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Local signaling by the EGF receptor.

Kempiak SJ, Yip SC, Backer JM, Segall JE - J. Cell Biol. (2003)

Bottom Line: We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression.The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function.Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY 10461, USA.

ABSTRACT
Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

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The EGF bead response is PI3-kinase and rho family GTPase independent. (A) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO or 100 nM Wortmannin for 15 min. Cells recovered for 1 h, before being treated with either EGF beads for 5 min (A) or 10 nM soluble EGF (B) for 3 min. (A, left) The percentage of cells responding to beads treated with Wortmannin or DMSO. Data represent the mean ± SEM from >120 cells in three separate experiments. (A, right) Wortmannin effects on actin polymerization and phosphoAkt near beads. BSA beads (white), or EGF beads (black and gray), were given to cells that were pretreated with DMSO (white, black) or 100 nM Wortmannin (gray). The relative increase in staining for F-actin and pAkt near the beads compared with plasma membranes far from the beads was measured. Data represent the mean ± SEM from >60 cells in two or more separate experiments. (B) Cells untreated or treated with 10 nM soluble EGF and Wortmannin were lysed, and the whole cell lysates were probed for pAkt. (C) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO (white) or 50 ng/ml Toxin B (gray) for 1 h. The cells were then stimulated for 80 s, and the response was then analyzed. Data represent the mean ± SEM from >120 cells in four or more separate experiments. (D) Cells untreated or treated for 1 min with 5 nM soluble EGF and Toxin B were lysed and probed for activated rac by GST-CRIB pulldown (top) or total rac content (bottom) as described in Materials and methods.
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fig4: The EGF bead response is PI3-kinase and rho family GTPase independent. (A) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO or 100 nM Wortmannin for 15 min. Cells recovered for 1 h, before being treated with either EGF beads for 5 min (A) or 10 nM soluble EGF (B) for 3 min. (A, left) The percentage of cells responding to beads treated with Wortmannin or DMSO. Data represent the mean ± SEM from >120 cells in three separate experiments. (A, right) Wortmannin effects on actin polymerization and phosphoAkt near beads. BSA beads (white), or EGF beads (black and gray), were given to cells that were pretreated with DMSO (white, black) or 100 nM Wortmannin (gray). The relative increase in staining for F-actin and pAkt near the beads compared with plasma membranes far from the beads was measured. Data represent the mean ± SEM from >60 cells in two or more separate experiments. (B) Cells untreated or treated with 10 nM soluble EGF and Wortmannin were lysed, and the whole cell lysates were probed for pAkt. (C) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO (white) or 50 ng/ml Toxin B (gray) for 1 h. The cells were then stimulated for 80 s, and the response was then analyzed. Data represent the mean ± SEM from >120 cells in four or more separate experiments. (D) Cells untreated or treated for 1 min with 5 nM soluble EGF and Toxin B were lysed and probed for activated rac by GST-CRIB pulldown (top) or total rac content (bottom) as described in Materials and methods.

Mentions: Chemotaxing leukocytes and Dictyostelium utilize PI3-kinase to polarize toward their respective ligands (Iijima et al., 2002; Weiner et al., 2002). EGF-induced lamellipods are also dependent on PI3-kinase (Hill et al., 2000). It is possible that a positive feedback loop composed of PI3-kinase and rho family proteins could lead to localized amplification of actin polymerization responses. Therefore, MTLn3:EGFR cells were treated with wortmannin, a PI3-kinase inhibitor. There was no decrease in the number of positive responses (determined by phase–contrast or phalloidin staining) with wortmannin treatment (Fig. 4 A, left). As controls, the levels of phosphoAkt induced by either soluble EGF (Fig. 4 B) or EGF beads (Fig. 4 A, right) were measured and found to be decreased in the presence of wortmannin, confirming inhibition of PI3-kinase. Similar results were obtained with MTLn3:PLXSN cells (unpublished data). To determine the contributions of rho family proteins to bead-induced actin polymerization, we treated cells with C. difficile toxin B, a potent inhibitor of Rho, Rac, and Cdc42 proteins. Toxin B treatment did not block the EGF bead–induced actin polymerization response (Fig. 4 C), although rac activation by EGF was blocked (Fig. 4 D). Introduction of dominant–negative rac or cdc42 by transfection of cDNA constructs or microinjection of protein also had no effect (unpublished data). Thus, rho family proteins, PI3-kinase, and phosphoAkt are not necessary for generation of a localized actin polymerization response.


Local signaling by the EGF receptor.

