Limits...
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.

Show MeSH
Disruption of the actin filament meshwork by cytochalasin D leads to mislocalization of Eps8 and of the Eps8 effector domain. Quiescent mouse embryo fibroblasts, transfected with the vectors encoding various GFP-tagged Eps8 fragments (amino acids boundaries are indicated on the left), were treated with cythocalasin D (100 nM) for 60 min (+Cyt. D) or with vehicle as a control (−Cyt. D). Cells were fixed and GFP was detected by epifluorescence (green). F-actin was evidenced by phalloidin staining (red). Note the colocalization of the Eps8 full length (1–821) and of the Eps8 fragments containing the effector domain (amino acids 648–821) with F-actin aggregates induced by treatment with cythocalasin D (yellow). Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196181&req=5

fig10: Disruption of the actin filament meshwork by cytochalasin D leads to mislocalization of Eps8 and of the Eps8 effector domain. Quiescent mouse embryo fibroblasts, transfected with the vectors encoding various GFP-tagged Eps8 fragments (amino acids boundaries are indicated on the left), were treated with cythocalasin D (100 nM) for 60 min (+Cyt. D) or with vehicle as a control (−Cyt. D). Cells were fixed and GFP was detected by epifluorescence (green). F-actin was evidenced by phalloidin staining (red). Note the colocalization of the Eps8 full length (1–821) and of the Eps8 fragments containing the effector domain (amino acids 648–821) with F-actin aggregates induced by treatment with cythocalasin D (yellow). Bar, 10 μM.

Mentions: The interaction in vivo between Eps8 and F-actin may dictate the proper intracellular localization of Eps8. In this case, disruption of the actin filaments should lead to Eps8 mislocalization. Treatment of cells with cytochalasin D, an inhibitor of actin polymerization (Goddette and Frieden, 1986; Sampath and Pollard, 1991), caused the disruption of the F-actin meshwork (Fig. 10) . This resulted in the disruption of membrane ruffles and the scattered accumulation of short bundles of F-actin aggregates, which colocalized (Fig. 10) with Eps8 or with those Eps8 fragments containing an intact actin binding domain (Fig. 10). Thus, the integrity of the F-actin meshwork is required to direct Eps8 to its proper subcellular localization.


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)

Disruption of the actin filament meshwork by cytochalasin D leads to mislocalization of Eps8 and of the Eps8 effector domain. Quiescent mouse embryo fibroblasts, transfected with the vectors encoding various GFP-tagged Eps8 fragments (amino acids boundaries are indicated on the left), were treated with cythocalasin D (100 nM) for 60 min (+Cyt. D) or with vehicle as a control (−Cyt. D). Cells were fixed and GFP was detected by epifluorescence (green). F-actin was evidenced by phalloidin staining (red). Note the colocalization of the Eps8 full length (1–821) and of the Eps8 fragments containing the effector domain (amino acids 648–821) with F-actin aggregates induced by treatment with cythocalasin D (yellow). Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Disruption of the actin filament meshwork by cytochalasin D leads to mislocalization of Eps8 and of the Eps8 effector domain. Quiescent mouse embryo fibroblasts, transfected with the vectors encoding various GFP-tagged Eps8 fragments (amino acids boundaries are indicated on the left), were treated with cythocalasin D (100 nM) for 60 min (+Cyt. D) or with vehicle as a control (−Cyt. D). Cells were fixed and GFP was detected by epifluorescence (green). F-actin was evidenced by phalloidin staining (red). Note the colocalization of the Eps8 full length (1–821) and of the Eps8 fragments containing the effector domain (amino acids 648–821) with F-actin aggregates induced by treatment with cythocalasin D (yellow). Bar, 10 μM.
Mentions: The interaction in vivo between Eps8 and F-actin may dictate the proper intracellular localization of Eps8. In this case, disruption of the actin filaments should lead to Eps8 mislocalization. Treatment of cells with cytochalasin D, an inhibitor of actin polymerization (Goddette and Frieden, 1986; Sampath and Pollard, 1991), caused the disruption of the F-actin meshwork (Fig. 10) . This resulted in the disruption of membrane ruffles and the scattered accumulation of short bundles of F-actin aggregates, which colocalized (Fig. 10) with Eps8 or with those Eps8 fragments containing an intact actin binding domain (Fig. 10). Thus, the integrity of the F-actin meshwork is required to direct Eps8 to its proper subcellular localization.

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