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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 activates Rac and Rac- dependent biochemical pathways. (A) The expression vectors indicated on the top, and encoding GFP-Eps8 (648–821), GFP-Eps8 (586–701), GFP, and HA-Rac were transfected, alone or in combination, into mouse embryo fibroblasts. After 24 h of serum deprivation, Rac-GTP levels were determined by an in vitro binding assay using the CRIB domain of PAK65 fused to GST (top). Aliquots of the lysates were also analyzed for the expression of the GFP or the GFP-tagged Eps8 fragments and of HA-Rac by direct immunoblot (bottom). (B) Mouse embryo fibroblasts were cotransfected with expression vectors for HA-PAK65 together with the indicated (top) GFP-tagged Eps8 fragments (1–535 or 648–821), an activated Rac mutant (RacQL), or an empty vector as a control (ctr). After 24 h of serum deprivation, PAK65 kinase activity was determined by immunocomplex kinase assays using MBP as a substrate (MBP). An aliquot of the immunoprecipitates used for each kinase assay was immunoblotted with anti-HA to detect PAK65 (PAK65). Aliquots of the lysates were also analyzed for the expression of the GFP-tagged Eps8 fragments and of RacQL by direct immunoblot (GFP and RacQL).
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fig6: The effector region of Eps8 activates Rac and Rac- dependent biochemical pathways. (A) The expression vectors indicated on the top, and encoding GFP-Eps8 (648–821), GFP-Eps8 (586–701), GFP, and HA-Rac were transfected, alone or in combination, into mouse embryo fibroblasts. After 24 h of serum deprivation, Rac-GTP levels were determined by an in vitro binding assay using the CRIB domain of PAK65 fused to GST (top). Aliquots of the lysates were also analyzed for the expression of the GFP or the GFP-tagged Eps8 fragments and of HA-Rac by direct immunoblot (bottom). (B) Mouse embryo fibroblasts were cotransfected with expression vectors for HA-PAK65 together with the indicated (top) GFP-tagged Eps8 fragments (1–535 or 648–821), an activated Rac mutant (RacQL), or an empty vector as a control (ctr). After 24 h of serum deprivation, PAK65 kinase activity was determined by immunocomplex kinase assays using MBP as a substrate (MBP). An aliquot of the immunoprecipitates used for each kinase assay was immunoblotted with anti-HA to detect PAK65 (PAK65). Aliquots of the lysates were also analyzed for the expression of the GFP-tagged Eps8 fragments and of RacQL by direct immunoblot (GFP and RacQL).

Mentions: The Rac-dependent ruffling activity of the effector region of Eps8 suggests that it may act by activating Rac. Thus, we tested the ability of the 648–821 region of Eps8 to induce the activation of Rac and of PAK65, a direct downstream effector of Rac (for review see Lim et al., 1996). As shown in Fig. 6 A, the expression in quiescent fibroblasts of GFP-Eps8 (648–821), but not of GFP or GFP-Eps8 (586–701), increased the levels of activated GTP-bound Rac. This was mirrored by the activation of PAK65 to levels similar to those induced by a constitutively active Rac mutant. These results indicate that the effector region of Eps8 activates Rac in vivo.


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 activates Rac and Rac- dependent biochemical pathways. (A) The expression vectors indicated on the top, and encoding GFP-Eps8 (648–821), GFP-Eps8 (586–701), GFP, and HA-Rac were transfected, alone or in combination, into mouse embryo fibroblasts. After 24 h of serum deprivation, Rac-GTP levels were determined by an in vitro binding assay using the CRIB domain of PAK65 fused to GST (top). Aliquots of the lysates were also analyzed for the expression of the GFP or the GFP-tagged Eps8 fragments and of HA-Rac by direct immunoblot (bottom). (B) Mouse embryo fibroblasts were cotransfected with expression vectors for HA-PAK65 together with the indicated (top) GFP-tagged Eps8 fragments (1–535 or 648–821), an activated Rac mutant (RacQL), or an empty vector as a control (ctr). After 24 h of serum deprivation, PAK65 kinase activity was determined by immunocomplex kinase assays using MBP as a substrate (MBP). An aliquot of the immunoprecipitates used for each kinase assay was immunoblotted with anti-HA to detect PAK65 (PAK65). Aliquots of the lysates were also analyzed for the expression of the GFP-tagged Eps8 fragments and of RacQL by direct immunoblot (GFP and RacQL).
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Related In: Results  -  Collection

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fig6: The effector region of Eps8 activates Rac and Rac- dependent biochemical pathways. (A) The expression vectors indicated on the top, and encoding GFP-Eps8 (648–821), GFP-Eps8 (586–701), GFP, and HA-Rac were transfected, alone or in combination, into mouse embryo fibroblasts. After 24 h of serum deprivation, Rac-GTP levels were determined by an in vitro binding assay using the CRIB domain of PAK65 fused to GST (top). Aliquots of the lysates were also analyzed for the expression of the GFP or the GFP-tagged Eps8 fragments and of HA-Rac by direct immunoblot (bottom). (B) Mouse embryo fibroblasts were cotransfected with expression vectors for HA-PAK65 together with the indicated (top) GFP-tagged Eps8 fragments (1–535 or 648–821), an activated Rac mutant (RacQL), or an empty vector as a control (ctr). After 24 h of serum deprivation, PAK65 kinase activity was determined by immunocomplex kinase assays using MBP as a substrate (MBP). An aliquot of the immunoprecipitates used for each kinase assay was immunoblotted with anti-HA to detect PAK65 (PAK65). Aliquots of the lysates were also analyzed for the expression of the GFP-tagged Eps8 fragments and of RacQL by direct immunoblot (GFP and RacQL).
Mentions: The Rac-dependent ruffling activity of the effector region of Eps8 suggests that it may act by activating Rac. Thus, we tested the ability of the 648–821 region of Eps8 to induce the activation of Rac and of PAK65, a direct downstream effector of Rac (for review see Lim et al., 1996). As shown in Fig. 6 A, the expression in quiescent fibroblasts of GFP-Eps8 (648–821), but not of GFP or GFP-Eps8 (586–701), increased the levels of activated GTP-bound Rac. This was mirrored by the activation of PAK65 to levels similar to those induced by a constitutively active Rac mutant. These results indicate that the effector region of Eps8 activates Rac in vivo.

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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