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

Related in: MedlinePlus

The COOH-terminal region (535–821) of Eps8 is necessary and sufficient for Eps8 localization to the cell cortex and its accumulation into PDGF-induced membrane ruffles. (A) Mouse embryo fibroblasts were serum starved for 24 h and then treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF). Fibroblasts were fixed and stained with rhodamine-conjugated phalloidin (red, left) to detect F-actin or anti-Eps8 (green, right). Ruffling was detected in >90% of the PDGF-treated cells. (B) Mouse embryo fibroblasts were transfected with expression vectors encoding Eps8 full length or the indicated Eps8 fragments fused to GFP (FL, full length; amino acid boundaries of the other constructs are given in parentheses). After serum starvation, cells were treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF), followed by fixation and detection of GFP-Eps8 fusion proteins by epifluorescence. Representative pictures are shown. In B, ruffles were identified by costaining with phalloidin (not shown). Arrowheads point to ruffles. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196181&req=5

fig1: The COOH-terminal region (535–821) of Eps8 is necessary and sufficient for Eps8 localization to the cell cortex and its accumulation into PDGF-induced membrane ruffles. (A) Mouse embryo fibroblasts were serum starved for 24 h and then treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF). Fibroblasts were fixed and stained with rhodamine-conjugated phalloidin (red, left) to detect F-actin or anti-Eps8 (green, right). Ruffling was detected in >90% of the PDGF-treated cells. (B) Mouse embryo fibroblasts were transfected with expression vectors encoding Eps8 full length or the indicated Eps8 fragments fused to GFP (FL, full length; amino acid boundaries of the other constructs are given in parentheses). After serum starvation, cells were treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF), followed by fixation and detection of GFP-Eps8 fusion proteins by epifluorescence. Representative pictures are shown. In B, ruffles were identified by costaining with phalloidin (not shown). Arrowheads point to ruffles. Bars, 10 μm.

Mentions: The biochemical function of Eps8, which mediates Ras to Rac signaling, leading to actin cytoskeleton reorganization (Scita et al., 1999, 2000), is mirrored by its subcellular localization. In serum-starved fibroblasts, Eps8 displays a punctuate, cytoplasmic perinuclear distribution with some staining at the plasma membrane corresponding to sites of cortical actin accumulation (Provenzano et al., 1998; Scita et al., 1999; Fig. 1 A). A substantial relocalization of Eps8 occurs upon treatment with growth factors within sites of actin polymerization, at the dorsal surface of cells, and in membrane ruffles (Provenzano et al., 1998; Scita et al., 1999; Fig. 1 A).


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 COOH-terminal region (535–821) of Eps8 is necessary and sufficient for Eps8 localization to the cell cortex and its accumulation into PDGF-induced membrane ruffles. (A) Mouse embryo fibroblasts were serum starved for 24 h and then treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF). Fibroblasts were fixed and stained with rhodamine-conjugated phalloidin (red, left) to detect F-actin or anti-Eps8 (green, right). Ruffling was detected in >90% of the PDGF-treated cells. (B) Mouse embryo fibroblasts were transfected with expression vectors encoding Eps8 full length or the indicated Eps8 fragments fused to GFP (FL, full length; amino acid boundaries of the other constructs are given in parentheses). After serum starvation, cells were treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF), followed by fixation and detection of GFP-Eps8 fusion proteins by epifluorescence. Representative pictures are shown. In B, ruffles were identified by costaining with phalloidin (not shown). Arrowheads point to ruffles. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: The COOH-terminal region (535–821) of Eps8 is necessary and sufficient for Eps8 localization to the cell cortex and its accumulation into PDGF-induced membrane ruffles. (A) Mouse embryo fibroblasts were serum starved for 24 h and then treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF). Fibroblasts were fixed and stained with rhodamine-conjugated phalloidin (red, left) to detect F-actin or anti-Eps8 (green, right). Ruffling was detected in >90% of the PDGF-treated cells. (B) Mouse embryo fibroblasts were transfected with expression vectors encoding Eps8 full length or the indicated Eps8 fragments fused to GFP (FL, full length; amino acid boundaries of the other constructs are given in parentheses). After serum starvation, cells were treated with 10 ng/ml of PDGF (+PDGF) for 10 min or mock treated (−PDGF), followed by fixation and detection of GFP-Eps8 fusion proteins by epifluorescence. Representative pictures are shown. In B, ruffles were identified by costaining with phalloidin (not shown). Arrowheads point to ruffles. Bars, 10 μm.
Mentions: The biochemical function of Eps8, which mediates Ras to Rac signaling, leading to actin cytoskeleton reorganization (Scita et al., 1999, 2000), is mirrored by its subcellular localization. In serum-starved fibroblasts, Eps8 displays a punctuate, cytoplasmic perinuclear distribution with some staining at the plasma membrane corresponding to sites of cortical actin accumulation (Provenzano et al., 1998; Scita et al., 1999; Fig. 1 A). A substantial relocalization of Eps8 occurs upon treatment with growth factors within sites of actin polymerization, at the dorsal surface of cells, and in membrane ruffles (Provenzano et al., 1998; Scita et al., 1999; Fig. 1 A).

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