PDK1 regulates MRCKα kinase activity. (A) MCF10A, infected with lentiviral vectors coding for shScr, shPDK1 #79, or shPDK1 #81, were assayed for the phosphorylation of MLC2 on S19 or on T18/S19 and for the phosphorylation of MyPT1 on T696 in the absence or presence of 5 ng/ml EGF stimulation for 200 s. Band intensity quantification of pS19MLC2 pT18/S19MLC2 and pT696MYPT1 normalized to the total protein are represented as the mean ± standard error of the mean (error bars) of at least three independent experiments. (B) The same cells as in A were analyzed by immunofluorescence for MyPT1 phosphorylation: pT696MyPT1 antibody (green) and DAPI (blue). Total pixel intensity in cellular region of interest was quantified and expressed as normalization to shScr unstimulated cells. (C) MCF10A were infected with lentiviral vectors coding for PDK1_WT, PDK1_KD, PDK1_ΔPH, PDK1_L155E, or the empty vector and tested for the phosphorylation of MLC2 on S19 or on T18/S19. Band intensity quantification of pS19MLC2 and pT18/S19MLC2 normalized to the total protein are represented as the mean ± standard error of the mean (error bars) of three independent experiments. (D) The same cells as in C were analyzed by immunofluorescence for MLC2 phosphorylation: pT18/S19MLC2 antibody (green) and DAPI (blue) and quantification of pixel intensity. (E and F) GST-MRCKα or GST alone were pulled down and used for a kinase assay from HeLa cells infected with the following lentivirus: empty vector, PDK1_WT, shScr, shPDK1 #79, and shPDK1 #81. Each error bar represents the mean and standard error of the mean of five independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.