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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 Eps8 effector region binds to Sos-1 and stimulates Rac-specific, Sos-1–dependent GEF activity. (A) Total cellular lysates from Cos-7 cells were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated at the top) or GST as a negative control, or GST-E3b1 as a positive control (Scita et al., 1999). Detection was with anti–Sos-1 Ab. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Left, eukaryotically produced, affinity-purified Sos-1 was resolved by SDS-PAGE and stained with Coomassie brilliant blue. Sos-1 was estimated to be >90% pure (see Materials and methods). Right, purified Sos-1 (1 μg) was incubated with 5 μg of the indicated GST fusion proteins (all Eps8 fragments; amino acids boundaries are indicated on the top). Detection was with anti–Sos-1 Ab. The binding of Sos-1 to full length Eps8 (1–821) was estimated to be ∼10 less than the binding to GST-Eps8 (586–821) or GST-Eps8 (648–821) by densitometric analysis. (C) Lysates of 293T cells transfected with the indicated cDNAs (tfx) were immunoprecipitated (IP) with anti-Eps8 (Eps8) or anti-HA (HA), or the corresponding preimmune sera (ctr) followed by detection (Western blot, WB) with anti–Sos-1, anti-Eps8, anti-E3b1, or anti-HA antibodies. Native Sos-1 could also be recovered in immunoprecipitates of the Eps8 fragments 648–821 (not shown). The lanes “lysate” were loaded with 50 μg of total cellular lysates. (D) Top, Rac-specific GEF activity was measured in lysates of cells expressing the indicated Eps8 fragments (indicated underneath the bar graph. Ctr, control vector). Lysates were immunodepleted with an irrelevant (mock ID, solid bars) or by an anti–Sos-1 (Sos-1 ID, empty bars) antibody. The same immunodepleted lysates used for the GEF assays were analyzed by immunoblotting (WB, bottom) with the indicated antibodies. No Cdc42-specific GEF activity could be detected in the same lysates (not shown). (E) Top, Rac-specific GEF activity was measured in immunoprecipitates performed with anti-HA (IP HA) or an irrelevant antibody (IP ctr) on lysates of 293T cells transfected with the indicated expression vectors (tfx; the Eps8-based constructs were HA tagged). Bottom, aliquots of the immunoprecipitates (IP) used in the GEF assay, or total cellular lysates (50 μg) transfected with the expression vectors indicated at the bottom (tfx), were immunoblotted (WB) with the indicated antibodies.
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fig7: The Eps8 effector region binds to Sos-1 and stimulates Rac-specific, Sos-1–dependent GEF activity. (A) Total cellular lysates from Cos-7 cells were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated at the top) or GST as a negative control, or GST-E3b1 as a positive control (Scita et al., 1999). Detection was with anti–Sos-1 Ab. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Left, eukaryotically produced, affinity-purified Sos-1 was resolved by SDS-PAGE and stained with Coomassie brilliant blue. Sos-1 was estimated to be >90% pure (see Materials and methods). Right, purified Sos-1 (1 μg) was incubated with 5 μg of the indicated GST fusion proteins (all Eps8 fragments; amino acids boundaries are indicated on the top). Detection was with anti–Sos-1 Ab. The binding of Sos-1 to full length Eps8 (1–821) was estimated to be ∼10 less than the binding to GST-Eps8 (586–821) or GST-Eps8 (648–821) by densitometric analysis. (C) Lysates of 293T cells transfected with the indicated cDNAs (tfx) were immunoprecipitated (IP) with anti-Eps8 (Eps8) or anti-HA (HA), or the corresponding preimmune sera (ctr) followed by detection (Western blot, WB) with anti–Sos-1, anti-Eps8, anti-E3b1, or anti-HA antibodies. Native Sos-1 could also be recovered in immunoprecipitates of the Eps8 fragments 648–821 (not shown). The lanes “lysate” were loaded with 50 μg of total cellular lysates. (D) Top, Rac-specific GEF activity was measured in lysates of cells expressing the indicated Eps8 fragments (indicated underneath the bar graph. Ctr, control vector). Lysates were immunodepleted with an irrelevant (mock ID, solid bars) or by an anti–Sos-1 (Sos-1 ID, empty bars) antibody. The same immunodepleted lysates used for the GEF assays were analyzed by immunoblotting (WB, bottom) with the indicated antibodies. No Cdc42-specific GEF activity could be detected in the same lysates (not shown). (E) Top, Rac-specific GEF activity was measured in immunoprecipitates performed with anti-HA (IP HA) or an irrelevant antibody (IP ctr) on lysates of 293T cells transfected with the indicated expression vectors (tfx; the Eps8-based constructs were HA tagged). Bottom, aliquots of the immunoprecipitates (IP) used in the GEF assay, or total cellular lysates (50 μg) transfected with the expression vectors indicated at the bottom (tfx), were immunoblotted (WB) with the indicated antibodies.

