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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.

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p85 interacts with Abi1. (A) Left; lysates (5 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, preimmune serum). Abi1 could also be detected in p85 immunoprecipitates (supplemental materials). About 1% of total p85 could be found associated with Abi1 immunoprecipitates. Right; lysates (5 mg; p85 = 0.3 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (top). The immunocomplexes were tested for PI3K lipid kinase activity by TLC (bottom; Domin et al., 1996). (B) Binding of GST-p85 fragments (amino acid boundaries or domains are indicated) to Abi1 from lysates of HA-Abi1–transfected 293T cells. The altered electrophoretic mobility of the Abi1 band in the 320–724 lane is due its co-migration with the GST-fusion protein. (C) Lysates (2 mg) from 293T cells transfected with HA-Abi1 were immunoprecipitated with anti-HA antibody. Immunoprecipitates, treated with alkaline phosphatase in the presence (+) or absence (−) of inhibitors (API), were immunoblotted (IB) with anti-HA (left) or anti-pY (middle), or analyzed in Far-western with the proteins indicated on the top. (D) Lysates (1 mg) from 293T cells, transfected with HA-tagged wild-type (WT) or Y407F Abi1, were subjected to in vitro binding with the GST-NH2-terminal SH2 domain of p85 (SH2-N) or GST alone. Similar results were also obtained using the COOH-terminal SH2 domain of p85, indicating that the apparent affinities of the SH2 domains of p85 for Abi1 were similar (not depicted). (E) Lysates (5 mg) from Cos-7 cells, transfected with HA-Abi1 (WT) or HA-Abi1Y407F, were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, anti-flag antibody used as a control).
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fig1: p85 interacts with Abi1. (A) Left; lysates (5 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, preimmune serum). Abi1 could also be detected in p85 immunoprecipitates (supplemental materials). About 1% of total p85 could be found associated with Abi1 immunoprecipitates. Right; lysates (5 mg; p85 = 0.3 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (top). The immunocomplexes were tested for PI3K lipid kinase activity by TLC (bottom; Domin et al., 1996). (B) Binding of GST-p85 fragments (amino acid boundaries or domains are indicated) to Abi1 from lysates of HA-Abi1–transfected 293T cells. The altered electrophoretic mobility of the Abi1 band in the 320–724 lane is due its co-migration with the GST-fusion protein. (C) Lysates (2 mg) from 293T cells transfected with HA-Abi1 were immunoprecipitated with anti-HA antibody. Immunoprecipitates, treated with alkaline phosphatase in the presence (+) or absence (−) of inhibitors (API), were immunoblotted (IB) with anti-HA (left) or anti-pY (middle), or analyzed in Far-western with the proteins indicated on the top. (D) Lysates (1 mg) from 293T cells, transfected with HA-tagged wild-type (WT) or Y407F Abi1, were subjected to in vitro binding with the GST-NH2-terminal SH2 domain of p85 (SH2-N) or GST alone. Similar results were also obtained using the COOH-terminal SH2 domain of p85, indicating that the apparent affinities of the SH2 domains of p85 for Abi1 were similar (not depicted). (E) Lysates (5 mg) from Cos-7 cells, transfected with HA-Abi1 (WT) or HA-Abi1Y407F, were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, anti-flag antibody used as a control).

Mentions: We searched for interaction(s) of the Eps8–Abi1–Sos-1 complex with class I PI3K. Endogenous p85 and Abi1 could be coimmunoprecipitated (Fig. 1 A, left, and supplemental materials), and a wortmannin-sensitive (Fig S1, available at http://www.jcb.org/cgi/content/full/jcb.200206079/DC1) PI3K enzymatic activity was detected in Abi1 immunocomplexes (Fig. 1 A, right), suggesting that the p110–p85 complex binds to Abi1.


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)

p85 interacts with Abi1. (A) Left; lysates (5 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, preimmune serum). Abi1 could also be detected in p85 immunoprecipitates (supplemental materials). About 1% of total p85 could be found associated with Abi1 immunoprecipitates. Right; lysates (5 mg; p85 = 0.3 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (top). The immunocomplexes were tested for PI3K lipid kinase activity by TLC (bottom; Domin et al., 1996). (B) Binding of GST-p85 fragments (amino acid boundaries or domains are indicated) to Abi1 from lysates of HA-Abi1–transfected 293T cells. The altered electrophoretic mobility of the Abi1 band in the 320–724 lane is due its co-migration with the GST-fusion protein. (C) Lysates (2 mg) from 293T cells transfected with HA-Abi1 were immunoprecipitated with anti-HA antibody. Immunoprecipitates, treated with alkaline phosphatase in the presence (+) or absence (−) of inhibitors (API), were immunoblotted (IB) with anti-HA (left) or anti-pY (middle), or analyzed in Far-western with the proteins indicated on the top. (D) Lysates (1 mg) from 293T cells, transfected with HA-tagged wild-type (WT) or Y407F Abi1, were subjected to in vitro binding with the GST-NH2-terminal SH2 domain of p85 (SH2-N) or GST alone. Similar results were also obtained using the COOH-terminal SH2 domain of p85, indicating that the apparent affinities of the SH2 domains of p85 for Abi1 were similar (not depicted). (E) Lysates (5 mg) from Cos-7 cells, transfected with HA-Abi1 (WT) or HA-Abi1Y407F, were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, anti-flag antibody used as a control).
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fig1: p85 interacts with Abi1. (A) Left; lysates (5 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, preimmune serum). Abi1 could also be detected in p85 immunoprecipitates (supplemental materials). About 1% of total p85 could be found associated with Abi1 immunoprecipitates. Right; lysates (5 mg; p85 = 0.3 mg) from MEFs were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (top). The immunocomplexes were tested for PI3K lipid kinase activity by TLC (bottom; Domin et al., 1996). (B) Binding of GST-p85 fragments (amino acid boundaries or domains are indicated) to Abi1 from lysates of HA-Abi1–transfected 293T cells. The altered electrophoretic mobility of the Abi1 band in the 320–724 lane is due its co-migration with the GST-fusion protein. (C) Lysates (2 mg) from 293T cells transfected with HA-Abi1 were immunoprecipitated with anti-HA antibody. Immunoprecipitates, treated with alkaline phosphatase in the presence (+) or absence (−) of inhibitors (API), were immunoblotted (IB) with anti-HA (left) or anti-pY (middle), or analyzed in Far-western with the proteins indicated on the top. (D) Lysates (1 mg) from 293T cells, transfected with HA-tagged wild-type (WT) or Y407F Abi1, were subjected to in vitro binding with the GST-NH2-terminal SH2 domain of p85 (SH2-N) or GST alone. Similar results were also obtained using the COOH-terminal SH2 domain of p85, indicating that the apparent affinities of the SH2 domains of p85 for Abi1 were similar (not depicted). (E) Lysates (5 mg) from Cos-7 cells, transfected with HA-Abi1 (WT) or HA-Abi1Y407F, were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies (ctr, anti-flag antibody used as a control).
Mentions: We searched for interaction(s) of the Eps8–Abi1–Sos-1 complex with class I PI3K. Endogenous p85 and Abi1 could be coimmunoprecipitated (Fig. 1 A, left, and supplemental materials), and a wortmannin-sensitive (Fig S1, available at http://www.jcb.org/cgi/content/full/jcb.200206079/DC1) PI3K enzymatic activity was detected in Abi1 immunocomplexes (Fig. 1 A, right), suggesting that the p110–p85 complex binds to Abi1.

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