Fig. 5: AXL interacts with PEAK1 and modulates its phosphorylation and localization.

a PEAK1 localizes with AXL in TNBC cells. Representative confocal images of MDA-MB-231 cells. PLA was used to analyze the proximity localization of PEAK1 and AXL. Scale bar, 20 μm. b PEAK1 interacts with AXL. Lysates of 293T cells expressing the indicated plasmids were used for anti-Myc immunoprecipitation. Levels of AXL is detected via western blotting. c PEAK1 is phosphorylated by AXL. Lysates of 293T cells expressing the indicated plasmids and treated or not with 1 μM R428 for 1 h were subjected to anti-Myc immunoprecipitation. Levels of Myc-PEAK1 tyrosine phosphorylation was detected via western blotting. d PEAK1 is localized at PXN FA sites. Representative confocal images of MDA-MB-231 cells treated with DMSO or 1 μM R428, GAS6, or GAS6 and R428. PLA was used to analyze proximity localization of PEAK1 at PXN-positive FAs. Scale bar, 20 μm. e Quantifications of the number of PLA puncta per cell per condition. ***P = 0.000014; ****P < 0.000001 (n = 3 experiments, 15 cells per condition per experiment). f PEAK1 recruitment to the cell membrane is AXL regulated. Representative confocal images of Hs578T cells treated with DMSO or 1 μM R428. Cells were stained for PEAK1 (green) and FAK (magenta). Boxes are used to depict the location of the zoomed images. Scale bar, 20 μm. g Quantification of mean fluorescence intensity (MFI) of PEAK1 at periphery of membrane shown in f. ****P < 0.0000001 (15 cells per condition per experiment). h Network layout of the PEAK1 BioID dataset. Surrounding subnetworks in zoom boxes exhibit selected and relevant functions, such as “FA” and “Actin” where PEAK1 seems to play a role. Node sizes represent the relative amount of GAS6-modulated phosphosites. Edge thickness represents the relative protein abundance depicted by the SAF (Spectral Abundance Factor) metric. The color of the node indicates if a PEAK1 prey has been phospho-modulated or not in the GAS6 phosphoproteomic screen (see Fig. 2). Data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value, and the lower whisker extends from the hinge to the smallest value (e, g). Two-tailed Mann–Whitney test was used to calculate P value. Source data are provided as a Source data file.