P52L mutation results in loss of PAK6 autoinhibition.

<p><b>A)</b> Alignment of the pseudosubstrate autoinhibitory domain (AID) of the type II PAK family members is shown. Pseudosubstrate region indicated by a green bar. Peptide used in panel B indicated in red. Conservation shown with “*” indicating identical, “:” indicating highly conserved and “.” Indicating semi-conserved. <b>B–D)</b> Dose-dependent relationship of the inhibition of wild-type (RRPKPVVDP), P52L (RRPK<u>L</u>VVDP) mutant, and control (APRTPGGRR) peptides on PAK6 kinase activity. <b>B)</b> Inhibition of PAK6 by wild-type (WT) peptide shown on a log scale. Data shows a mean and S.E.M error bar with 3 independent experiments. Concentration range from 0.06 mM to 5.85 mM of peptides was tested. <b>C)</b> Inhibition of PAK6 by P52L peptide shown on a log scale. Data shows a mean and S.E.M error bar with 3 independent experiments. Concentration range from 1.8 mM to 5.85 mM of peptides was tested. <b>D)</b> Inhibition of PAK6 by a control peptide. Data show a mean and S.E.M error bar with 3 independent experiments. Concentration range from 0.5 mM to 4.5 mM of peptide was tested. <b>E)</b> Inhibition plots from panels B-D shown on the same graph. <b>F)</b> Co-transfection of PAK6 with PACSIN1. Co-transfection of myc-PACSIN1 and GFP-PAK6 constructs probed for PACSIN1 phosphorylation using a phospho-specific PACSIN1 antibody. M indicates molecular weight marker. <b>G)</b> Quantification of E. Ratio of phosphorylated PACSIN1 to total PACSIN1 expressed is shown, normalized for Phospho PACSIN1/Total PACSIN1 (myc). PACSIN1 shows significantly increased phosphorylation when co-transfected with the P52L mutant compared to the wild-type PAK6 (* indicates p<0.05; ** indicates p<0.01; Student’s t-test). n = 4. Error bars indicate S.E.M.