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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 Akt/PKB and Ras. The effects of RasGS17N on Akt/PKB, Ras activation, and production of PI(3,4,5)P3 in rasG (A) and aleA (B)  cells were assayed as described in the legends to Figs. 1–4. The results were similar in five independent assays.
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fig7: Activation of Akt/PKB and Ras. The effects of RasGS17N on Akt/PKB, Ras activation, and production of PI(3,4,5)P3 in rasG (A) and aleA (B) cells were assayed as described in the legends to Figs. 1–4. The results were similar in five independent assays.

Mentions: Dominant-negative forms of small GTPases inhibit downstream function by blocking the activity of their cognate exchange factor (GDP/GTP exchange factor [GEF]). To further examine the roles of Ras in chemotaxis, we expressed dominant-negative RasGS17N in rasG cells, which should block the activation of other Ras proteins that are regulated by GEFs sensitive to inhibition by RasGS17N. As expected if expression of RasGS17N were to inhibit Ras activation and thus decrease the activity of a downstream effector such as PI3K, we observed that activation of Ras, Akt/PKB, and PI(3,4,5)P3 production (as illustrated by PhdA membrane localization) in RasGS17N/rasG cells was significantly reduced (but not eliminated) compared with rasG cells (Fig. 7 A). Fig. 7 A also shows that as one titrates down Ras function (wild-type, rasG , and RasGS17N/rasG cells), there is a decrease in chemoattractant-mediated F-actin polymerization. When chemotaxis was assayed, RasGS17N/rasG cells produced numerous small pseudopodia at right or oblique angles to the direction of the cAMP gradient and displayed severe polarization and migration defects with very little forward movement (Fig. 6 C and Video 7, available at http:www.jcb.org/cgi/content/full/jcb.200406177/DC1). These results suggest that, in addition to RasG, other Ras proteins play an important role in controlling the ability of cells to polarize and move. Our observation that these cells produce multiple pseudopodia on all sides when placed in a chemoattractant gradient suggests that the cells cannot effectively sense direction. These cells are able to polymerize F-actin, as illustrated by the ability to extend pseudopodia, but the absolute level of chemoattractant-mediated F-actin polymerization is reduced (Fig. 7 A).


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 Akt/PKB and Ras. The effects of RasGS17N on Akt/PKB, Ras activation, and production of PI(3,4,5)P3 in rasG (A) and aleA (B)  cells were assayed as described in the legends to Figs. 1–4. The results were similar in five independent assays.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172490&req=5

fig7: Activation of Akt/PKB and Ras. The effects of RasGS17N on Akt/PKB, Ras activation, and production of PI(3,4,5)P3 in rasG (A) and aleA (B) cells were assayed as described in the legends to Figs. 1–4. The results were similar in five independent assays.
Mentions: Dominant-negative forms of small GTPases inhibit downstream function by blocking the activity of their cognate exchange factor (GDP/GTP exchange factor [GEF]). To further examine the roles of Ras in chemotaxis, we expressed dominant-negative RasGS17N in rasG cells, which should block the activation of other Ras proteins that are regulated by GEFs sensitive to inhibition by RasGS17N. As expected if expression of RasGS17N were to inhibit Ras activation and thus decrease the activity of a downstream effector such as PI3K, we observed that activation of Ras, Akt/PKB, and PI(3,4,5)P3 production (as illustrated by PhdA membrane localization) in RasGS17N/rasG cells was significantly reduced (but not eliminated) compared with rasG cells (Fig. 7 A). Fig. 7 A also shows that as one titrates down Ras function (wild-type, rasG , and RasGS17N/rasG cells), there is a decrease in chemoattractant-mediated F-actin polymerization. When chemotaxis was assayed, RasGS17N/rasG cells produced numerous small pseudopodia at right or oblique angles to the direction of the cAMP gradient and displayed severe polarization and migration defects with very little forward movement (Fig. 6 C and Video 7, available at http:www.jcb.org/cgi/content/full/jcb.200406177/DC1). These results suggest that, in addition to RasG, other Ras proteins play an important role in controlling the ability of cells to polarize and move. Our observation that these cells produce multiple pseudopodia on all sides when placed in a chemoattractant gradient suggests that the cells cannot effectively sense direction. These cells are able to polymerize F-actin, as illustrated by the ability to extend pseudopodia, but the absolute level of chemoattractant-mediated F-actin polymerization is reduced (Fig. 7 A).

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