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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 is required for Abi1-induced activation of Rac in vivo. Cos-7 cells, transfected (tfx) with HA-Rac together with HA-Abi1, HA-Abi1Y407F, HA-Abi1DY, or the empty vector (ctr) were treated with 100 ng/ml of EGF for 3 min (+) or mock-treated (−). Lysates were either incubated with GST-CRIB and detected with anti-Rac antibodies (RacGTP), or directly immunoblotted with the indicated antibody. (B) p85α/p85β-double (p85 −/−) and p85β-single (p85 −/− [p85α]) knockout MEFs, transfected (tfx) with HA-Rac together with either an HA-Abi1 (+) vector or the empty vector as control (−), were serum-starved for 24 h. Lysates were incubated with GST-CRIB as described above and detected with anti–HA-Rac antibody (RacGTP) or directly immunoblotted with the indicated antibody. (C) Cos-7 cells, transfected (tfx) with HA-Rac together with Abi1 (Abi WT) or the empty vector (ctr), were incubated for 1 h with 100 nM of wortmannin (Wort, + lanes) or vehicle (Wort, − lanes), as a control, and treated with EGF (EGF, + lanes) or mock-treated (EGF, − lanes). Rac-GTP levels, total Rac, and the expression of Abi1 were determined as in A.
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fig4: p85 is required for Abi1-induced activation of Rac in vivo. Cos-7 cells, transfected (tfx) with HA-Rac together with HA-Abi1, HA-Abi1Y407F, HA-Abi1DY, or the empty vector (ctr) were treated with 100 ng/ml of EGF for 3 min (+) or mock-treated (−). Lysates were either incubated with GST-CRIB and detected with anti-Rac antibodies (RacGTP), or directly immunoblotted with the indicated antibody. (B) p85α/p85β-double (p85 −/−) and p85β-single (p85 −/− [p85α]) knockout MEFs, transfected (tfx) with HA-Rac together with either an HA-Abi1 (+) vector or the empty vector as control (−), were serum-starved for 24 h. Lysates were incubated with GST-CRIB as described above and detected with anti–HA-Rac antibody (RacGTP) or directly immunoblotted with the indicated antibody. (C) Cos-7 cells, transfected (tfx) with HA-Rac together with Abi1 (Abi WT) or the empty vector (ctr), were incubated for 1 h with 100 nM of wortmannin (Wort, + lanes) or vehicle (Wort, − lanes), as a control, and treated with EGF (EGF, + lanes) or mock-treated (EGF, − lanes). Rac-GTP levels, total Rac, and the expression of Abi1 were determined as in A.

Mentions: The above observations indicate that the recruitment of PI3K by Abi1 into an Eps8–Abi1–Sos-1 complex is necessary and sufficient to activate, in vitro, the Rac-GEF capability of the latter. They further highlight a regulatory role exerted by PIP3 on this complex. If these in vitro findings were to translate into physiologically relevant events, then one would predict that (1) interference with the formation of the Eps8–Abi1–p85–Sos-1 complex either by preventing the binding between Abi1 and p85 (Fig. 3 A) or by genetically removing p85; and (2) pharmacological inhibition of PI3-K by wortmannin should affect Rac activation mediated by the complex in vivo. As show in Fig. 4 A, both the basal and the EGF-induced levels of Rac-GTP were increased by the expression of wild-type Abi1, consistent with the notion that Abi1 is rate-limiting in Rac activation (Innocenti et al., 2002). However, no Rac activity could be detected when the Abi1Y407F, or a mutant of Abi1 (Abi1DY), which does not associate with Eps8, were used (Fig. 4 A). These results strongly suggest that Abi1 mutants, defective in their ability to assemble to PI3K or Eps8, are not only biologically inactive, but act as dominant-negatives, most likely by sequestering the other endogenous components in inactive complexes. Moreover, a formal proof of the requirement of p85 for Abi1-dependent activation of Rac was obtained by using fibroblasts in which both p85 isoforms (α and β) were genetically removed (unpublished data). In these cells, expression of Abi1 failed to induce Rac activation, which was, however, restored by reintroduction of p85α (Fig. 4 B). Finally, treatment with wortmannin reduced (but did not abrogate) EGF-dependent and EGF-independent Rac activation induced by Abi1 (Fig. 4 C). Similarly, the Rac-GEF activity in the Eps8–Abi1–p85–Sos-1 immunocomplex was only reduced by pretreatment of the cells with wortmannin (unpublished data). Thus, all together, these data support the notion that PI3K recruitment to the Eps8–Abi1–Sos-1 complex is physically required to elicit a basal Rac-GEF activity, which is further increased by PIP3.


