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Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation.

Mouneimne G, Soon L, DesMarais V, Sidani M, Song X, Yip SC, Ghosh M, Eddy R, Backer JM, Condeelis J - J. Cell Biol. (2004)

Bottom Line: Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient.Phosphoinositide-3 kinase selectively regulates the late barbed end transient.Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. gmouneim@aecom.yu.edu

ABSTRACT
The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

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PLCγ activity peaks at 60 s after EGF stimulation in MTLn3 cells. (A) The plot of p(Y)-PLCγ levels, standardized over total IgG levels (from the same blot), versus time after EGF addition. Error bars are SEM of averages of three independent experiments. (B) Representative Western blot of PLCγ [pY783]. (C) Effect of the PLC inhibitor (U73122) as compared with control (the inactive isoform, U73343).
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fig2: PLCγ activity peaks at 60 s after EGF stimulation in MTLn3 cells. (A) The plot of p(Y)-PLCγ levels, standardized over total IgG levels (from the same blot), versus time after EGF addition. Error bars are SEM of averages of three independent experiments. (B) Representative Western blot of PLCγ [pY783]. (C) Effect of the PLC inhibitor (U73122) as compared with control (the inactive isoform, U73343).

Mentions: Previous studies indicated that PLCγ-dependent signaling pathways are important for cell motility (Kassis et al., 1999; Piccolo et al., 2002). This finding induced us to study the role of PLCγ in the EGF-induced free barbed end transients. We started by examining the activation of PLCγ in response to EGF stimulation. PLC activity is regulated by tyrosine phosphorylation (Rebecchi and Pentyala, 2000). Therefore, to measure the amount of activated PLC at several time points after EGF addition, we performed an immunoprecipitation using antiphosphotyrosine antibodies. The levels of PLC in the anti-P(Y) immunoprecipitates were then detected by Western blot using an anti-PLCγ1 antibody. The immunoblot band intensities were quantitated and standardized over the intensities of the corresponding IgG bands from the same blot (Fig. 2 A). These results show that PLCγ phosphorylation increases, by twofold, in response to EGF, peaks at 1 min, and drops to unstimulated levels by 2 min after stimulation. Treatment with U73122, a synthetic inhibitor drug specific for PLC (Bleasdale et al., 1989), suppresses the activity of PLC to basal levels (Fig. 2 C). Alternatively, anti-PLCγ [pY783] Western blotting was performed, and standardization over total PLCγ revealed that the levels of PLCγ [pY783] follow the same pattern observed by immunoprecipitation (Fig. 2 B). In both experiments, there was temporal concurrence between PLCγ activation and the early barbed end transient.


Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation.

Mouneimne G, Soon L, DesMarais V, Sidani M, Song X, Yip SC, Ghosh M, Eddy R, Backer JM, Condeelis J - J. Cell Biol. (2004)

PLCγ activity peaks at 60 s after EGF stimulation in MTLn3 cells. (A) The plot of p(Y)-PLCγ levels, standardized over total IgG levels (from the same blot), versus time after EGF addition. Error bars are SEM of averages of three independent experiments. (B) Representative Western blot of PLCγ [pY783]. (C) Effect of the PLC inhibitor (U73122) as compared with control (the inactive isoform, U73343).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172433&req=5

fig2: PLCγ activity peaks at 60 s after EGF stimulation in MTLn3 cells. (A) The plot of p(Y)-PLCγ levels, standardized over total IgG levels (from the same blot), versus time after EGF addition. Error bars are SEM of averages of three independent experiments. (B) Representative Western blot of PLCγ [pY783]. (C) Effect of the PLC inhibitor (U73122) as compared with control (the inactive isoform, U73343).
Mentions: Previous studies indicated that PLCγ-dependent signaling pathways are important for cell motility (Kassis et al., 1999; Piccolo et al., 2002). This finding induced us to study the role of PLCγ in the EGF-induced free barbed end transients. We started by examining the activation of PLCγ in response to EGF stimulation. PLC activity is regulated by tyrosine phosphorylation (Rebecchi and Pentyala, 2000). Therefore, to measure the amount of activated PLC at several time points after EGF addition, we performed an immunoprecipitation using antiphosphotyrosine antibodies. The levels of PLC in the anti-P(Y) immunoprecipitates were then detected by Western blot using an anti-PLCγ1 antibody. The immunoblot band intensities were quantitated and standardized over the intensities of the corresponding IgG bands from the same blot (Fig. 2 A). These results show that PLCγ phosphorylation increases, by twofold, in response to EGF, peaks at 1 min, and drops to unstimulated levels by 2 min after stimulation. Treatment with U73122, a synthetic inhibitor drug specific for PLC (Bleasdale et al., 1989), suppresses the activity of PLC to basal levels (Fig. 2 C). Alternatively, anti-PLCγ [pY783] Western blotting was performed, and standardization over total PLCγ revealed that the levels of PLCγ [pY783] follow the same pattern observed by immunoprecipitation (Fig. 2 B). In both experiments, there was temporal concurrence between PLCγ activation and the early barbed end transient.

Bottom Line: Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient.Phosphoinositide-3 kinase selectively regulates the late barbed end transient.Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA. gmouneim@aecom.yu.edu

ABSTRACT
The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

Show MeSH
Related in: MedlinePlus