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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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Expression and biological activity of dominant negative mutants of Ras, Cdc42, Rho, Rac, PI-3K, and of the PI-3K inhibitor, wortmannin. (A) Mouse embryo fibroblasts were cotransfected with an expression vector encoding RasN17 (Ras N17) or an empty vector as a control (Ctr), together with pCDNA-HA-MAPK. Cells were serum starved for 24 h and treated with 100 ng/ml of EGF (+) or mock treated (−) for 10 min. MAPK kinase activity was determined in immunocomplex kinase assays using myelin basic protein (MBP) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-MAPK antibodies (MAPK). The doublet MAPK band, detected in the EGF-treated control sample, represents the active phosphorylated form. (B) Mouse embryo fibroblasts were cotransfected with expression vectors encoding a dominant negative HA-tagged Cdc42 (Cdc42N17) or an empty vector as a control (Ctr) together with pCDNA-HA-JNK, serum starved for 24 h, and treated with 100 ng/ml of EGF (+) or mock treated for 10 min (−). JNK kinase activity was determined in immunocomplex kinase assays, using the COOH-terminal region of c-JUN (Jun79) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-JNK antibodies (JNK). The expression of RasN17 in A and HA-Cdc42N17 in B was determined by immunoblot analysis with anti-Ras and anti-HA antibodies, respectively (not shown). (C–E). Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors encoding RhoN19 (C), RacN17 (D), or p85ΔiSH2 (E). After 3 h, cells were stimulated with either 10% serum for 60 min (C) or PDGF (10 ng/ml) for 10 min (D and E) and fixed and stained with rhodamine-conjugated phalloidin (red) to detect F-actin, anti-Rho (C, green), anti-Rac (D, green) or anti-p85 (E, green) antibodies. (F) Quiescent mouse embryo fibroblasts were treated with wortmannin (100 nM) (+Wrt) or vehicle as a control (−Wrt) for 1 h before adding PDGF for 10 min. Cells were then fixed and stained with phalloidin (red). Arrows point to ruffles. Bar, 10 μm.
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fig3: Expression and biological activity of dominant negative mutants of Ras, Cdc42, Rho, Rac, PI-3K, and of the PI-3K inhibitor, wortmannin. (A) Mouse embryo fibroblasts were cotransfected with an expression vector encoding RasN17 (Ras N17) or an empty vector as a control (Ctr), together with pCDNA-HA-MAPK. Cells were serum starved for 24 h and treated with 100 ng/ml of EGF (+) or mock treated (−) for 10 min. MAPK kinase activity was determined in immunocomplex kinase assays using myelin basic protein (MBP) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-MAPK antibodies (MAPK). The doublet MAPK band, detected in the EGF-treated control sample, represents the active phosphorylated form. (B) Mouse embryo fibroblasts were cotransfected with expression vectors encoding a dominant negative HA-tagged Cdc42 (Cdc42N17) or an empty vector as a control (Ctr) together with pCDNA-HA-JNK, serum starved for 24 h, and treated with 100 ng/ml of EGF (+) or mock treated for 10 min (−). JNK kinase activity was determined in immunocomplex kinase assays, using the COOH-terminal region of c-JUN (Jun79) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-JNK antibodies (JNK). The expression of RasN17 in A and HA-Cdc42N17 in B was determined by immunoblot analysis with anti-Ras and anti-HA antibodies, respectively (not shown). (C–E). Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors encoding RhoN19 (C), RacN17 (D), or p85ΔiSH2 (E). After 3 h, cells were stimulated with either 10% serum for 60 min (C) or PDGF (10 ng/ml) for 10 min (D and E) and fixed and stained with rhodamine-conjugated phalloidin (red) to detect F-actin, anti-Rho (C, green), anti-Rac (D, green) or anti-p85 (E, green) antibodies. (F) Quiescent mouse embryo fibroblasts were treated with wortmannin (100 nM) (+Wrt) or vehicle as a control (−Wrt) for 1 h before adding PDGF for 10 min. Cells were then fixed and stained with phalloidin (red). Arrows point to ruffles. Bar, 10 μm.

Mentions: Upon microinjection in mouse fibroblasts, all the dominant negative mutants were readily expressed (Fig. 3) and biologically active, as shown by the ability of: (a) RasN17 to inhibit EGF-induced mitogen-activated protein kinase (MAPK) activation (Fig. 3 A); (b) Cdc42N17 to reduce growth factor–induced JNK activity (Fig. 3 B); (c) RhoN19 to inhibit the formation of serum-induced stress fibers (Fig. 3 C); and (d) RacN17 (Fig. 3 D) and p85ΔiSH2 (Fig. 3 E) to inhibit PDGF-mediated actin cytoskeleton reorganization. Wortmannin also abrogated completely PDGF-induced actin remodeling (Fig. 3 F).


