Activation of LIMK1 (A and B) and LIMK2 (C) kinase activity by Rho family GTPases. A, COS-7 cells were coexpressed with HA-tagged full-length LIMK1, and empty vector (Mock), Myc-tagged active, or the inactive form of Rho, Rac, and Cdc42, respectively. B, COS-7 cells were coexpressed with HA-tagged LIMK1 and empty vector (Mock), Myc-tagged Cdc42V12, RacN17, respectively, or increasing doses of Myc-tagged RacN17 (5, 10 μg), together with HA-tagged LIMK1 and Myc-tagged Cdc42V12. C, COS-7 cells were coexpressed with HA-tagged LIMK2 and empty vector (Mock), Myc-tagged active, or the inactive form of Rho, Rac, and Cdc42, respectively. After transient expression, LIMK1 and LIMK2 were respectively immunoprecipitated from cell lysates, using anti-HA antibody, run on SDS-PAGE, and immunoblotted with anti-LIMK1, anti-LIMK2, and anti-HA antibody. The immunoprecipitates were subjected to in vitro kinase reaction with γ[³²P]ATP, using GST-fused cofilin as substrate. The phosphorylated cofilin was visualized by autoradiography after SDS-PAGE. The initial extracts (50 μg) were also subjected to anti-Myc or anti-Cdc42 immunoblot. The arrowheads respectively indicate autophosphorylated LIMK1 or LIMK2. Cofilin phosphorylation was estimated using an image analyzer (model BAS-2000; Fuji), and the amount of cofilin phosphorylation by LIMK1 or LIMK2 expressed with Mock was respectively taken as 1.0. Each value represents the mean ± standard error (SE) of three independent experiments.

Formation of stress fibers (A) and focal complexes (B) by LIMK1 and LIMK2 in HeLa cells. Serum-starved HeLa cells were transfected with DNA construct encoding HA-tagged full-length LIMK1 (a and b), LIMK2 (e and f), LIMK1/KD (c and d), or LIMK2/KD (g and h). A, The cells were fixed and double stained with anti-HA antibody (a, c, e, and g) and phalloidin (b, d, f, and h). B, The cells were fixed and double stained with anti-HA antibody (a, c, e, and g) and antivinculin antibody (b, d, f, and h). Confocal images were obtained, as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Inhibition of LIMK2-induced actin reorganization (A) and focal complex formation (B) by cofilin inactivation. Expression vectors for FLAG-tagged cofilin-S3A (100 μg/ml) and HA-tagged LIMK2 (25 μg/ml) were microinjected into serum-starved HeLa cells. A, 2 h later, cells were fixed and double stained with anti-FLAG antibody (c), anti-HA antibody (a and e), and phalloidin (b, d, and f). B, Two hours later, cells were fixed and double stained with anti-HA antibody (a and c) and antivinculin antibody (b and d). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Specific role of LIMK1 and LIMK2 in actin reorganization induced by Rho family GTPases. A, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-HA antibody (c and e), anti-Myc antibody (a), and phalloidin (b, d, and f). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Specific role of LIMK1 and LIMK2 in actin reorganization induced by Rho family GTPases. A, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-HA antibody (c and e), anti-Myc antibody (a), and phalloidin (b, d, and f). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Specific role of LIMK1 and LIMK2 in focal complex formation induced by Rho family GTPases. A, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-HA antibody (c and e), anti-Myc antibody (a), and antivinculin antibody (b, d, and f). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Effect of the active form of cofilin on actin reorganization induced by Rho family GTPases. A, Expression vectors for FLAG-tagged cofilin-S3A (50 μg/ml) and the Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for FLAG-tagged cofilin-S3A (50 μg/ml) and the Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for FLAG-tagged cofilin-S3A (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-Myc antibody (a), anti-FLAG antibody (c), and phalloidin (b and d). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Protein kinase activities of LIMK2 and its mutants. A, Schematic structure of LIMK2 mutants. Numbers indicate amino acid residues of the NH₂ and COOH termini. Position of point mutations are indicated by an asterisk. B, Cofilin phosphorylation by LIMK2 and its mutants. COS-7 cells were transfected with expression vectors for HA-tagged LIMK2 and its mutants. After transient expression, protein kinase activity was measured as described for Fig. 1. Top, autoradiograph of the phosphorylated cofilin; middle, corresponding immunoblot analysis of the immunoprecipitated proteins; bottom, substrate phosphorylation was estimated, using an image analyzer (model BAS-2000; Fuji). Protein kinase activity of wild-type LIMK2 was taken as 1.0. Each value represents the means ± SE for experiments done in triplicate. The arrowheads indicate autophosphorylated LIMK2 and ΔLIM, respectively.