Mentions: Therefore, the effector region of Eps8 seems to recapitulate the physiological function of Eps8, behaving as a dominant active mutant and bypassing the need for growth factor stimulation. However, at variance with Eps8, the effector region does not require a physical interaction with E3b1 (Fig. 2, and Fig. 7, A and C) and the ensuing formation of a trimeric complex with Sos-1. On the other hand, the COOH terminus of Eps8 has been shown to bind in vitro to Sos-1 independently of E3b1 (Scita et al., 1999), albeit with low stoichiometry. Thus, this interaction may be sufficient to elicit Sos-1 guanine nucleotide exchange activity, leading to the activation of 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 Eps8 effector region binds to Sos-1 and stimulates Rac-specific, Sos-1–dependent GEF activity. (A) Total cellular lysates from Cos-7 cells were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated at the top) or GST as a negative control, or GST-E3b1 as a positive control (Scita et al., 1999). Detection was with anti–Sos-1 Ab. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Left, eukaryotically produced, affinity-purified Sos-1 was resolved by SDS-PAGE and stained with Coomassie brilliant blue. Sos-1 was estimated to be >90% pure (see Materials and methods). Right, purified Sos-1 (1 μg) was incubated with 5 μg of the indicated GST fusion proteins (all Eps8 fragments; amino acids boundaries are indicated on the top). Detection was with anti–Sos-1 Ab. The binding of Sos-1 to full length Eps8 (1–821) was estimated to be ∼10 less than the binding to GST-Eps8 (586–821) or GST-Eps8 (648–821) by densitometric analysis. (C) Lysates of 293T cells transfected with the indicated cDNAs (tfx) were immunoprecipitated (IP) with anti-Eps8 (Eps8) or anti-HA (HA), or the corresponding preimmune sera (ctr) followed by detection (Western blot, WB) with anti–Sos-1, anti-Eps8, anti-E3b1, or anti-HA antibodies. Native Sos-1 could also be recovered in immunoprecipitates of the Eps8 fragments 648–821 (not shown). The lanes “lysate” were loaded with 50 μg of total cellular lysates. (D) Top, Rac-specific GEF activity was measured in lysates of cells expressing the indicated Eps8 fragments (indicated underneath the bar graph. Ctr, control vector). Lysates were immunodepleted with an irrelevant (mock ID, solid bars) or by an anti–Sos-1 (Sos-1 ID, empty bars) antibody. The same immunodepleted lysates used for the GEF assays were analyzed by immunoblotting (WB, bottom) with the indicated antibodies. No Cdc42-specific GEF activity could be detected in the same lysates (not shown). (E) Top, Rac-specific GEF activity was measured in immunoprecipitates performed with anti-HA (IP HA) or an irrelevant antibody (IP ctr) on lysates of 293T cells transfected with the indicated expression vectors (tfx; the Eps8-based constructs were HA tagged). Bottom, aliquots of the immunoprecipitates (IP) used in the GEF assay, or total cellular lysates (50 μg) transfected with the expression vectors indicated at the bottom (tfx), were immunoblotted (WB) with the indicated antibodies.
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fig7: The Eps8 effector region binds to Sos-1 and stimulates Rac-specific, Sos-1–dependent GEF activity. (A) Total cellular lysates from Cos-7 cells were incubated with 5 μg of each Eps8 fragment (all engineered as GST fusions; amino acid boundaries are indicated at the top) or GST as a negative control, or GST-E3b1 as a positive control (Scita et al., 1999). Detection was with anti–Sos-1 Ab. The lane “lysate” was loaded with 50 μg of total cellular lysate. (B) Left, eukaryotically produced, affinity-purified Sos-1 was resolved by SDS-PAGE and stained with Coomassie brilliant blue. Sos-1 was estimated to be >90% pure (see Materials and methods). Right, purified Sos-1 (1 μg) was incubated with 5 μg of the indicated GST fusion proteins (all Eps8 fragments; amino acids boundaries are indicated on the top). Detection was with anti–Sos-1 Ab. The binding of Sos-1 to full length Eps8 (1–821) was estimated to be ∼10 less than the binding to GST-Eps8 (586–821) or GST-Eps8 (648–821) by densitometric analysis. (C) Lysates of 293T cells transfected with the indicated cDNAs (tfx) were immunoprecipitated (IP) with anti-Eps8 (Eps8) or anti-HA (HA), or the corresponding preimmune sera (ctr) followed by detection (Western blot, WB) with anti–Sos-1, anti-Eps8, anti-E3b1, or anti-HA antibodies. Native Sos-1 could also be recovered in immunoprecipitates of the Eps8 fragments 648–821 (not shown). The lanes “lysate” were loaded with 50 μg of total cellular lysates. (D) Top, Rac-specific GEF activity was measured in lysates of cells expressing the indicated Eps8 fragments (indicated underneath the bar graph. Ctr, control vector). Lysates were immunodepleted with an irrelevant (mock ID, solid bars) or by an anti–Sos-1 (Sos-1 ID, empty bars) antibody. The same immunodepleted lysates used for the GEF assays were analyzed by immunoblotting (WB, bottom) with the indicated antibodies. No Cdc42-specific GEF activity could be detected in the same lysates (not shown). (E) Top, Rac-specific GEF activity was measured in immunoprecipitates performed with anti-HA (IP HA) or an irrelevant antibody (IP ctr) on lysates of 293T cells transfected with the indicated expression vectors (tfx; the Eps8-based constructs were HA tagged). Bottom, aliquots of the immunoprecipitates (IP) used in the GEF assay, or total cellular lysates (50 μg) transfected with the expression vectors indicated at the bottom (tfx), were immunoblotted (WB) with the indicated antibodies.
Mentions: Therefore, the effector region of Eps8 seems to recapitulate the physiological function of Eps8, behaving as a dominant active mutant and bypassing the need for growth factor stimulation. However, at variance with Eps8, the effector region does not require a physical interaction with E3b1 (Fig. 2, and Fig. 7, A and C) and the ensuing formation of a trimeric complex with Sos-1. On the other hand, the COOH terminus of Eps8 has been shown to bind in vitro to Sos-1 independently of E3b1 (Scita et al., 1999), albeit with low stoichiometry. Thus, this interaction may be sufficient to elicit Sos-1 guanine nucleotide exchange activity, leading to the activation of 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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