Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM-kinase 2 gene.

LIM-Kinase (LIMK), including LIMK1 and LIMK2, is the only known catalytic protein among LIM-family molecules. It is well known that LIMK phosphorylates and inactivates cofilin, an actin-depolymerizing factor regulating actin reorganization, while in vivo functions have remained to be elucidated. In the present study, we generated Limk2 gene-deficient mice in which three LIMK2 isoforms were disrupted in a Cre-mediated fashion. Impaired cofilin phosphorylation was clearly observed in Limk2-/- fibroblasts stimulated with bradykinin or lysophosphatidic acid, thereby suggesting that Cdc42 or Rho-dependent LIMK activation did not occur. However, Limk2-/- mice did not exhibit embryonic lethality or any phenotypic abnormalities in postnatal growth and development, except for spermatogenesis in the testis. The testes of Limk2-/- mice were smaller in size and partial degeneration of spermatogenic cells in the seminiferous tubules was apparent in association with increased apoptosis. In addition, the viability of Limk2-/- spermatogenic cells, when cultured under stressed conditions, was diminished. Furthermore, the potential for germ cells to differentiate in a regenerative state was severely impaired in Limk2-/- testis. Experimental hyperthermia induced impairment of ADF/cofilin phosphorylation and the formation of intranuclear cofilin inclusions in mutant germ cells. Based on these findings, we propose that LIMK2, especially the testis-specific isoform tLIMK2, plays an important role in proper progression of spermatogenesis by regulation of cofilin activity and/or localization in germ cells.     

Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo

Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2.     

Protein kinase D regulates cofilin activity through p21-activated kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.     

LIM kinase 1 is essential for the invasive growth of prostate epithelial cells: implications in prostate cancer

Mammalian LIM kinase 1 (LIMK1) is involved in reorganization of actin cytoskeleton through inactivating phosphorylation of the ADF family protein cofilin, which depolymerizes actin filaments. Maintenance of the actin dynamics in an ordered fashion is essential for stabilization of cell shape or promotion of cell motility depending on the cell type. These are the two key phenomena that may become altered during acquisition of the metastatic phenotype by cancer cells. Here we show that LIMK1 is overexpressed in prostate tumors and in prostate cancer cell lines, that the concentration of phosphorylated cofilin is higher in metastatic prostate cancer cells, and that a partial reduction of LIMK1 altered cell proliferation by arresting cells at G2/M, changed cell shape, and abolished the invasiveness of metastatic prostate cancer cells. We also show that the ectopic expression of LIMK1 promotes acquisition of invasive phenotype by the benign prostate epithelial cells. Our data provide evidence of a novel role of LIMK1 in regulating cell division and invasive property of prostate cancer cells and indicate that the effect is not mediated by phosphorylation of cofilin. Our study correlates with the recent observations showing a metastasis-associated chromosomal gain on 7q11.2 in prostate cancer, suggesting a possible gain in LIMK1 DNA (7q11.23).     

Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

Mitosis-specific activation of LIM motif-containing protein kinase and roles of cofilin phosphorylation and dephosphorylation in mitosis

Actin filament dynamics play a critical role in mitosis and cytokinesis. LIM motif-containing protein kinase 1 (LIMK1) regulates actin reorganization by phosphorylating and inactivating cofilin, an actin-depolymerizing and -severing protein. To examine the role of LIMK1 and cofilin during the cell cycle, we measured cell cycle-associated changes in the kinase activity of LIMK1 and in the level of cofilin phosphorylation. Using synchronized HeLa cells, we found that LIMK1 became hyperphosphorylated and activated in prometaphase and metaphase, then gradually returned to the basal level as cells entered into telophase and cytokinesis. Although Rho-associated kinase and p21-activated protein kinase phosphorylate and activate LIMK1, they are not likely to be involved in mitosis-specific activation and phosphorylation of LIMK1. Immunoblot and immunofluorescence analyses using an anti-phosphocofilin-specific antibody revealed that the level of cofilin phosphorylation, similar to levels of LIMK1 activity, increased during prometaphase and metaphase then gradually declined in telophase and cytokinesis. Ectopic expression of LIMK1 increased the level of cofilin phosphorylation throughout the cell cycle and induced the formation of multinucleate cells. These results suggest that LIMK1 is involved principally in control of mitosis-specific cofilin phosphorylation and that dephosphorylation and reactivation of cofilin at later stages of mitosis play a critical role in cytokinesis of mammalian cells.     