Regulation of protein kinase activity of LIMK2 and its mutants by Rho and Cdc42. A, Regulation of LIMK2 and mutant LIMK2 activities by Cdc42. COS-7 cells were coexpressed with HA-tagged wild-type LIMK2, ΔLIM, or ΔN, and empty vector (Mock), Myc-tagged Cdc42V12, or Cdc42N17. B, Regulation of LIMK2 and mutant LIMK2 activities by Rho. COS-7 cells were coexpressed with HA-tagged wild-type LIMK2, ΔLIM, or ΔN, and empty vector (Mock), Myc-tagged RhoV14, or RhoN19. After transient expression, LIMK2 or mutant LIMK2 kinase activity was measured as described for Fig. 1. The initial extracts (50 μg) were also subjected to anti-Myc immunoblot. The arrowheads indicate autophosphorylated LIMK2 and ΔLIM. Cofilin phosphorylation was estimated, using an image analyzer (model BAS-2000; Fuji), and the amount of cofilin phosphorylation by LIMK2 expressed with Mock was respectively taken as 1.0. The results shown are representative of three independent experiments. Each value represents the mean ± SE of three independent experiments.

Activation of LIMK1 (A and B) and LIMK2 (C) kinase activity by Rho family GTPases. A, COS-7 cells were coexpressed with HA-tagged full-length LIMK1, and empty vector (Mock), Myc-tagged active, or the inactive form of Rho, Rac, and Cdc42, respectively. B, COS-7 cells were coexpressed with HA-tagged LIMK1 and empty vector (Mock), Myc-tagged Cdc42V12, RacN17, respectively, or increasing doses of Myc-tagged RacN17 (5, 10 μg), together with HA-tagged LIMK1 and Myc-tagged Cdc42V12. C, COS-7 cells were coexpressed with HA-tagged LIMK2 and empty vector (Mock), Myc-tagged active, or the inactive form of Rho, Rac, and Cdc42, respectively. After transient expression, LIMK1 and LIMK2 were respectively immunoprecipitated from cell lysates, using anti-HA antibody, run on SDS-PAGE, and immunoblotted with anti-LIMK1, anti-LIMK2, and anti-HA antibody. The immunoprecipitates were subjected to in vitro kinase reaction with γ[³²P]ATP, using GST-fused cofilin as substrate. The phosphorylated cofilin was visualized by autoradiography after SDS-PAGE. The initial extracts (50 μg) were also subjected to anti-Myc or anti-Cdc42 immunoblot. The arrowheads respectively indicate autophosphorylated LIMK1 or LIMK2. Cofilin phosphorylation was estimated using an image analyzer (model BAS-2000; Fuji), and the amount of cofilin phosphorylation by LIMK1 or LIMK2 expressed with Mock was respectively taken as 1.0. Each value represents the mean ± standard error (SE) of three independent experiments.