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)

Expression and biological activity of dominant negative mutants of Ras, Cdc42, Rho, Rac, PI-3K, and of the PI-3K inhibitor, wortmannin. (A) Mouse embryo fibroblasts were cotransfected with an expression vector encoding RasN17 (Ras N17) or an empty vector as a control (Ctr), together with pCDNA-HA-MAPK. Cells were serum starved for 24 h and treated with 100 ng/ml of EGF (+) or mock treated (−) for 10 min. MAPK kinase activity was determined in immunocomplex kinase assays using myelin basic protein (MBP) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-MAPK antibodies (MAPK). The doublet MAPK band, detected in the EGF-treated control sample, represents the active phosphorylated form. (B) Mouse embryo fibroblasts were cotransfected with expression vectors encoding a dominant negative HA-tagged Cdc42 (Cdc42N17) or an empty vector as a control (Ctr) together with pCDNA-HA-JNK, serum starved for 24 h, and treated with 100 ng/ml of EGF (+) or mock treated for 10 min (−). JNK kinase activity was determined in immunocomplex kinase assays, using the COOH-terminal region of c-JUN (Jun79) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-JNK antibodies (JNK). The expression of RasN17 in A and HA-Cdc42N17 in B was determined by immunoblot analysis with anti-Ras and anti-HA antibodies, respectively (not shown). (C–E). Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors encoding RhoN19 (C), RacN17 (D), or p85ΔiSH2 (E). After 3 h, cells were stimulated with either 10% serum for 60 min (C) or PDGF (10 ng/ml) for 10 min (D and E) and fixed and stained with rhodamine-conjugated phalloidin (red) to detect F-actin, anti-Rho (C, green), anti-Rac (D, green) or anti-p85 (E, green) antibodies. (F) Quiescent mouse embryo fibroblasts were treated with wortmannin (100 nM) (+Wrt) or vehicle as a control (−Wrt) for 1 h before adding PDGF for 10 min. Cells were then fixed and stained with phalloidin (red). Arrows point to ruffles. Bar, 10 μm.
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fig3: Expression and biological activity of dominant negative mutants of Ras, Cdc42, Rho, Rac, PI-3K, and of the PI-3K inhibitor, wortmannin. (A) Mouse embryo fibroblasts were cotransfected with an expression vector encoding RasN17 (Ras N17) or an empty vector as a control (Ctr), together with pCDNA-HA-MAPK. Cells were serum starved for 24 h and treated with 100 ng/ml of EGF (+) or mock treated (−) for 10 min. MAPK kinase activity was determined in immunocomplex kinase assays using myelin basic protein (MBP) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-MAPK antibodies (MAPK). The doublet MAPK band, detected in the EGF-treated control sample, represents the active phosphorylated form. (B) Mouse embryo fibroblasts were cotransfected with expression vectors encoding a dominant negative HA-tagged Cdc42 (Cdc42N17) or an empty vector as a control (Ctr) together with pCDNA-HA-JNK, serum starved for 24 h, and treated with 100 ng/ml of EGF (+) or mock treated for 10 min (−). JNK kinase activity was determined in immunocomplex kinase assays, using the COOH-terminal region of c-JUN (Jun79) as a substrate. An aliquot of the immunoprecipitates was also immunoblotted with anti-JNK antibodies (JNK). The expression of RasN17 in A and HA-Cdc42N17 in B was determined by immunoblot analysis with anti-Ras and anti-HA antibodies, respectively (not shown). (C–E). Nuclei of quiescent mouse embryo fibroblasts were microinjected with expression vectors encoding RhoN19 (C), RacN17 (D), or p85ΔiSH2 (E). After 3 h, cells were stimulated with either 10% serum for 60 min (C) or PDGF (10 ng/ml) for 10 min (D and E) and fixed and stained with rhodamine-conjugated phalloidin (red) to detect F-actin, anti-Rho (C, green), anti-Rac (D, green) or anti-p85 (E, green) antibodies. (F) Quiescent mouse embryo fibroblasts were treated with wortmannin (100 nM) (+Wrt) or vehicle as a control (−Wrt) for 1 h before adding PDGF for 10 min. Cells were then fixed and stained with phalloidin (red). Arrows point to ruffles. Bar, 10 μm.
Mentions: Upon microinjection in mouse fibroblasts, all the dominant negative mutants were readily expressed (Fig. 3) and biologically active, as shown by the ability of: (a) RasN17 to inhibit EGF-induced mitogen-activated protein kinase (MAPK) activation (Fig. 3 A); (b) Cdc42N17 to reduce growth factor–induced JNK activity (Fig. 3 B); (c) RhoN19 to inhibit the formation of serum-induced stress fibers (Fig. 3 C); and (d) RacN17 (Fig. 3 D) and p85ΔiSH2 (Fig. 3 E) to inhibit PDGF-mediated actin cytoskeleton reorganization. Wortmannin also abrogated completely PDGF-induced actin remodeling (Fig. 3 F).

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