PDK1 controls cell migration through its PIF-binding pocket and PH domain in a kinase-independent manner. (A) Schematic structure of PDK1_WT and its functional mutants: PDK1_KD (kinase dead, K111N), PDK1_K465E (point mutation that impairs the ability of the PH domain to bind PIP3), PDK1_L155E (point mutation in the PIF-binding pocket of PDK1 that abrogates the binding to the HM of its substrates), PDK1_ΔPH (lacking the PH domain, amino acids from 451 to 556), PDK1_Δ50 (lacking amino acids from 1 to 50), and PDK1_ K465EL155E (which harbors point mutations of both the PIF-binding pocket and PH domain). (B) MCF10A, transduced with lentiviral vectors coding for PDK1 functional mutants, were assayed for the migration toward 5 ng/ml EGF in comparison to empty vector–transduced cells. PDK1 mutant expression was checked by immunoblotting. Each error bar represents the mean and standard error of the mean of 10 independent experiments. (C) MCF10A cells overexpressing PDK1_WT, PDK1_KD, PDK1_L155E, PDK1_ΔPH, and PDK1_Δ50 or infected with the empty vector were tracked during the wound healing assay. (D and E) Effects of PDK1 mutants overexpression on the FMI (D) and persistence (E). FMI and persistence are represented as box plot distribution of >100 cells in three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.