Figure 6: Phosphorylation of EphA2 at Ser897 is necessary for HGF-driven EphA2 trafficking and cell scattering. | Nature Communications

Figure 6: Phosphorylation of EphA2 at Ser897 is necessary for HGF-driven EphA2 trafficking and cell scattering.

From: Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion

Figure 6

(a) H1299 cells were transfected with a siRNA targeting EphA2 in combination with siRNA-resistant forms of EphA2-GFP or EphA2897A-GFP. Internalization of EphA2-GFPs in the presence and absence of HGF was then determined as for Fig. 3b, but with the ELISA plate coated with anti-GFP to specifically detect only the GFP-tagged receptor. Values are mean±s.e.m. n=4 biological replicates from two independent experiments. ***P<0.001 (EphA2897A-GFP+HGF versus EphA2-GFP+HGF); two-way ANOVA, Bonferroni post-test. (b) H1299 cells were transfected with EphA2-GFP or EphA2897A-GFP in combination with mCherry-RCP. Trafficking of EphA2-GFP and mCherry-RCPs in the presence and absence of HGF (added 30 min before collecting the movies) was visualized by fluorescence confocal time-lapse microscopy and co-localization was quantified as for Fig. 3e. Stills were extracted from movies at the indicated time points, and these display the region of interest indicated by the white box. Bar, 10 μm. Values are mean±s.e.m. from three separate experiments incorporating >10 cells per condition per experiment. ***P<0.001; one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparison Test). (c) H1299 cells were transfected with GFP-RCP or GFP-RCP435A, in the presence or absence of siRNAs targeting Rab14 (si-Rab14), LMTK3 (si-LMTK3) or non-targeting control (si-Nt) as indicated. Cells were incubated in the presence or absence of HGF for 30 min and then fixed and phospho-Ser897-EphA2 (EphA2pS897) and nuclei were visualized by immunofluorescence followed by confocal microscopy. Bar, 10 μm. Co-localization of GFP-RCPs and EphA2pS897 was determined as for Fig. 3e. Values are mean±s.e.m. from three separate experiments incorporating >60 cells per condition. ***P<0.001; one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparison Test). (d) H1299 cells were transfected with a non-targeting siRNA or with an siRNA targeting EphA2 (si-EphA2) in combination with siRNA-resistant forms of EphA2-GFP or EphA2897A-GFP. Cell scattering and the migration of subconfluent, non-contacting cells in the presence and absence of HGF was then determined and quantified as for Fig. 5c. Data are represented as box and whiskers plots (whiskers: 10–90 percentile,+represents the mean). ***P<0.001; one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparison Test). Data are from three independent experiments with >50 cells being tracked per condition. The expression levels of the siRNA-resistant forms of EphA2-GFP and EphA2897A-GFP were determined in EphA2 knockdown cells by western blotting with antibodies recognizing EphA2 or pSer897-EphA2. The band marked with an asterisk is recognized non-specifically by the anti- pSer897-EphA2 antibody. Uncropped blots corresponding to the experiment presented in d are displayed in Supplementary Fig. 9.

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