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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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Biochemical characterization of the interaction between the Eps8 and actin. (A) Total cellular lysates from mouse embryo fibroblasts were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated on the top) or GST or GST-E3b1, used as negative controls. Detection was with antiactin antibodies. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Actin cosedimentation assay. The indicated fragments of Eps8 (left, Eps8 [733–821]; right, Eps8 [586–821]) fused to GST (2 μg) were mixed with increasing amounts of F-actin (0.5, 1.0, 2.0, and 10 μg, respectively), as described in Materials and methods, and the samples were ultracentrifuged. The input lanes (inp) show the supernatant (S) and the pellet (P) of a control tube, in which the GST fusion proteins were subjected to ultracentrifugation in the absence of F-actin. Detection was with anti-GST (top) or antiactin (bottom). (C) Purified, globular, nonmuscle actin (2 μg) was incubated with 5 μg of Sepharose-conjugated GST as a negative control, or GST-Scar-2-WA-domain (Scar2-WA) as a positive control (left; Miki et al., 1998), or GST-Eps8 fragments (amino acid boundaries are indicated in the top right panels). Bound proteins were recovered by low speed centrifugation and analyzed by immunoblotting (WB) with antiactin (Actin) and anti-GST (GST) antibodies. The lane “input” was loaded with 100 ng of G-actin. Molecular weight markers are indicated in kD. (D) Left, total cellular lysates from mouse embryo fibroblasts were immunoprecipitated with antiactin (Actin) or an irrelevant (ctr) antibody, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies. Right, total cellular lysates from Eps8  mouse embryo fibroblasts (−/−), or from the same cells in which Eps8 was stably reintroduced (−/− Eps8), were immunoprecipitated with anti-Eps8 (Eps8) or the preimmune (ctr) sera, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies.
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fig9: Biochemical characterization of the interaction between the Eps8 and actin. (A) Total cellular lysates from mouse embryo fibroblasts were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated on the top) or GST or GST-E3b1, used as negative controls. Detection was with antiactin antibodies. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Actin cosedimentation assay. The indicated fragments of Eps8 (left, Eps8 [733–821]; right, Eps8 [586–821]) fused to GST (2 μg) were mixed with increasing amounts of F-actin (0.5, 1.0, 2.0, and 10 μg, respectively), as described in Materials and methods, and the samples were ultracentrifuged. The input lanes (inp) show the supernatant (S) and the pellet (P) of a control tube, in which the GST fusion proteins were subjected to ultracentrifugation in the absence of F-actin. Detection was with anti-GST (top) or antiactin (bottom). (C) Purified, globular, nonmuscle actin (2 μg) was incubated with 5 μg of Sepharose-conjugated GST as a negative control, or GST-Scar-2-WA-domain (Scar2-WA) as a positive control (left; Miki et al., 1998), or GST-Eps8 fragments (amino acid boundaries are indicated in the top right panels). Bound proteins were recovered by low speed centrifugation and analyzed by immunoblotting (WB) with antiactin (Actin) and anti-GST (GST) antibodies. The lane “input” was loaded with 100 ng of G-actin. Molecular weight markers are indicated in kD. (D) Left, total cellular lysates from mouse embryo fibroblasts were immunoprecipitated with antiactin (Actin) or an irrelevant (ctr) antibody, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies. Right, total cellular lysates from Eps8 mouse embryo fibroblasts (−/−), or from the same cells in which Eps8 was stably reintroduced (−/− Eps8), were immunoprecipitated with anti-Eps8 (Eps8) or the preimmune (ctr) sera, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies.

Mentions: We mapped the minimal interaction surface of Eps8 with actin to a stretch of amino acids extending from position 648 to 821 (Fig. 9 A). Notably, this actin binding domain coincided with the minimal region required to elicit ruffling, bind to Sos-1, and colocalize with F-actin. Attempts to define determinants within this region individually responsible for binding to actin or to Sos-1 failed, suggesting that the integrity of the whole domain is required for both functions.


