Fig. 7: PLEKHH2 binds β-arrestin1 through its FERM domain, competitively inhibits β-arrestin1 interaction with FAK, and promotes FAK phosphorylation.

A, B Schematic diagram of PLEKHH2 and PLEKHH2-FERM splicing mutants. Compared to the full-length PLEKHH2, PLEKHH2-FERM only has the isolated FERM domain. C PLEKHH2 overexpression attenuates the binding between β-arrestin1 and FAK. In the A549 cell line, the PLEKHH2 plasmid (0 μg, 0.5 μg, 1.5 μg, 3 μg, and 5 μg) was transfected in a dose-dependent manner. β-arrestin1 was used for immunoprecipitation. The binding of β-arrestin1 to PLEKHH2 gradually increased, while the association of β-arrestin1 and FAK was gradually decreased (Statistical analysis is shown in Supplementary Fig. 7E). Moreover, PLEKHH2 overexpression increased FAK phosphorylation gradually. D FERM domain (0 μg, 0.5 μg, 1.5 μg, 3 μg, and 5 μg) was transfected into A549 cells. It exhibited gradually increase in binding with β-arrestin1 and gradually decrease in binding with β-arrestin1 and FAK (Statistical analysis is shown in Supplementary Fig. 7F). It also promoted p-FAK level in a dose gradient manner. Both PLEKHH2 and its FERM had no effect on FAK expression. Similar results are seen in H1299 cell line (E, F, Supplementary Fig. 7G and H). Increased p-FAK was compared to the cells with no transfection. P < 0.05 indicates statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001.