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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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PI3K recruitment to the Eps8–Abi1–Sos-1 complex is required for Rac-GEF activity, which is further increased by PIP3. (A) Cos-7 cells were transfected (tfx) with mycEps8 and Sos-1, together with either Abi1wt (WT) or the Y407F mutant (Y407F). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. Control immunoprecipitates were obtained using an irrelevant antibody. (B) Rac-GEF activity was measured in aliquots of the immunoprecipitates shown in A, in the absence or presence of the indicated water-soluble phosphoinositides (Han et al., 1998; supplemental materials). The difference in the samples marked by the asterisks was significant (P < 0.01) in a paired t test. (C) Cos-7 cells were transfected (tfx) with mycEps8 and Abi1, together with either Sos-1 (WT) or a dominant-negative mutant of Sos-1 (DH−). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (D) Rac-GEF activity was measured on aliquots of the immunoprecipitate containing equal amounts of wild-type Sos-1 and the Sos-1 DH− mutant shown in C, in the absence or presence of the indicated water-soluble phosphoinositides. The difference in the samples marked by the asterisks was significant (P < 0.005) in a paired t test.
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fig3: PI3K recruitment to the Eps8–Abi1–Sos-1 complex is required for Rac-GEF activity, which is further increased by PIP3. (A) Cos-7 cells were transfected (tfx) with mycEps8 and Sos-1, together with either Abi1wt (WT) or the Y407F mutant (Y407F). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. Control immunoprecipitates were obtained using an irrelevant antibody. (B) Rac-GEF activity was measured in aliquots of the immunoprecipitates shown in A, in the absence or presence of the indicated water-soluble phosphoinositides (Han et al., 1998; supplemental materials). The difference in the samples marked by the asterisks was significant (P < 0.01) in a paired t test. (C) Cos-7 cells were transfected (tfx) with mycEps8 and Abi1, together with either Sos-1 (WT) or a dominant-negative mutant of Sos-1 (DH−). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (D) Rac-GEF activity was measured on aliquots of the immunoprecipitate containing equal amounts of wild-type Sos-1 and the Sos-1 DH− mutant shown in C, in the absence or presence of the indicated water-soluble phosphoinositides. The difference in the samples marked by the asterisks was significant (P < 0.005) in a paired t test.

Mentions: The immunoprecipitated Eps8–Abi1–Sos-1 complex displays Rac-specific GEF activity (Scita et al., 1999). This, and the results presented here, raise the possibility that the recruitment in vivo of PI3K to the complex is also required. Cells were transfected with a combination of Sos-1–mycEps8–Abi1wt or Sos-1–mycEps8–Abi1Y407F, and Rac-GEF assays were performed on anti-myc immunoprecipitates. Trimeric Eps8–Abi1–Sos-1 complexes were readily detected that contained similar amounts of the catalytic subunit Sos-1 (Fig. 3 A). Endogenous p85 (Fig. 3 A) and Rac-GEF activity (Fig. 3 B) were present only in the complexes containing Abi1wt, but not in those with Abi1Y407F.


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)

PI3K recruitment to the Eps8–Abi1–Sos-1 complex is required for Rac-GEF activity, which is further increased by PIP3. (A) Cos-7 cells were transfected (tfx) with mycEps8 and Sos-1, together with either Abi1wt (WT) or the Y407F mutant (Y407F). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. Control immunoprecipitates were obtained using an irrelevant antibody. (B) Rac-GEF activity was measured in aliquots of the immunoprecipitates shown in A, in the absence or presence of the indicated water-soluble phosphoinositides (Han et al., 1998; supplemental materials). The difference in the samples marked by the asterisks was significant (P < 0.01) in a paired t test. (C) Cos-7 cells were transfected (tfx) with mycEps8 and Abi1, together with either Sos-1 (WT) or a dominant-negative mutant of Sos-1 (DH−). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (D) Rac-GEF activity was measured on aliquots of the immunoprecipitate containing equal amounts of wild-type Sos-1 and the Sos-1 DH− mutant shown in C, in the absence or presence of the indicated water-soluble phosphoinositides. The difference in the samples marked by the asterisks was significant (P < 0.005) in a paired t test.
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Related In: Results  -  Collection

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fig3: PI3K recruitment to the Eps8–Abi1–Sos-1 complex is required for Rac-GEF activity, which is further increased by PIP3. (A) Cos-7 cells were transfected (tfx) with mycEps8 and Sos-1, together with either Abi1wt (WT) or the Y407F mutant (Y407F). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. Control immunoprecipitates were obtained using an irrelevant antibody. (B) Rac-GEF activity was measured in aliquots of the immunoprecipitates shown in A, in the absence or presence of the indicated water-soluble phosphoinositides (Han et al., 1998; supplemental materials). The difference in the samples marked by the asterisks was significant (P < 0.01) in a paired t test. (C) Cos-7 cells were transfected (tfx) with mycEps8 and Abi1, together with either Sos-1 (WT) or a dominant-negative mutant of Sos-1 (DH−). Lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (D) Rac-GEF activity was measured on aliquots of the immunoprecipitate containing equal amounts of wild-type Sos-1 and the Sos-1 DH− mutant shown in C, in the absence or presence of the indicated water-soluble phosphoinositides. The difference in the samples marked by the asterisks was significant (P < 0.005) in a paired t test.
Mentions: The immunoprecipitated Eps8–Abi1–Sos-1 complex displays Rac-specific GEF activity (Scita et al., 1999). This, and the results presented here, raise the possibility that the recruitment in vivo of PI3K to the complex is also required. Cells were transfected with a combination of Sos-1–mycEps8–Abi1wt or Sos-1–mycEps8–Abi1Y407F, and Rac-GEF assays were performed on anti-myc immunoprecipitates. Trimeric Eps8–Abi1–Sos-1 complexes were readily detected that contained similar amounts of the catalytic subunit Sos-1 (Fig. 3 A). Endogenous p85 (Fig. 3 A) and Rac-GEF activity (Fig. 3 B) were present only in the complexes containing Abi1wt, but not in those with Abi1Y407F.

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