Activation of LIMK1 (A and B) and LIMK2 (C) kinase activity by Rho family GTPases. A, COS-7 cells were coexpressed with HA-tagged full-length LIMK1, and empty vector (Mock), Myc-tagged active, or the inactive form of Rho, Rac, and Cdc42, respectively. B, COS-7 cells were coexpressed with HA-tagged LIMK1 and empty vector (Mock), Myc-tagged Cdc42V12, RacN17, respectively, or increasing doses of Myc-tagged RacN17 (5, 10 μg), together with HA-tagged LIMK1 and Myc-tagged Cdc42V12. C, COS-7 cells were coexpressed with HA-tagged LIMK2 and empty vector (Mock), Myc-tagged active, or the inactive form of Rho, Rac, and Cdc42, respectively. After transient expression, LIMK1 and LIMK2 were respectively immunoprecipitated from cell lysates, using anti-HA antibody, run on SDS-PAGE, and immunoblotted with anti-LIMK1, anti-LIMK2, and anti-HA antibody. The immunoprecipitates were subjected to in vitro kinase reaction with γ[³²P]ATP, using GST-fused cofilin as substrate. The phosphorylated cofilin was visualized by autoradiography after SDS-PAGE. The initial extracts (50 μg) were also subjected to anti-Myc or anti-Cdc42 immunoblot. The arrowheads respectively indicate autophosphorylated LIMK1 or LIMK2. Cofilin phosphorylation was estimated using an image analyzer (model BAS-2000; Fuji), and the amount of cofilin phosphorylation by LIMK1 or LIMK2 expressed with Mock was respectively taken as 1.0. Each value represents the mean ± standard error (SE) of three independent experiments.

Formation of stress fibers (A) and focal complexes (B) by LIMK1 and LIMK2 in HeLa cells. Serum-starved HeLa cells were transfected with DNA construct encoding HA-tagged full-length LIMK1 (a and b), LIMK2 (e and f), LIMK1/KD (c and d), or LIMK2/KD (g and h). A, The cells were fixed and double stained with anti-HA antibody (a, c, e, and g) and phalloidin (b, d, f, and h). B, The cells were fixed and double stained with anti-HA antibody (a, c, e, and g) and antivinculin antibody (b, d, f, and h). Confocal images were obtained, as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Inhibition of LIMK2-induced actin reorganization (A) and focal complex formation (B) by cofilin inactivation. Expression vectors for FLAG-tagged cofilin-S3A (100 μg/ml) and HA-tagged LIMK2 (25 μg/ml) were microinjected into serum-starved HeLa cells. A, 2 h later, cells were fixed and double stained with anti-FLAG antibody (c), anti-HA antibody (a and e), and phalloidin (b, d, and f). B, Two hours later, cells were fixed and double stained with anti-HA antibody (a and c) and antivinculin antibody (b and d). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Specific role of LIMK1 and LIMK2 in actin reorganization induced by Rho family GTPases. A, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-HA antibody (c and e), anti-Myc antibody (a), and phalloidin (b, d, and f). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Specific role of LIMK1 and LIMK2 in focal complex formation induced by Rho family GTPases. A, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-HA antibody (c and e), anti-Myc antibody (a), and antivinculin antibody (b, d, and f). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Specific role of LIMK1 and LIMK2 in focal complex formation induced by Rho family GTPases. A, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for HA-tagged LIMK1/KD or LIMK2/KD (50 μg/ml) and Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-HA antibody (c and e), anti-Myc antibody (a), and antivinculin antibody (b, d, and f). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Effect of the active form of cofilin on actin reorganization induced by Rho family GTPases. A, Expression vectors for FLAG-tagged cofilin-S3A (50 μg/ml) and the Myc-tagged active form of Cdc42 (Cdc42V12; 50 μg/ml) were microinjected into serum-starved HeLa cells. B, Expression vectors for FLAG-tagged cofilin-S3A (50 μg/ml) and the Myc-tagged active form of Rac (RacV12; 50 μg/ml) were microinjected into serum-starved HeLa cells. C, Expression vectors for FLAG-tagged cofilin-S3A (50 μg/ml) and the Myc-tagged active form of Rho (RhoV14; 50 μg/ml) were microinjected into serum-starved HeLa cells. 4 h later, these cells were fixed and double stained with anti-Myc antibody (a), anti-FLAG antibody (c), and phalloidin (b and d). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bars, 20 μm.

Effects of LIMK2 mutants on actin reorganization. Serum-starved HeLa cells were transfected with DNA constructs encoding HA-tagged wild-type LIMK2 (a and b), ΔLIM (c and d), ΔN (e and f), and ΔPK (g and h). Cells were fixed 24 h after DNA transfection and double stained with anti-HA antibody (a, c, e, and g) and phalloidin (b, d, f, and h). Confocal images were obtained as described in Materials and Methods. The results shown are representative of three independent experiments. Bar, 20 μm.