Figure 1.
Palladin is phosphorylated upon growth factor stimulation.
(A) Indicated cells were serum-starved and stimulated with EGF (20 ng/ml) or PDGF (10 ng/ml). Five min after the stimulation, cells were lysed and probed with anti-palladin or anti-HA antibody. Arrows indicate mobility shifted palladin. (B) Serum-starved MCF10A and 293T/HA-wt cells were treated with EGF and 5 min later cells were lysed with TNE buffer. The lysates were immunoprecipitated with either anti-palladin or anti-HA antibody and incubated with or without alkaline phosphatase for 30 min. Immunoprecipitates were subjected to immunoblotting with the indicated antibody. Arrows indicate mobility shifted palladin. (C) 293T/HA-wt cells were serum-starved and treated or non-treated with EGF for 5 min and then immmunoprecipitated with anti-HA antibody. The immunoprecipitates were probed with the indicated antibodies. Whole cell extract of v-Src transformed cells (SR3Y1) was used as a control for anti-phospho-tyrosine antibody (PY20).
Figure 2.
Palladin phosphorylation is dependent on the MEK/ERK pathway.
(A) Serum-starved Vero cells were treated with EGF in the presence or absence of the indicated inhibitors. Five minutes after stimulation, cells were lysed and immunoblotted with anti-palladin antibody. (B) HeLa cells were treated as in (A) and immunblotted with anti-palladin antibody. Arrows indicate 90 kDa and 140 kDa form of palladin. (C) Cos7 cells were transfected with HA-palladin together with or without active MEK (MEK1/EEΔN3). Twenty-four hours later, cells were lysed and immunoblotted with the indicated antibodies. (D) MDA-MB-231 cells were cultured on glass coverslips and stimulated with EGF for 5 min and then fixed to immunostain for palladin (green) and phospho-ERK (red). Pictures were taken using an A1Rsi confocal microscopy (Nikon). Images in the bottom panel are partially enlarged images of the squared fields of the pictures. (Upper panels; scale bar = 50 µm, Lower panels; scale bar = 15 µm).
Figure 3.
Ser197 is phosphorylated by EGF stimulation.
(A) Schematic representation of palladin deletion constructs. (B) Mobility shift of mutant palladin after EGF stimulation was examined by western blot. Vero cells were transfected with Myc-tagged mutant palladin. Twenty-four hours later, serum-starved cells were stimulated with or without EGF for 5 min and lysed for western blot analysis. Lower panel shows ERK phosphorylation. (C) Mobility shift of mutant palladin after EGF stimulation was examined by western blotting. (D) Mobility shifts of Δ190-wt and Δ190-S190G after EGF stimulation were examined by western blotting. Amino acid sequence from 190–197 is indicated.
Figure 4.
Ser77 is phosphorylated by EGF stimulation.
(A) Vero cells were transfected with the indicated mutant palladin. Twenty-four hours later, serum-starved cells were stimulated with EGF for 5 min. Mobility shifts were examined by western blotting. (B) Mobility shifts of Δ44-S197G and Δ102-S197G were examined by western blotting. (C) Amino acid sequence from 45 to 102 is indicated. Mobility shift of the indicated mutant palladin was examined by western blotting. (D) Mobility shift of the indicated mutant palladin was examined by western blotting. (E) Vero cells were transfected with either FL-wt or FL-S77.197G palladin. Twenty-four hours later, cells were stimulated with EGF for 5 min. Cells were lysed and immunoprecipited with anti-Myc antibody and blotted with anti-MPM2 antibody.
Figure 5.
ERK phosphorylates palladin in an in vitro kinase assay.
GST-fused residues 45–249 of wild-type or mutant palladin was incubated with active ERK for 10 min at 37°C in the presence of radioactive [γ32−P]-ATP and separated by SDS-PAGE. Left panel shows the phosphorylation of recombinant proteins, and the right panel shows Coomassie blue staining of recombinant proteins.
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.
Figure 7.
Phosphorylation of Ser77 and Ser197 regulates palladin association with Abl.
(A) Serum-starved 293T/HA-wt cells were either stimulated or non-stimulated with EGF for 5 min, and cell lysates were affinity precipitated with the indicated recombinant proteins (SH3-Abl; aa66–118, SH-ArgBP2; aa432–666). Lower panels show the Coomassie blue staining of recombinant proteins. (B) HEK 293T cells were transfected with either HA-tagged palladin (HA-wt) or HA-tagged S77.197G palladin (HA-S77.197G). Twenty-four hours later, cells were lysed and affinity precipitated with GST-SH3-Abl protein bound to glutathione agarose. Immunoprecipitates were blotted with anti-HA antibody. Lower panel shows the Coomassie blue staining of the GST-SH3 Abl protein. (C) Serum-starved 293T/HA-S77.197G cells were either stimulated or non-stimulated with EGF for 5 min, and cell lysates were affinity precipitated with GST-SH3-Abl protein bound to glutathione agarose. Immunoprecipitates were blotted with anti-HA antibody. (D) MDA-MB-231 cells were lysed and immunprecipitated with either control IgG or anti-palladin antibody. The lysates were blotted with anti-Abl or anti-palladin antibody. The arrow indicates Abl and the arrowheads indicate palladin. The band around 140 kDa is an alternative form of palladin. (E) MDA-MB-231 cells were serum-starved and stimulated with or without EGF and lysed for immunopreciptation. The lyates were blotted with anti-Abl and anti-palladin antibodies. The graph shows the relative band intensity of immunoprecipitated Abl from three independent experiments. (F) Migration of shPal/GFP-wt and shPal/GFP-S77.197G cells (MDA-MB-231) was examined in the presence of indicated concentrations of STI571. Relative ratio of migrated cells was normalized by the number of migrating cells in the absence of STI571. Three independent experiments were performed and the data are shown as mean ±SD. (G) shPal/GFP-wt and shPal/GFP-S77.197G cells (MDA-MB-231) were transfected with either luciferase (Luc) or Abl siRNA and 48 h later, cell migration was examined. Relative ratio of migrated cells was normalized by the number of migrating shPal/GFP-wt cells transfected with Luc siRNA. Three independent experiments were performed and the data are shown mean ±SD (*P<0.01). Expression of Abl after siRNA transfection was examined by immunoblot. (H) Schematic presentation of regulation of cell migration by palladin phosphorylation.