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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 by MALDI-MS of novel interactors of the effector domain of Eps8. (A) Various immobilized GST fusion fragments of the COOH-terminal region of Eps8 (amino acid boundaries are indicated on the top), GST-SH3 domain of Eps8 (SH3), or GST alone were incubated in the presence of lysis buffer as a control or with total cellular lysates (+lysates lanes) from mouse embryo fibroblasts. Specifically bound proteins were eluted in sample buffer and resolved by SDS-PAGE, followed by detection by silver staining. The marked (asterisk and arrowhead) bands were unequivocally identified by MALDI-MS, as described in Materials and methods, as the myosin II heavy chain and actin, respectively. Molecular weight markers are indicated in kD. (B) Total cellular lysates (1 mg) from mouse embryo fibroblasts were incubated with 5 μg of GST or GST-Eps8 (586–821). Detection was with antimyosin II heavy chain and antiactin antibodies as shown. The lane “lysate” was loaded with 100 μg of total cellular lysate.
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fig8: Identification by MALDI-MS of novel interactors of the effector domain of Eps8. (A) Various immobilized GST fusion fragments of the COOH-terminal region of Eps8 (amino acid boundaries are indicated on the top), GST-SH3 domain of Eps8 (SH3), or GST alone were incubated in the presence of lysis buffer as a control or with total cellular lysates (+lysates lanes) from mouse embryo fibroblasts. Specifically bound proteins were eluted in sample buffer and resolved by SDS-PAGE, followed by detection by silver staining. The marked (asterisk and arrowhead) bands were unequivocally identified by MALDI-MS, as described in Materials and methods, as the myosin II heavy chain and actin, respectively. Molecular weight markers are indicated in kD. (B) Total cellular lysates (1 mg) from mouse embryo fibroblasts were incubated with 5 μg of GST or GST-Eps8 (586–821). Detection was with antimyosin II heavy chain and antiactin antibodies as shown. The lane “lysate” was loaded with 100 μg of total cellular lysate.

Mentions: The effector domain of Eps8 was also responsible for its localization within F-actin–containing structures (Fig. 2 A). Thus, we looked for interactors with the Eps8 effector region. We used various GST-fused fragments of Eps8 in order to specifically recover proteins from cell lysates. As shown in Fig. 8 A, several cellular proteins were detected by silver staining. We concentrated our attention on those bands that were recovered by the 586–821 fragment (which is biologically active and properly localized), but not by the 733–821 or by the SH3 fragments (which are biologically inactive and delocalized), and subjected them to MALDI mass spectrometry and NanoElectrospray. Two proteins were unequivocally identified: actin and the myosin II heavy chain (Fig. 8 A). Both associations were confirmed by in vitro binding experiments (Fig. 8 B). Of note, the low stoichiometry of interaction between Eps8 and myosin II heavy chain suggested an indirect interaction, possibly mediated by actin. Therefore, we characterized the Eps8/actin association.


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 by MALDI-MS of novel interactors of the effector domain of Eps8. (A) Various immobilized GST fusion fragments of the COOH-terminal region of Eps8 (amino acid boundaries are indicated on the top), GST-SH3 domain of Eps8 (SH3), or GST alone were incubated in the presence of lysis buffer as a control or with total cellular lysates (+lysates lanes) from mouse embryo fibroblasts. Specifically bound proteins were eluted in sample buffer and resolved by SDS-PAGE, followed by detection by silver staining. The marked (asterisk and arrowhead) bands were unequivocally identified by MALDI-MS, as described in Materials and methods, as the myosin II heavy chain and actin, respectively. Molecular weight markers are indicated in kD. (B) Total cellular lysates (1 mg) from mouse embryo fibroblasts were incubated with 5 μg of GST or GST-Eps8 (586–821). Detection was with antimyosin II heavy chain and antiactin antibodies as shown. The lane “lysate” was loaded with 100 μg of total cellular lysate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196181&req=5

fig8: Identification by MALDI-MS of novel interactors of the effector domain of Eps8. (A) Various immobilized GST fusion fragments of the COOH-terminal region of Eps8 (amino acid boundaries are indicated on the top), GST-SH3 domain of Eps8 (SH3), or GST alone were incubated in the presence of lysis buffer as a control or with total cellular lysates (+lysates lanes) from mouse embryo fibroblasts. Specifically bound proteins were eluted in sample buffer and resolved by SDS-PAGE, followed by detection by silver staining. The marked (asterisk and arrowhead) bands were unequivocally identified by MALDI-MS, as described in Materials and methods, as the myosin II heavy chain and actin, respectively. Molecular weight markers are indicated in kD. (B) Total cellular lysates (1 mg) from mouse embryo fibroblasts were incubated with 5 μg of GST or GST-Eps8 (586–821). Detection was with antimyosin II heavy chain and antiactin antibodies as shown. The lane “lysate” was loaded with 100 μg of total cellular lysate.
Mentions: The effector domain of Eps8 was also responsible for its localization within F-actin–containing structures (Fig. 2 A). Thus, we looked for interactors with the Eps8 effector region. We used various GST-fused fragments of Eps8 in order to specifically recover proteins from cell lysates. As shown in Fig. 8 A, several cellular proteins were detected by silver staining. We concentrated our attention on those bands that were recovered by the 586–821 fragment (which is biologically active and properly localized), but not by the 733–821 or by the SH3 fragments (which are biologically inactive and delocalized), and subjected them to MALDI mass spectrometry and NanoElectrospray. Two proteins were unequivocally identified: actin and the myosin II heavy chain (Fig. 8 A). Both associations were confirmed by in vitro binding experiments (Fig. 8 B). Of note, the low stoichiometry of interaction between Eps8 and myosin II heavy chain suggested an indirect interaction, possibly mediated by actin. Therefore, we characterized the Eps8/actin association.

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