Fig. 4: PDZ ___domain mutants suppress yeast growth and increase cofilin phosphorylation.

a Serial dilutions of cof1Δ yeast expressing human cofilin and the indicated human LIMK1 mutants. Controls of human LIMK1 constructs, full-length (FL), kinase ___domain (CAT), unphosphorylatable cofilin S3A (S3A) and empty vector (EV). Mutants of full-length LIMK1: E225A, D221A, R222A, L165A, Q251A. Corresponding LIMK2 residue shown in parentheses. 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 30 °C for 2 days (glucose plate) or 4 days (galactose plate). Representative of 3 independent experiments. b Mutants assessed are shown on the cartoon and surface representations of the conservation map of LIMK2 PDZ ___domain. Residues shown and equivalent human LIMK1 residue numbers: L152 (L165 in LIMK1), Q232 (Q251 in LIMK1), D202 (D221 in LIMK1), R203 (R222 in LIMK1) and E206 (E225 in LIMK1). c Immunoblots of lysates corresponding to yeast plated in (a). Cofilin species are separated based on phosphorylation state by Phos-tag SDS-PAGE. KSS1 serves as a loading control. Images are representative of N = 4. d Quantification of immunoblots measuring the percentage of total unphosphorylated cofilin. Statistical analysis was carried out using a non-parametric unpaired two-sided Mann-Whitney test. Data are mean values (bar graph) +/− SD (error bars), and individual measurements are plotted (dots, N = 4). One star (*) indicates p < 0.05 for all samples compared. p-values: FL vs CAT: p = 0.0286, FL vs L165A: p = 0.0286, FL vs D221A: p = 0.0286, FL vs R222A: p = 0.0286, FL vs E225A: p = 0.0286, FL vs Q251A: p = 0.0286. A total of 4 replicates were analyzed using GraphPad Prism.