Figure 4: Rab14 controls cell repulsion and LMTK3 phosphorylates RCP at Ser435. | Nature Communications

Figure 4: Rab14 controls cell repulsion and LMTK3 phosphorylates RCP at Ser435.

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

Figure 4

(a) H1299 cells were transfected with siRNAs targeting Rab14 (si-Rab14), Rab11 (si-Rab11) or a non-targeting control (si-Nt) and cell scattering was determined as for Fig. 2d,e. Representative trackplots from these experiments are displayed and cell scattering was quantified as for Fig. 2e and is expressed as the accumulated distance travelled over 6 h. 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. Bar, 50 μm. (b) PC3 cells were transfected with siRNAs targeting Rab14, Rab11 or a non-targeting control (si-Nt) and the contact time during collisions (left panel) and migration speed between collisions (right panel) was determined as for Fig. 2a,b. 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 collisions being tracked per condition. (c,d) H1299 cells were transfected with GFP, GFP-RCP or GFP-RCP435A in combination with siRNAs targeting LMTK3 (si-LMTK3), LMTK1 (si-LMTK1) or non-targeting control (si-Nt) and plated onto 15 cm plastic dishes. In the right panel of d cells were transfected with siRNA targeting LMTK3 (si-LMTK3) or non-targeting control in the absence of GFP or GFP-tagged RCPs. Forty-eight hours following transfection, cells were treated with HGF (10 ng ml−1) for the indicated times or were left untreated (0 min). Cells were lysed in a buffer containing 0.15% Tween-20 and GFP or EphA2 immunoprecipitated (IP) as for Fig. 1c. The immunoprecipitates were analysed by immunoblotting using antibodies recognizing EphA2, Rab14, Rab11, phosphoSer435RCP and RCP. The quantity of phosphoSer435RCP in the immunoblots was estimated by densitometric scanning (c; right panel). Data are mean±s.e.m. from six independent experiments. ***P<0.001, Mann-Whitney test. Uncropped blots corresponding to the experiments presented in c,d are displayed in Supplementary Fig. 9.

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