Autoregulation of the LIM kinases by their PDZ domain

Fig. 1. LIMK domain architecture. a LIM domain kinase family architecture showing human LIMK1 (UniProt ID: P53667), and human LIMK2 (UniProt ID: P53671). LIM1: first LIM domain, LIM2: second LIM domain, PDZ: PDZ domain, Kinase: kinase domain. Activation loop phosphorylation residues indicated, Thr508/Thr505 for LIMK1 and LIMK2, respectively. Residue numbers are shown. b Sequence alignment of PDZ domains. Alignment was created using PROMALS74. Uniprot ID for LIMK1_HUMAN, P53667; LIMK1_MOUSE, P53668; LIMK1_XENLA, O42565; LIMK1_DANRE, B3DIV5; LIMK1_DROME, Q8IR79; LIMK2_HUMAN, P53671; LIMK2_MOUSE, O54785; LIMK2_XENTR, F7AFJ1; LIMK2_DANRE, Q6DG29; GRIP1_PDZ6, P97879; PSD95_PDZ3, P31016. GRIP1_PDZ6 is a Class I PDZ domain, PSD95_PDZ3 is a Class II PDZ domain. ‘x-Φ-G-Φ’ sequence is inside a black lined box. The conserved arginine residue equivalent to Arg163 in human LIMK1 is colored red. Conserved amino acid residues targeted in mutagenesis studies are in bold and under a black arrow.

Fig. 2. Structure of LIMK2 PDZ domain. a Human LIMK2 PDZ domain determined to 2.0 Å resolution shown in cartoon format. Secondary structure named. The ‘x-Φ-G-Φ’ loop, βA-βB loop, αB-βF loop and βB-βC loop are indicated and the x-Φ-G-Φ’ loop colored green. b Electron density map of Arg163. 2Fobs–Fcalc electron density map contoured at 1σ (blue). Fobs–Fcalc electron density map contoured at +3σ (green) and −3σ (red). c Hydrogen bonds of Arg163. d Inward orientation of the αA-βF loop. Comparison of the αA-βF loop orientation of LIMK2 PDZ crystal structure (orange), and the most similar PDZ domains structures from Dali75; spinophilin, PDB ID: 3EGG50 (pink), disks large homolog 4, PDB ID: 5HEY52 (light blue), harmonin, PDB ID: 3K1R53 (purple), and syntenin-1, PDB ID: 5G1E51 (teal). Inward orientation of helix αB helps create a somewhat shallow binding grove between the βB strand and αB helix compared to these most similar PDZ domains. Images generated using CCP4mg76.

Fig. 3. Surface analysis of LIMK2 PDZ domain. a Conservation of the LIMK2 PDZ domain. PDZ domain conservation mapped onto the structure of LIMK2 PDZ for 421 aligned LIMK sequences from mammals, birds, fish, and insects. Dashed oval indicates the canonical PDZ binding cleft. b Surface electrostatics of the LIMK2 PDZ domain calculated by CCP4mg76. Red indicates negatively charged surfaces, blue indicates positively charged surfaces, and white surfaces indicate neutrally charged surfaces. Dashed oval indicates the canonical PDZ binding cleft.

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…

Fig. 5. Increased in vitro kinase activity for LIMK1 PDZ mutants. Quantified autoradiography from radiolabel cofilin kinase activity of FLAG-LIMK1 constructs purified from yeast. a Full-length wild type (WT), kinase domain alone (CAT), and catalytically inactive kinase domain (CAT D460N) were used as positive and negative controls, respectively. PDZ domain mutants of conserved residues in FL LIMK1 are shown (equivalent residue in LIMK2 shown in parentheses). b Graph focused on full-length mutant constructs compared to WT. c Lower panel shows representative autoradiography reading of cofilin phosphorylation for kinase assays with corresponding Coomassie staining. 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 = 5). Two stars (**) indicates p < 0.005 for all samples compared. p-values: FL vs CAT: p = 0.0079, FL vs L165A: p = 0.0079, FL vs D221A: p = 0.0079, FL vs R222A: p = 0.0079, FL vs E225A: p = 0.0079, FL vs Q251A: p = 0.0079, FL vs K175D: p = 0.6825. A total of 5 replicates (4 for D221A) were analyzed using GraphPad Prism.

Fig. 6. Assessment of LIMK expression and activation loop phosphorylation in yeast. a Western Blot assessing activation loop phosphorylation in LIMK1 constructs from our yeast growth assays. The top panel shows yeast lysates blotted with anti-pLIMK antibody and the bottom panel purified yeast protein blotted with anti-FLAG. CAT indicates catalytic domain, FL indicates full-length LIMK1. Mutations of full-length LIMK1 are indicated. b Quantification of Western blot signal. For each construct, phospho-LIMK1 signal was normalized to FLAG signal then compared to full-length LIMK1 (FL). c Quantification of FLAG signal in all four replicates, normalized to FLAG-LIMK1 FL signal. All mutant constructs signals are non-significantly different from each other. 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). p-values: FL vs CAT: p = 0.0143, FL vs L165A: p = 0.0143, FL vs D221A: p = 0.0143, FL vs R222A: p = 0.0143, FL vs E225A: p = 0.0143, FL vs Q251A: p = 0.0143. One star (*) indicates p < 0.05. A total of 4 replicates were analyzed using…

Fig. 7. Impact of LIMK1 activation loop and PDZ domain mutations on activity. a Serial dilutions of cof1Δ 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 30 °C for 2 days (glucose plate) or 4 days (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…