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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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Feedback loop–mediated Ras/PI3K activation. (A) Fluorescent images of indicated GFP-protein expressing pten  cells before or 5 min after 50-μM LY294002 treatment or 25 min after 5 μM LatA treatment, and 20 min after the removal of LatA. (B) Fluorescent images of GFP-PhdA– and RasGQ61L-expressing rasG  cells.
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fig5: Feedback loop–mediated Ras/PI3K activation. (A) Fluorescent images of indicated GFP-protein expressing pten cells before or 5 min after 50-μM LY294002 treatment or 25 min after 5 μM LatA treatment, and 20 min after the removal of LatA. (B) Fluorescent images of GFP-PhdA– and RasGQ61L-expressing rasG cells.

Mentions: Although initial Ras activation requires neither PI3K activity nor F-actin polymerization, the Ras activation level was reduced in the presence of LY294002 or LatA (Figs. 3 and 4). Furthermore, Ras was spontaneously activated at multiple sites in randomly moving (no chemoattractant) pten cells. Those observations suggest that Ras itself may be activated, in part, by events involving PI(3,4,5)P3 and F-actin polymerization. To examine this hypothesis, we treated randomly moving pten cells with LY294002. After the drug treatment, both spontaneous PhdA accumulation at the membrane and the formation of multiple pseudopodia were completely abolished (Fig. 5 A). The result is consistent with high PI(3,4,5)P3 inducing F-actin polymerization in pten cells PI3K (Iijima et al., 2004). Strikingly, spontaneous PI3K localization and Ras activation at multiple pseudopodia also completely disappeared after LY294002 treatment. The results suggest that, under some conditions such as “stochastic activation” during random movement, PI3K may regulate Ras activation and PI3K localization through PI(3,4,5)P3-mediated signaling. We further examined the possible involvement of F-actin polymerization in activating Ras and PI3K in randomly moving pten cells. The “spontaneous” (no external stimulation) Ras activation, PIP3 accumulation, and PI3K localization gradually decreased in pten cells after LatA addition. The loss of these responses coincided with an inability to make new F-actin, as judged by a complete smoothness of the cell surface. After removal of the LatA, cells regained the spontaneous Ras activation, PI(3,4,5)P3 accumulation, and PI3K localization at sites of F-actin accumulation (protrusions; Fig. 5 A). The findings suggest that F-actin is important for observable autonomous Ras and PI3K activation. As Ras is required for PI3K activation, we suggest that Ras and PI3K activation are tightly connected through a positive feedback loop.


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)

Feedback loop–mediated Ras/PI3K activation. (A) Fluorescent images of indicated GFP-protein expressing pten  cells before or 5 min after 50-μM LY294002 treatment or 25 min after 5 μM LatA treatment, and 20 min after the removal of LatA. (B) Fluorescent images of GFP-PhdA– and RasGQ61L-expressing rasG  cells.
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fig5: Feedback loop–mediated Ras/PI3K activation. (A) Fluorescent images of indicated GFP-protein expressing pten cells before or 5 min after 50-μM LY294002 treatment or 25 min after 5 μM LatA treatment, and 20 min after the removal of LatA. (B) Fluorescent images of GFP-PhdA– and RasGQ61L-expressing rasG cells.
Mentions: Although initial Ras activation requires neither PI3K activity nor F-actin polymerization, the Ras activation level was reduced in the presence of LY294002 or LatA (Figs. 3 and 4). Furthermore, Ras was spontaneously activated at multiple sites in randomly moving (no chemoattractant) pten cells. Those observations suggest that Ras itself may be activated, in part, by events involving PI(3,4,5)P3 and F-actin polymerization. To examine this hypothesis, we treated randomly moving pten cells with LY294002. After the drug treatment, both spontaneous PhdA accumulation at the membrane and the formation of multiple pseudopodia were completely abolished (Fig. 5 A). The result is consistent with high PI(3,4,5)P3 inducing F-actin polymerization in pten cells PI3K (Iijima et al., 2004). Strikingly, spontaneous PI3K localization and Ras activation at multiple pseudopodia also completely disappeared after LY294002 treatment. The results suggest that, under some conditions such as “stochastic activation” during random movement, PI3K may regulate Ras activation and PI3K localization through PI(3,4,5)P3-mediated signaling. We further examined the possible involvement of F-actin polymerization in activating Ras and PI3K in randomly moving pten cells. The “spontaneous” (no external stimulation) Ras activation, PIP3 accumulation, and PI3K localization gradually decreased in pten cells after LatA addition. The loss of these responses coincided with an inability to make new F-actin, as judged by a complete smoothness of the cell surface. After removal of the LatA, cells regained the spontaneous Ras activation, PI(3,4,5)P3 accumulation, and PI3K localization at sites of F-actin accumulation (protrusions; Fig. 5 A). The findings suggest that F-actin is important for observable autonomous Ras and PI3K activation. As Ras is required for PI3K activation, we suggest that Ras and PI3K activation are tightly connected through a positive feedback loop.

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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