Kempiak SJ, Yip SC, Backer JM, Segall JE - J. Cell Biol. (2003)

The EGF bead response is PI3-kinase and rho family GTPase independent. (A) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO or 100 nM Wortmannin for 15 min. Cells recovered for 1 h, before being treated with either EGF beads for 5 min (A) or 10 nM soluble EGF (B) for 3 min. (A, left) The percentage of cells responding to beads treated with Wortmannin or DMSO. Data represent the mean ± SEM from >120 cells in three separate experiments. (A, right) Wortmannin effects on actin polymerization and phosphoAkt near beads. BSA beads (white), or EGF beads (black and gray), were given to cells that were pretreated with DMSO (white, black) or 100 nM Wortmannin (gray). The relative increase in staining for F-actin and pAkt near the beads compared with plasma membranes far from the beads was measured. Data represent the mean ± SEM from >60 cells in two or more separate experiments. (B) Cells untreated or treated with 10 nM soluble EGF and Wortmannin were lysed, and the whole cell lysates were probed for pAkt. (C) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO (white) or 50 ng/ml Toxin B (gray) for 1 h. The cells were then stimulated for 80 s, and the response was then analyzed. Data represent the mean ± SEM from >120 cells in four or more separate experiments. (D) Cells untreated or treated for 1 min with 5 nM soluble EGF and Toxin B were lysed and probed for activated rac by GST-CRIB pulldown (top) or total rac content (bottom) as described in Materials and methods.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172819&req=5

fig4: The EGF bead response is PI3-kinase and rho family GTPase independent. (A) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO or 100 nM Wortmannin for 15 min. Cells recovered for 1 h, before being treated with either EGF beads for 5 min (A) or 10 nM soluble EGF (B) for 3 min. (A, left) The percentage of cells responding to beads treated with Wortmannin or DMSO. Data represent the mean ± SEM from >120 cells in three separate experiments. (A, right) Wortmannin effects on actin polymerization and phosphoAkt near beads. BSA beads (white), or EGF beads (black and gray), were given to cells that were pretreated with DMSO (white, black) or 100 nM Wortmannin (gray). The relative increase in staining for F-actin and pAkt near the beads compared with plasma membranes far from the beads was measured. Data represent the mean ± SEM from >60 cells in two or more separate experiments. (B) Cells untreated or treated with 10 nM soluble EGF and Wortmannin were lysed, and the whole cell lysates were probed for pAkt. (C) MTLn3:EGFR cells were starved for 1 h, and then treated with DMSO (white) or 50 ng/ml Toxin B (gray) for 1 h. The cells were then stimulated for 80 s, and the response was then analyzed. Data represent the mean ± SEM from >120 cells in four or more separate experiments. (D) Cells untreated or treated for 1 min with 5 nM soluble EGF and Toxin B were lysed and probed for activated rac by GST-CRIB pulldown (top) or total rac content (bottom) as described in Materials and methods.
Mentions: Chemotaxing leukocytes and Dictyostelium utilize PI3-kinase to polarize toward their respective ligands (Iijima et al., 2002; Weiner et al., 2002). EGF-induced lamellipods are also dependent on PI3-kinase (Hill et al., 2000). It is possible that a positive feedback loop composed of PI3-kinase and rho family proteins could lead to localized amplification of actin polymerization responses. Therefore, MTLn3:EGFR cells were treated with wortmannin, a PI3-kinase inhibitor. There was no decrease in the number of positive responses (determined by phase–contrast or phalloidin staining) with wortmannin treatment (Fig. 4 A, left). As controls, the levels of phosphoAkt induced by either soluble EGF (Fig. 4 B) or EGF beads (Fig. 4 A, right) were measured and found to be decreased in the presence of wortmannin, confirming inhibition of PI3-kinase. Similar results were obtained with MTLn3:PLXSN cells (unpublished data). To determine the contributions of rho family proteins to bead-induced actin polymerization, we treated cells with C. difficile toxin B, a potent inhibitor of Rho, Rac, and Cdc42 proteins. Toxin B treatment did not block the EGF bead–induced actin polymerization response (Fig. 4 C), although rac activation by EGF was blocked (Fig. 4 D). Introduction of dominant–negative rac or cdc42 by transfection of cDNA constructs or microinjection of protein also had no effect (unpublished data). Thus, rho family proteins, PI3-kinase, and phosphoAkt are not necessary for generation of a localized actin polymerization response.

Bottom Line: We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression.The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function.Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY 10461, USA.

ABSTRACT
Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

Show MeSH
Related in: MedlinePlus