Role of Palladin Phosphorylation by Extracellular Signal-Regulated Kinase in Cell Migration
Figure 6
Phosphorylation of Ser77 and Ser197 controls cell migration.
(A) Cells were cultured on glass coverslips for 24 h and fixed with paraformaldehyde. Cells were stained with rhodamine-conjugated phalloidin to visualize the actin cytoskeleton. (Scale bar = 15 µm) To quantitate cells with stress fiber formation, 50 cells for each cell line were evaluated and the graph shows the percentage of cells with stress fiber formation. Three independent experiments were performed. Each bar represents mean ± SD. (N.S; P>0.05, *P<0.01) (B) Cos7 cells were transfected with either GFP-wt or GFP-S77.197G palladin. Twenty-four hours later, cells were fixed and stained with rhodamine-conjugated phalloidin. (Scale bar = 15 µm) To quantitate cells with thick stress fiber formation, 30 transfected cells were evaluated and the graph shows the percentage of cells with thick stress fiber formation. Three independent experiments were performed. Each bar represents mean ± SD. (N.S; P>0.05) (C) Cell migration was examined using a modified Boyden chamber. Three independent experiments were performed, and relative ratios of migrated cells are indicated (means±SD; *P<0.01). (D) Cell migration was examined using a modified Boyden chamber. Three independent experiments were performed, and relative ratios of migrated cells are indicated (means ±SD; *P<0.01). (E) shPal/GFP-wt and shPal/GFP-S77.197G cells (MDA-MB-231) were serum-starved and stimulated with EGF for the indicated time points. Cells were lysed and incubated with GST-fused GST-PAK-PBD fusion protein bound to glutathione-agarose beads. Beads were subjected to western blot with anti-Rac or anti-Cdc42 antibody. Total Rac or cdc42 protein was detected by immunoblotting of cell lysates.