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 is required for Abi1-induced activation of Rac in vivo. Cos-7 cells, transfected (tfx) with HA-Rac together with HA-Abi1, HA-Abi1Y407F, HA-Abi1DY, or the empty vector (ctr) were treated with 100 ng/ml of EGF for 3 min (+) or mock-treated (−). Lysates were either incubated with GST-CRIB and detected with anti-Rac antibodies (RacGTP), or directly immunoblotted with the indicated antibody. (B) p85α/p85β-double (p85 −/−) and p85β-single (p85 −/− [p85α]) knockout MEFs, transfected (tfx) with HA-Rac together with either an HA-Abi1 (+) vector or the empty vector as control (−), were serum-starved for 24 h. Lysates were incubated with GST-CRIB as described above and detected with anti–HA-Rac antibody (RacGTP) or directly immunoblotted with the indicated antibody. (C) Cos-7 cells, transfected (tfx) with HA-Rac together with Abi1 (Abi WT) or the empty vector (ctr), were incubated for 1 h with 100 nM of wortmannin (Wort, + lanes) or vehicle (Wort, − lanes), as a control, and treated with EGF (EGF, + lanes) or mock-treated (EGF, − lanes). Rac-GTP levels, total Rac, and the expression of Abi1 were determined as in A.
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Related In: Results  -  Collection

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fig4: p85 is required for Abi1-induced activation of Rac in vivo. Cos-7 cells, transfected (tfx) with HA-Rac together with HA-Abi1, HA-Abi1Y407F, HA-Abi1DY, or the empty vector (ctr) were treated with 100 ng/ml of EGF for 3 min (+) or mock-treated (−). Lysates were either incubated with GST-CRIB and detected with anti-Rac antibodies (RacGTP), or directly immunoblotted with the indicated antibody. (B) p85α/p85β-double (p85 −/−) and p85β-single (p85 −/− [p85α]) knockout MEFs, transfected (tfx) with HA-Rac together with either an HA-Abi1 (+) vector or the empty vector as control (−), were serum-starved for 24 h. Lysates were incubated with GST-CRIB as described above and detected with anti–HA-Rac antibody (RacGTP) or directly immunoblotted with the indicated antibody. (C) Cos-7 cells, transfected (tfx) with HA-Rac together with Abi1 (Abi WT) or the empty vector (ctr), were incubated for 1 h with 100 nM of wortmannin (Wort, + lanes) or vehicle (Wort, − lanes), as a control, and treated with EGF (EGF, + lanes) or mock-treated (EGF, − lanes). Rac-GTP levels, total Rac, and the expression of Abi1 were determined as in A.
Mentions: The above observations indicate that the recruitment of PI3K by Abi1 into an Eps8–Abi1–Sos-1 complex is necessary and sufficient to activate, in vitro, the Rac-GEF capability of the latter. They further highlight a regulatory role exerted by PIP3 on this complex. If these in vitro findings were to translate into physiologically relevant events, then one would predict that (1) interference with the formation of the Eps8–Abi1–p85–Sos-1 complex either by preventing the binding between Abi1 and p85 (Fig. 3 A) or by genetically removing p85; and (2) pharmacological inhibition of PI3-K by wortmannin should affect Rac activation mediated by the complex in vivo. As show in Fig. 4 A, both the basal and the EGF-induced levels of Rac-GTP were increased by the expression of wild-type Abi1, consistent with the notion that Abi1 is rate-limiting in Rac activation (Innocenti et al., 2002). However, no Rac activity could be detected when the Abi1Y407F, or a mutant of Abi1 (Abi1DY), which does not associate with Eps8, were used (Fig. 4 A). These results strongly suggest that Abi1 mutants, defective in their ability to assemble to PI3K or Eps8, are not only biologically inactive, but act as dominant-negatives, most likely by sequestering the other endogenous components in inactive complexes. Moreover, a formal proof of the requirement of p85 for Abi1-dependent activation of Rac was obtained by using fibroblasts in which both p85 isoforms (α and β) were genetically removed (unpublished data). In these cells, expression of Abi1 failed to induce Rac activation, which was, however, restored by reintroduction of p85α (Fig. 4 B). Finally, treatment with wortmannin reduced (but did not abrogate) EGF-dependent and EGF-independent Rac activation induced by Abi1 (Fig. 4 C). Similarly, the Rac-GEF activity in the Eps8–Abi1–p85–Sos-1 immunocomplex was only reduced by pretreatment of the cells with wortmannin (unpublished data). Thus, all together, these data support the notion that PI3K recruitment to the Eps8–Abi1–Sos-1 complex is physically required to elicit a basal Rac-GEF activity, which is further increased by PIP3.

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