Fig. 7: Impact of LIMK1 activation loop and PDZ ___domain mutations on activity.
From: Autoregulation of the LIM kinases by their PDZ ___domain
a Serial dilutions of cof1Î yeast expressing human cofilin and the indicated human LIMK1 and constitutively active mutants. Controls of human LIMK1 constructs, full-length (FL), kinase ___domain (CAT) and empty vector (EV); controls of unphosphorylatable cofilin S3A (S3A). Activation loop mutants indicated by T508EE, PDZ mutants indicated by E225A. Five-fold dilutions of yeast cultures were plated on solid media in the presence of glucose (-Gal) or galactose and raffinose (+Gal/Raff) to induce LIMK1 expression. Plates were grown at 30â°C for 2âdays (glucose plate) or 4âdays (galactose plate). Representative of 3 independent experiments. b Immunoblot analysis of FLAG-LIMK1 constructs expressed in yeast. Blots for FLAG, KSS1 loading control, His-cofilin (anti-His), and cofilin phospho-Ser3 (anti-p-Cofilin). FL indicates full-length LIMK1, and CAT indicates LIMK1 catalytic ___domain. Mutants as indicated. c Quantified autoradiography of LIMK1 kinase activity towards cofilin. Activity towards cofilin monitored for full-length T508EE mutant LIMK1 (FL T508EE), T508EE mutant kinase ___domain alone (CAT T508EE), catalytically inactive kinase ___domain (CAT D460N) and full-length PDZ E225A and activation loop T508EE mutant LIMK1 (FL E225A/T508EE). Equivalent LIMK1 and LIMK2 residues, respectively: E225/E206, D460/D451, T508/T505. Right graph shows only on full-length LIMK1 T508EE and full-length LIMK1 E225A/T508EE. Left lower panel shows representative autoradiography reading of cofilin phosphorylation for kinase assays and corresponding Coomassie staining. Statistical analysis was carried out using a non-parametric unpaired two-sided Mann-Whitney test. Data are mean values (bar graph)â+/ââSD (error bars), and individual measurements are plotted (dots, Nâ=â4). One star (*) indicates pâ<â0.05 for all samples compared. p-values: FL T508EE vs CAT T508EE: pâ=â0.0286, FL T508EE vs FL E225A/T508EEA: pâ=â0.0286. A total of 4 replicates were analyzed using GraphPad Prism.
