOH-terminal fragment (amino acids 535–821) displayed intense localization along the cell cortex within structures reminiscent of ruffles. These structures corresponded to sites of actin polymerization (not shown; see also Fig. 2) . The differential cellular localization of the Eps8 mutants was not due to the GFP moiety, since GFP alone was diffusely distributed in the nucleus and the cytoplasm (not shown). Furthermore, similar levels of expression of the various Eps8 constructs were obtained, as determined by immunoblotting (not shown) and immunofluorescence analysis (Fig. 1 B). Thus, our data indicate that determinants in the COOH terminus are responsible for the subcellular localization of Eps8.


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)

Identification of the minimal effector region of Eps8 capable of inducing actin polymerization into ruffles. (A) Schematic of the various Eps8 fragments, all engineered as GFP fusion proteins (amino acid boundaries are given on the left of the constructs. The SH3 domain is indicated by a solid box), and their biological characterization. Mouse embryo fibroblasts, transfected with the indicated GFP-tagged expression vectors, were serum starved and treated with 10 ng/ml of PDGF for 10 min (+PDGF) or mock treated (−PDGF). Cells were then fixed for subsequent detection of GFP by epifluorescence and F-actin by rhodamine-conjugated phalloidin. (B) Mouse embryo fibroblasts, transfected with the indicated expression vectors, were serum-starved and fixed for subsequent detection of GFP by epifluorescence (green), and F-actin by rhodamine-conjugated phalloidin (red). Actin polymerization into ruffles induced by the biologically active Eps8 fragments was detected in >80% of the transfected cells. The effect of selected constructs is shown. Arrows point to ruffles. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196181&req=5

fig2: Identification of the minimal effector region of Eps8 capable of inducing actin polymerization into ruffles. (A) Schematic of the various Eps8 fragments, all engineered as GFP fusion proteins (amino acid boundaries are given on the left of the constructs. The SH3 domain is indicated by a solid box), and their biological characterization. Mouse embryo fibroblasts, transfected with the indicated GFP-tagged expression vectors, were serum starved and treated with 10 ng/ml of PDGF for 10 min (+PDGF) or mock treated (−PDGF). Cells were then fixed for subsequent detection of GFP by epifluorescence and F-actin by rhodamine-conjugated phalloidin. (B) Mouse embryo fibroblasts, transfected with the indicated expression vectors, were serum-starved and fixed for subsequent detection of GFP by epifluorescence (green), and F-actin by rhodamine-conjugated phalloidin (red). Actin polymerization into ruffles induced by the biologically active Eps8 fragments was detected in >80% of the transfected cells. The effect of selected constructs is shown. Arrows point to ruffles. Bar, 10 μm.
Mentions: To identify the region of Eps8 that mediates its localization, we used deletion mutants of Eps8 fused to the green fluorescence protein (GFP). A GFP full length Eps8 (amino acids 1–821) displayed a sub-cellular localization indistinguishable from that of the endogenous protein (Fig. 1 B). In contrast, a NH2-terminal fragment (amino acids 1–535) showed a diffuse cytoplasmic and nuclear staining unaltered by treatment with growth factors, suggesting that the COOH terminus contains residues important for Eps8 localization (Fig. 1 B). Accordingly, a COOH-terminal fragment (amino acids 535–821) displayed intense localization along the cell cortex within structures reminiscent of ruffles. These structures corresponded to sites of actin polymerization (not shown; see also Fig. 2) . The differential cellular localization of the Eps8 mutants was not due to the GFP moiety, since GFP alone was diffusely distributed in the nucleus and the cytoplasm (not shown). Furthermore, similar levels of expression of the various Eps8 constructs were obtained, as determined by immunoblotting (not shown) and immunofluorescence analysis (Fig. 1 B). Thus, our data indicate that determinants in the COOH terminus are responsible for the subcellular localization of Eps8.

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