Activation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alpha

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through cofilin phosphorylation downstream of distinct Rho family GTPases. Pak1 and ROCK, respectively, activate LIMK1 and LIMK2 downstream of Rac and Rho; however, an effector protein kinase for LIMKs downstream of Cdc42 remains to be defined. We now report evidence that LIMK1 and LIMK2 activities toward cofilin phosphorylation are stimulated in cells by the co-expression of myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), an effector protein kinase of Cdc42. In vitro, MRCKalpha phosphorylated the protein kinase domain of LIM kinases, and the site in LIMK2 phosphorylated by MRCKalpha proved to be threonine 505 within the activation segment. Expression of MRCKalpha induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in cells, whereas MRCKalpha-induced ADF/cofilin phosphorylation was inhibited by the co-expression with the protein kinase-deficient form of LIM kinases. These results indicate that MRCKalpha phosphorylates and activates LIM kinases downstream of Cdc42, which in turn regulates the actin cytoskeletal reorganization through the phosphorylation and inactivation of ADF/cofilin.     

Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase

LIM-kinase 1 (LIMK1) and LIM-kinase 2 (LIMK2) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases. We report our findings that ROCK, a downstream protein kinase of Rho, specifically activates LIMK2 but not LIMK1 downstream of RhoA. LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK (ROCK-Delta3), whereas full-length ROCK selectively activates LIMK2 but not LIMK1. Activation of LIMK2 by RhoA was inhibited by Y-27632, a specific inhibitor of ROCK, but Rac1-mediated activation of LIMK1 was not. ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity. A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes, whereas replacement by glutamate enhanced the protein kinase activity and stress fiber formation by LIMK2. These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization. Together with the finding that LIMK1 is regulated by Pak1, LIMK1 and LIMK2 are regulated by different protein kinases downstream of distinct Rho family GTPases.     

Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells

Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization.     

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

Stromal Cell-Derived Factor 1α Activates LIM Kinase 1 and Induces Cofilin Phosphorylation for T-Cell Chemotaxis

Stromal cell-derived factor 1 alpha (SDF-1alpha), the ligand for G-protein-coupled receptor CXCR4, is a chemotactic factor for T lymphocytes. LIM kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing and -severing protein, at Ser-3 and regulates actin reorganization. We investigated the role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes. SDF-1alpha significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1alpha-induced LIMK1 activation, thus indicating that Gi protein is involved in LIMK1 activation. Expression of dominant negative Rac (DN-Rac), but not DN-Rho or DN-Cdc42, blocked SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. We used a cell-permeable peptide (S3 peptide) that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibited the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibited the SDF-1alpha-induced cofilin phosphorylation, actin reorganization, and chemotactic response of Jurkat cells. These results suggest that the phosphorylation of cofilin by LIMK1 plays a critical role in the SDF-1alpha-induced chemotactic response of T lymphocytes.     

LIM-kinase as a regulator of actin dynamics in spermatogenesis

We have identified LIM-kinase (LIMK1 and LIMK2), the only known catalytic protein among LIM-family molecules. Both LIMK1 and LIMK2 phosphorylate (inactivate) cofilin, an actin depolymerizing factor, and induce actin cytoskeleton reorganization. We as well as others concurrently demonstrated that LIMK activation was regulated by the Rho family of GTPases, and LIMK1 and LIMK2 share the distinct functional portion in Rho GTPases-mediated actin dynamics. Although evidence for their physiological significance is tenuous, several studies have focused on understanding LIMK functions, particularly in meiosis and mitosis. This review addresses recent LIMK findings, with emphasis on spermatogenesis studies, upon which future studies can shed light on the role of the LIMK/actin system in meiosis and mitosis.     

LIM kinase and slingshot are critical for neurite extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.     

LIM-kinase1

LIM-kinase1 (LIMK1) is a serine-only protein kinase that contains LIM and PDZ protein-protein interaction domains which is highly expressed in neurons. Overexpression of LIMK1 in cultured cells results in accumulation of filamentous (F-) actin. LIMK1 phosphorylates cofilin, an actin depolymerisation factor, which is then unable to bind and depolymerise F-actin. Rac-GTP enhances phosphorylation of LIMK1 and cofilin, which leads to accumulation of F-actin, while Rac-GDP and PMA reduce these effects. LIMK1 is therefore a key component of a signal transduction network that connects extracellular stimuli to changes in cytoskeletal structure. Control of cell morphology and mobility via LIMK1 activity may provide novel approaches to cancer therapy.     

Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.     

Adaptor Protein LRAP25 Mediates Myotonic Dystrophy Kinase-related Cdc42-binding Kinase (MRCK) Regulation of LIMK1 Protein in Lamellipodial F-actin Dynamics

Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) has been shown to localize to the lamella of mammalian cells through its interaction with an adaptor protein, leucine repeat adaptor protein 35a (LRAP35a), which links it with myosin 18A (MYO18A) for activation of the lamellar actomyosin network essential for cell migration. Here, we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIM kinase 1 (LIMK1). The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIMK1 and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility. These biochemical and functional characterizations thus underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.     

Nox4-dependent activation of cofilin mediates VSMC reorientation in response to cyclic stretching

Vascular smooth muscle cells (VSMCs) are subjected to various types of mechanical forces within the vessel wall. Although it is known that VSMCs undergo cell body reorientation in response to mechanical stimulation, how this mechanical stretch is transduced within the cell into biochemical signals causing cytoskeleton reorganization remains unclear. Cofilin, a protein that controls actin dynamics, is activated by Slingshot phosphatase-dependent serine 3 dephosphorylation by redox-dependent mechanisms. Nox4 is a main source of reactive oxygen species (ROS) in the vessel wall that localizes in association with the cytoskeleton. Therefore, we hypothesize that Nox4 mediates redox-dependent activation of cofilin, which is required for cytoskeletal reorganization and cell reorientation after mechanical stimulation. In this study, we found that mechanical stretch stimulates ROS production in VSMCs and that the signaling that leads to cell reorientation requires hydrogen peroxide but not superoxide. Indeed, mechanical stretch induces cofilin activation and stretch-induced cytoskeletal reorganization, and cell reorientation is inhibited in cells where cofilin activity has been downregulated. Importantly, Nox4-deficient cells fail to activate cofilin and to undergo cell reorientation, a phenotype rescued by the expression of a constitutively active cofilin mutant. Our results demonstrate that in VSMCs mechanical stimulation activates cofilin by a Nox4-dependent mechanism and that this pathway is required for cytoskeleton reorganization and cell reorientation.     

Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin

The ADF (actin-depolymerizing factor)/cofilin family is a stimulus-responsive mediator of actin dynamics. In contrast to the mechanisms of inactivation of ADF/cofilin by kinases such as LIM-kinase 1 (LIMK1), much less is known about its reactivation through dephosphorylation. Here we report Slingshot (SSH), a family of phosphatases that have the property of F actin binding. In Drosophila, loss of ssh function dramatically increased levels of both F actin and phospho-cofilin (P cofilin) and disorganized epidermal cell morphogenesis. In mammalian cells, human SSH homologs (hSSHs) suppressed LIMK1-induced actin reorganization. Furthermore, SSH and the hSSHs dephosphorylated P cofilin in cultured cells and in cell-free assays. Our results strongly suggest that the SSH family plays a pivotal role in actin dynamics by reactivating ADF/cofilin in vivo.     

LIM Kinase 1 (LIMK1) Interacts with Tropomyosin-related Kinase B (TrkB) and Mediates Brain-derived Neurotrophic Factor (BDNF)-induced Axonal Elongation

BDNF/TrkB signaling plays critical roles in axonal outgrowth of neurons, the process of which requires the remodeling of the cytoskeleton structure, including microtubules and filamentous actin. However, the mechanism by which BDNF/TrkB signaling regulates cytoskeleton reorganization is still unclear. Here, we identified a novel interaction between LIMK1 and TrkB, which is required for the BDNF-induced axonal elongation. We demonstrated that BDNF-induced TrkB dimerization led to LIMK1 dimerization and transphosphorylation independent of TrkB kinase activity, which could further enhance the activation and stabilization of LIMK1. Moreover, activated LIMK1 translocated to the membrane fraction and phosphorylated its substrate cofilin, thus promoting actin polymerization and axonal elongation. Our findings provided evidence of a novel mechanism for the BDNF-mediated signal transduction leading to axonal elongation.