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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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Ras regulates PI3K signaling and distribution along the membrane. (A and B) Activation of Akt/PKB is shown. Aggregation-stage cells (see Materials and methods) were treated with 10 μM cAMP for the indicated time (A) or at 10 s (B) and then lysed, Akt was immunoprecipitated with anti-Akt antibody, and Akt activity was assayed (Meili et al., 1999). Akt protein levels were determined in each sample by Western blot analysis (bottom panels). (C) Fluorescent images of GFP-RasG/rasG  cells exposed to a chemoattractant gradient (left) or to uniform chemoattractant stimulation (right). An asterisk indicates the position of the micropipette. The numbers in the top left corners represent the time after initiation when the image was captured. The data are representative of eight separate experiments.
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fig1: Ras regulates PI3K signaling and distribution along the membrane. (A and B) Activation of Akt/PKB is shown. Aggregation-stage cells (see Materials and methods) were treated with 10 μM cAMP for the indicated time (A) or at 10 s (B) and then lysed, Akt was immunoprecipitated with anti-Akt antibody, and Akt activity was assayed (Meili et al., 1999). Akt protein levels were determined in each sample by Western blot analysis (bottom panels). (C) Fluorescent images of GFP-RasG/rasG cells exposed to a chemoattractant gradient (left) or to uniform chemoattractant stimulation (right). An asterisk indicates the position of the micropipette. The numbers in the top left corners represent the time after initiation when the image was captured. The data are representative of eight separate experiments.

Mentions: To test whether or not RasG is involved in PI3K activation, we measured the activation of the PI3K effector Akt/PKB in rasG cells in response to chemoattractant stimulation. Fig. 1 A shows that chemoattractant-induced Akt/PKB activation was decreased in rasG cells, and this effect was complemented by expressing FLAG- or GFP-tagged wild-type RasG cDNA from a constitutive actin promoter in rasG cells (Fig. 1 B and not depicted). FLAG- and GFP-RasG also complemented the rasG cell cytokinesis defect (unpublished data). GFP-RasG displayed uniform localization along the plasma membrane and cytosol in chemotaxing cells, unstimulated cells, and cells that were stimulated globally with a uniform, rapidly applied, saturating level of chemoattractant (Fig. 1 C). As rasG cells only show a partial reduction in Akt/PKB activation, we expect other Ras proteins are involved in PI3K activation.


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)

Ras regulates PI3K signaling and distribution along the membrane. (A and B) Activation of Akt/PKB is shown. Aggregation-stage cells (see Materials and methods) were treated with 10 μM cAMP for the indicated time (A) or at 10 s (B) and then lysed, Akt was immunoprecipitated with anti-Akt antibody, and Akt activity was assayed (Meili et al., 1999). Akt protein levels were determined in each sample by Western blot analysis (bottom panels). (C) Fluorescent images of GFP-RasG/rasG  cells exposed to a chemoattractant gradient (left) or to uniform chemoattractant stimulation (right). An asterisk indicates the position of the micropipette. The numbers in the top left corners represent the time after initiation when the image was captured. The data are representative of eight separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Ras regulates PI3K signaling and distribution along the membrane. (A and B) Activation of Akt/PKB is shown. Aggregation-stage cells (see Materials and methods) were treated with 10 μM cAMP for the indicated time (A) or at 10 s (B) and then lysed, Akt was immunoprecipitated with anti-Akt antibody, and Akt activity was assayed (Meili et al., 1999). Akt protein levels were determined in each sample by Western blot analysis (bottom panels). (C) Fluorescent images of GFP-RasG/rasG cells exposed to a chemoattractant gradient (left) or to uniform chemoattractant stimulation (right). An asterisk indicates the position of the micropipette. The numbers in the top left corners represent the time after initiation when the image was captured. The data are representative of eight separate experiments.
Mentions: To test whether or not RasG is involved in PI3K activation, we measured the activation of the PI3K effector Akt/PKB in rasG cells in response to chemoattractant stimulation. Fig. 1 A shows that chemoattractant-induced Akt/PKB activation was decreased in rasG cells, and this effect was complemented by expressing FLAG- or GFP-tagged wild-type RasG cDNA from a constitutive actin promoter in rasG cells (Fig. 1 B and not depicted). FLAG- and GFP-RasG also complemented the rasG cell cytokinesis defect (unpublished data). GFP-RasG displayed uniform localization along the plasma membrane and cytosol in chemotaxing cells, unstimulated cells, and cells that were stimulated globally with a uniform, rapidly applied, saturating level of chemoattractant (Fig. 1 C). As rasG cells only show a partial reduction in Akt/PKB activation, we expect other Ras proteins are involved in PI3K activation.

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