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Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.

Sasaki AT, Chun C, Takeda K, Firtel RA - J. Cell Biol. (2004)

Bottom Line: Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass.These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis.A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P(3), the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P(3) asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

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Activation of Dictyostelium Ras. The FLAG-RasG or endogenous Ras activation level in response to cAMP was assayed by a GST-RBD pull-down assay (see Materials and methods). Cells were stimulated with cAMP for the indicated duration, and the amount of Ras protein bound to GST-RBD was determined by Western blotting with the indicated antibody. The activated Ras was quantified by densitometry and normalized with total Ras. Cells were treated with 50 μM LY294002 or DMSO as a control for 20 min before the addition of cAMP (C). Similar results were observed over at least three independent experiments.
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fig2: Activation of Dictyostelium Ras. The FLAG-RasG or endogenous Ras activation level in response to cAMP was assayed by a GST-RBD pull-down assay (see Materials and methods). Cells were stimulated with cAMP for the indicated duration, and the amount of Ras protein bound to GST-RBD was determined by Western blotting with the indicated antibody. The activated Ras was quantified by densitometry and normalized with total Ras. Cells were treated with 50 μM LY294002 or DMSO as a control for 20 min before the addition of cAMP (C). Similar results were observed over at least three independent experiments.

Mentions: The partial requirement of RasG for PI3K activation suggests that RasG and possibly other Dictyostelium Ras proteins are activated in response to chemoattractant stimulation. To examine this suggestion, we used a GST-RBD of Raf1 in a pull-down assay that has been used to determine Ras activation in mammalian cells (Taylor et al., 2001). The rationale for using the RBD of Raf1 was that Dictyostelium RasG (and RasB and RasD) and mammalian H-Ras and K-Ras have identical effector domains (Lim et al., 2002). The RBD of Raf1 bound strongly to Dictyostelium constitutively activated (Q61L) RasG, RasB, and RasD but not to their dominant negative forms (S17N) in yeast two-hybrid and in vitro binding assays (unpublished data). Interestingly, RasGQ61L bound more strongly to the Raf1 RBD than to either the PI3K1 or PI3K2 RBD (unpublished data). Fig. 2 A illustrates that the RasG activation level is rapidly stimulated in response to cAMP, peaking at 5 s, after which the level of RasG-GTP rapidly decreases. The same results were obtained by using rasG cells expressing GFP-tagged RasG (unpublished data). The kinetics of activation and subsequent adaptation are consistent with those of cAMP-stimulated PI3K activity (Huang et al., 2003). While we were preparing our manuscript for submission, an independent report also demonstrated chemoattractant-mediated RasG activation using the RBD of Byr2 (Kae et al., 2004; see Discussion).


Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.

Sasaki AT, Chun C, Takeda K, Firtel RA - J. Cell Biol. (2004)

Activation of Dictyostelium Ras. The FLAG-RasG or endogenous Ras activation level in response to cAMP was assayed by a GST-RBD pull-down assay (see Materials and methods). Cells were stimulated with cAMP for the indicated duration, and the amount of Ras protein bound to GST-RBD was determined by Western blotting with the indicated antibody. The activated Ras was quantified by densitometry and normalized with total Ras. Cells were treated with 50 μM LY294002 or DMSO as a control for 20 min before the addition of cAMP (C). Similar results were observed over at least three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172490&req=5

fig2: Activation of Dictyostelium Ras. The FLAG-RasG or endogenous Ras activation level in response to cAMP was assayed by a GST-RBD pull-down assay (see Materials and methods). Cells were stimulated with cAMP for the indicated duration, and the amount of Ras protein bound to GST-RBD was determined by Western blotting with the indicated antibody. The activated Ras was quantified by densitometry and normalized with total Ras. Cells were treated with 50 μM LY294002 or DMSO as a control for 20 min before the addition of cAMP (C). Similar results were observed over at least three independent experiments.
Mentions: The partial requirement of RasG for PI3K activation suggests that RasG and possibly other Dictyostelium Ras proteins are activated in response to chemoattractant stimulation. To examine this suggestion, we used a GST-RBD of Raf1 in a pull-down assay that has been used to determine Ras activation in mammalian cells (Taylor et al., 2001). The rationale for using the RBD of Raf1 was that Dictyostelium RasG (and RasB and RasD) and mammalian H-Ras and K-Ras have identical effector domains (Lim et al., 2002). The RBD of Raf1 bound strongly to Dictyostelium constitutively activated (Q61L) RasG, RasB, and RasD but not to their dominant negative forms (S17N) in yeast two-hybrid and in vitro binding assays (unpublished data). Interestingly, RasGQ61L bound more strongly to the Raf1 RBD than to either the PI3K1 or PI3K2 RBD (unpublished data). Fig. 2 A illustrates that the RasG activation level is rapidly stimulated in response to cAMP, peaking at 5 s, after which the level of RasG-GTP rapidly decreases. The same results were obtained by using rasG cells expressing GFP-tagged RasG (unpublished data). The kinetics of activation and subsequent adaptation are consistent with those of cAMP-stimulated PI3K activity (Huang et al., 2003). While we were preparing our manuscript for submission, an independent report also demonstrated chemoattractant-mediated RasG activation using the RBD of Byr2 (Kae et al., 2004; see Discussion).

Bottom Line: Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass.These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis.A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P(3), the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P(3) asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

Show MeSH