Limits...
Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1.

Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Brachmann SM, Di Fiore PP, Scita G - J. Cell Biol. (2003)

Bottom Line: Within this pathway, Rac is a key downstream target/effector of PI3K.On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation.Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.

ABSTRACT
Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

Show MeSH

Related in: MedlinePlus

The Abi1-p85 association is required for ruffling by activated RTK and Ras, but not by activated Rac. (A) Quiescent MEFs, microinjected with the indicated peptides together with rabbit IgG (supplemental materials), were treated with PDGF for 10 min. Cells were fixed and stained with rhodamine-conjugated phalloidin to detect F-actin (red) and anti–rabbit IgG (not depicted) to detect microinjected cells (indicated by asterisks). More than 100 cells were injected in each experiment. In the bar graph, a quantitation of the experiment is reported. (B) MEFs were transfected, as indicated on the top, with expression vectors for either RasV12 or RacQL, together with either ECFP-Abi1WT or ECFP-Abi1Y407F. After serum starvation, cells were fixed and stained with rhodamine-conjugated phalloidin (ph, red). Expression of the transfected proteins was detected (green) with either anti-Ras or anti-Rac antibodies or by epifluorescence (ECFP). The percentage of transfected cells undergoing ruffling is reported in Materials and methods. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172734&req=5

fig5: The Abi1-p85 association is required for ruffling by activated RTK and Ras, but not by activated Rac. (A) Quiescent MEFs, microinjected with the indicated peptides together with rabbit IgG (supplemental materials), were treated with PDGF for 10 min. Cells were fixed and stained with rhodamine-conjugated phalloidin to detect F-actin (red) and anti–rabbit IgG (not depicted) to detect microinjected cells (indicated by asterisks). More than 100 cells were injected in each experiment. In the bar graph, a quantitation of the experiment is reported. (B) MEFs were transfected, as indicated on the top, with expression vectors for either RasV12 or RacQL, together with either ECFP-Abi1WT or ECFP-Abi1Y407F. After serum starvation, cells were fixed and stained with rhodamine-conjugated phalloidin (ph, red). Expression of the transfected proteins was detected (green) with either anti-Ras or anti-Rac antibodies or by epifluorescence (ECFP). The percentage of transfected cells undergoing ruffling is reported in Materials and methods. Bars, 10 μm.

Mentions: The trimeric complex Eps8–Abi1–Sos-1 is essential for RTK-mediated actin cytoskeletal remodeling, as witnessed by the lack of PDGF-induced Rac activation and Rac-dependent membrane ruffling detected in Eps8−/− cells (Scita et al., 1999; Innocenti et al., 2002). To analyze the biological consequence of p85 recruitment by the tricomplex, two approaches were undertaken. First, a phosphorylated peptide encompassing Tyr407 of Abi, and corresponding to the Abi1 binding site of p85, was used. This phosphorylated peptide, but not its unphosphorylated version or one in which the tyrosine residue was replaced by phenylalanine, efficiently inhibited the binding of p85 to Abi1, but not to activated PDGFR (Fig. S2). Microinjection of the phosphorylated peptide (but not of the two control peptides) inhibited PDGF-induced ruffles by more than 70% (Fig. 5 A). Notably, inhibition of RTK-mediated ruffles could also be caused by microinjection of anti-Abi1 antibodies (Scita et al., 1999 and Fig. S4), which, however, did not affect TPA-induced actin remodeling, indicating that additional pathways leading to Rac activation (and possibly reflecting the simultaneous presence of several Rac GEFs in the cells) are at play (supplemental information, Fig. 4). Second, we cotransfected the activated version of either Ras (RasV12) or Rac (RacQL) together with either Abi1wt or the Abi1Y407F mutant, and scored the formation of ruffles. RasV12-induced (but not RacQL-induced) ruffles were efficiently inhibited by coexpression of Abi1Y407F, but not by Abi1wt (Fig. 5 B). Thus, the recruitment of PI3K by Abi1 plays a critical role in RTK-induced actin remodeling and is essential for the propagation of signals from Ras to Rac.


Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1.

Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Brachmann SM, Di Fiore PP, Scita G - J. Cell Biol. (2003)

The Abi1-p85 association is required for ruffling by activated RTK and Ras, but not by activated Rac. (A) Quiescent MEFs, microinjected with the indicated peptides together with rabbit IgG (supplemental materials), were treated with PDGF for 10 min. Cells were fixed and stained with rhodamine-conjugated phalloidin to detect F-actin (red) and anti–rabbit IgG (not depicted) to detect microinjected cells (indicated by asterisks). More than 100 cells were injected in each experiment. In the bar graph, a quantitation of the experiment is reported. (B) MEFs were transfected, as indicated on the top, with expression vectors for either RasV12 or RacQL, together with either ECFP-Abi1WT or ECFP-Abi1Y407F. After serum starvation, cells were fixed and stained with rhodamine-conjugated phalloidin (ph, red). Expression of the transfected proteins was detected (green) with either anti-Ras or anti-Rac antibodies or by epifluorescence (ECFP). The percentage of transfected cells undergoing ruffling is reported in Materials and methods. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172734&req=5

fig5: The Abi1-p85 association is required for ruffling by activated RTK and Ras, but not by activated Rac. (A) Quiescent MEFs, microinjected with the indicated peptides together with rabbit IgG (supplemental materials), were treated with PDGF for 10 min. Cells were fixed and stained with rhodamine-conjugated phalloidin to detect F-actin (red) and anti–rabbit IgG (not depicted) to detect microinjected cells (indicated by asterisks). More than 100 cells were injected in each experiment. In the bar graph, a quantitation of the experiment is reported. (B) MEFs were transfected, as indicated on the top, with expression vectors for either RasV12 or RacQL, together with either ECFP-Abi1WT or ECFP-Abi1Y407F. After serum starvation, cells were fixed and stained with rhodamine-conjugated phalloidin (ph, red). Expression of the transfected proteins was detected (green) with either anti-Ras or anti-Rac antibodies or by epifluorescence (ECFP). The percentage of transfected cells undergoing ruffling is reported in Materials and methods. Bars, 10 μm.
Mentions: The trimeric complex Eps8–Abi1–Sos-1 is essential for RTK-mediated actin cytoskeletal remodeling, as witnessed by the lack of PDGF-induced Rac activation and Rac-dependent membrane ruffling detected in Eps8−/− cells (Scita et al., 1999; Innocenti et al., 2002). To analyze the biological consequence of p85 recruitment by the tricomplex, two approaches were undertaken. First, a phosphorylated peptide encompassing Tyr407 of Abi, and corresponding to the Abi1 binding site of p85, was used. This phosphorylated peptide, but not its unphosphorylated version or one in which the tyrosine residue was replaced by phenylalanine, efficiently inhibited the binding of p85 to Abi1, but not to activated PDGFR (Fig. S2). Microinjection of the phosphorylated peptide (but not of the two control peptides) inhibited PDGF-induced ruffles by more than 70% (Fig. 5 A). Notably, inhibition of RTK-mediated ruffles could also be caused by microinjection of anti-Abi1 antibodies (Scita et al., 1999 and Fig. S4), which, however, did not affect TPA-induced actin remodeling, indicating that additional pathways leading to Rac activation (and possibly reflecting the simultaneous presence of several Rac GEFs in the cells) are at play (supplemental information, Fig. 4). Second, we cotransfected the activated version of either Ras (RasV12) or Rac (RacQL) together with either Abi1wt or the Abi1Y407F mutant, and scored the formation of ruffles. RasV12-induced (but not RacQL-induced) ruffles were efficiently inhibited by coexpression of Abi1Y407F, but not by Abi1wt (Fig. 5 B). Thus, the recruitment of PI3K by Abi1 plays a critical role in RTK-induced actin remodeling and is essential for the propagation of signals from Ras to Rac.

Bottom Line: Within this pathway, Rac is a key downstream target/effector of PI3K.On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation.Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.

ABSTRACT
Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8-Abi1-Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

Show MeSH
Related in: MedlinePlus