FIG. 6.

Acidic phospholipids bind the basic patch within the FAK FERM domain. (A) Normalized FRET/CFP emission ratios of the wild-type FAK biosensor and the basic patch mutant are shown. The mutant FRET/CFP ratio was normalized to FRET/CFP ratio of the wild-type biosensor. Shown is a representative experiment (n = 3). (B) The purified recombinant FERM domain was incubated with BODIPY-labeled phospholipids, and binding was measured by fluorescence polarization. Anisotropy (mP, millipolarization units) is plotted against FERM domain concentration. The average of three experiments ± standard deviation is shown. (C) PE/PC vesicles containing increasing amounts of PIP2 were incubated with a GST-FERM fusion protein. The vesicles were sedimented by centrifugation, and the amount of fusion protein in the vesicle containing pellet (P) and the supernatant (S) was determined by SDS-PAGE and Coomassie blue staining. (D) PE/PC vesicles containing 10% (mass ratio) of the indicated lipids were incubated with the GST-FERM fusion protein and analyzed as in panel A. Buf, buffer. (E) PE/PC vesicles containing PIP2 or phosphatidylinositol (PI) were incubated the wild-type GST-FERM domain or a basic patch mutant (KAKTLRK) and analyzed as in panel A. (F and G) HeLa cells expressing a YFP-FERM domain fusion protein or the basic patch mutant (KAKTLRK) were fixed, permeabilized, and then stained with rhodamine-phalloidin. Fixed cells were observed by laser-scanning confocal microscopy. (F) The percentages of cells containing the YFP-FERM constructs in ruffles or at the membrane at the edge of the cell were scored. wt, wild type. Results represent the average from three experiments ± standard deviation with >100 cells counted per experiment. **, P < 0.005; *, P < 0.05. (G) Representative images of cells expressing the YFP-FERM domain (two left panels) or the YFP-FERM KAKTLRK mutant (right panel) are shown. Arrows indicate YFP-FERM localization in ruffles (left panel) and at the membrane at the periphery of the cell (middle panel).