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)

Biochemical characterization of the interaction between the Eps8 and actin. (A) Total cellular lysates from mouse embryo fibroblasts were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated on the top) or GST or GST-E3b1, used as negative controls. Detection was with antiactin antibodies. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Actin cosedimentation assay. The indicated fragments of Eps8 (left, Eps8 [733–821]; right, Eps8 [586–821]) fused to GST (2 μg) were mixed with increasing amounts of F-actin (0.5, 1.0, 2.0, and 10 μg, respectively), as described in Materials and methods, and the samples were ultracentrifuged. The input lanes (inp) show the supernatant (S) and the pellet (P) of a control tube, in which the GST fusion proteins were subjected to ultracentrifugation in the absence of F-actin. Detection was with anti-GST (top) or antiactin (bottom). (C) Purified, globular, nonmuscle actin (2 μg) was incubated with 5 μg of Sepharose-conjugated GST as a negative control, or GST-Scar-2-WA-domain (Scar2-WA) as a positive control (left; Miki et al., 1998), or GST-Eps8 fragments (amino acid boundaries are indicated in the top right panels). Bound proteins were recovered by low speed centrifugation and analyzed by immunoblotting (WB) with antiactin (Actin) and anti-GST (GST) antibodies. The lane “input” was loaded with 100 ng of G-actin. Molecular weight markers are indicated in kD. (D) Left, total cellular lysates from mouse embryo fibroblasts were immunoprecipitated with antiactin (Actin) or an irrelevant (ctr) antibody, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies. Right, total cellular lysates from Eps8  mouse embryo fibroblasts (−/−), or from the same cells in which Eps8 was stably reintroduced (−/− Eps8), were immunoprecipitated with anti-Eps8 (Eps8) or the preimmune (ctr) sera, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196181&req=5

fig9: Biochemical characterization of the interaction between the Eps8 and actin. (A) Total cellular lysates from mouse embryo fibroblasts were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated on the top) or GST or GST-E3b1, used as negative controls. Detection was with antiactin antibodies. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Actin cosedimentation assay. The indicated fragments of Eps8 (left, Eps8 [733–821]; right, Eps8 [586–821]) fused to GST (2 μg) were mixed with increasing amounts of F-actin (0.5, 1.0, 2.0, and 10 μg, respectively), as described in Materials and methods, and the samples were ultracentrifuged. The input lanes (inp) show the supernatant (S) and the pellet (P) of a control tube, in which the GST fusion proteins were subjected to ultracentrifugation in the absence of F-actin. Detection was with anti-GST (top) or antiactin (bottom). (C) Purified, globular, nonmuscle actin (2 μg) was incubated with 5 μg of Sepharose-conjugated GST as a negative control, or GST-Scar-2-WA-domain (Scar2-WA) as a positive control (left; Miki et al., 1998), or GST-Eps8 fragments (amino acid boundaries are indicated in the top right panels). Bound proteins were recovered by low speed centrifugation and analyzed by immunoblotting (WB) with antiactin (Actin) and anti-GST (GST) antibodies. The lane “input” was loaded with 100 ng of G-actin. Molecular weight markers are indicated in kD. (D) Left, total cellular lysates from mouse embryo fibroblasts were immunoprecipitated with antiactin (Actin) or an irrelevant (ctr) antibody, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies. Right, total cellular lysates from Eps8 mouse embryo fibroblasts (−/−), or from the same cells in which Eps8 was stably reintroduced (−/− Eps8), were immunoprecipitated with anti-Eps8 (Eps8) or the preimmune (ctr) sera, followed by detection with the anti-Eps8 (top) or antiactin (bottom) antibodies.
Mentions: We mapped the minimal interaction surface of Eps8 with actin to a stretch of amino acids extending from position 648 to 821 (Fig. 9 A). Notably, this actin binding domain coincided with the minimal region required to elicit ruffling, bind to Sos-1, and colocalize with F-actin. Attempts to define determinants within this region individually responsible for binding to actin or to Sos-1 failed, suggesting that the integrity of the whole domain is required for both functions.

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
Related in: MedlinePlus