Fig. 6: CRKII/PEAK1 binding mediates PEAK1 phosphorylation by AXL and CRKII localization at FAs.

a Schematic of PEAK1 and CRKII domains. b CRKII binds PEAK1 proline-rich region (PRR). Lysates of 293T cells expressing the indicated plasmids were used for anti-GFP immunoprecipitation. Levels of Myc-PEAK1 is detected via western blotting. c Lysates of 293T cells transfected with the indicated plasmids were subjected to anti-Myc immunoprecipitation. Myc-PEAK1 phosphorylation levels were detected via western blotting. d Lysates of 293T cells transfected with the indicated plasmids were subjected to anti-GFP immunoprecipitation. Levels of Myc-PEAK1 were detected via western blotting. e PEAK1 regulates CRKII localization at FA sites. Representative confocal images of MDA-MB-231 cells transfected with either 100 nM siCTRL or siPEAK1. PLA was used to analyze proximity localization of CRKII at FAK-positive FAs. Scale bar, 20 μm. f Quantifications of the number of PLA puncta per cell per condition. ****P < 0.000001 (n = 3 experiments, 15 cells per condition per experiment). 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. Two-tailed Mann–Whitney test was used to calculate P value. Source data are provided as a Source data file. g AXL modulates CRKII binding to PXN. Lysates of 293T cells expressing the indicated plasmids were used for anti-Myc immunoprecipitation. Levels of GFP-PXN is detected via western blotting.