LIM Kinase Activity Research Articles

A role for LIM kinase in cancer invasion.

In this study, we show that LIM kinase 1 (LIMK1), a critical regulator of actin dynamics, plays a regulatory role in tumor cell invasion. We found that the level and activity of endogenous LIMK1 is increased in invasive breast and prostate cancer cell lines in comparison with less invasive cells. Overexpression of LIMK1 in MCF-7 and in MDA-MB-231 human breast cancer cell lines increased their motility, whereas the specific ROCK and Rho inhibitors Y-27632 and C3, respectively, attenuated this effect. In addition, inhibition of LIMK1 activity in the MDA-MB-231 cells by expression of dominant-negative LIMK1 resulted in decreased motility and formation of osteolytic bone lesions in an animal model of tumor invasion. This study shows an important role for LIMK1 signaling in invasion of cancer, demonstrating its potential as a therapeutic molecular target to decrease metastasis.

Regulation of growth cone actin dynamics by ADF/cofilin.

Nervous system development is reliant on neuronal pathfinding, the process in which axons are guided to their target cells by specific extracellular cues. The ability of neurons to extend over long distances in response to environmental guidance signals is made possible by the growth cone, a highly motile structure found at the end of neuronal processes. Growth cones detect directional cues and respond with either attractive or repulsive movements. The motility of growth cones is dependent on rapid reorganization of the actin cytoskeleton, presumably mediated by actin-associated proteins under the control of incoming guidance signals. This article reviews how one such family of proteins, the ADF/cofilins, are emerging as key regulators of growth cone actin dynamics. These proteins are essential for rapid actin turnover in a variety of different cell types. ADF/cofilins are heavily co-localized with actin in growth cones and are necessary for neurite outgrowth. ADF/cofilin activities are regulated through reversible phosphorylation by LIM kinases and slingshot phosphatases. LIM kinases are downstream effectors of the Rho GTPases Rho, Rac, and Cdc42. Growing evidence suggests that extracellular guidance cues may locally alter actin dynamics by regulating the activity of LIM kinase and ADF/cofilin phosphatases via the Rho GTPases. In this way, ADF/cofilins and their upstream effectors may be pivotal to our understanding of how guidance information is translated into physical alterations of the growth cone actin cytoskeleton.

Identification of the LIM kinase-1 as a ceramide-regulated gene in renal mesangial cells

Stimulation of rat renal mesangial cells with cell-permeable C6-ceramide for 6 and 24 h induces the expression of several genes as analyzed by a RNA fingerprinting arbitrarily primed-PCR method. Sequencing of the differentially expressed bands identified the serine/threonine protein kinase LIM kinase-1 (LIMK-1), which is involved in the regulation of cytoskeletal organization, as a ceramide-induced gene. The ceramide-triggered upregulation of LIMK-1 was verified by semiquantitative reverse transcriptase-PCR. A detailed time course reveals a first detectable increase in RNA level after 2 h of ceramide stimulation which reaches maximal levels after 6 h of stimulation and remains elevated up to 24 h. This ceramide-induced gene transcription of LIMK-1 is accompanied by enhanced LIMK-1 protein levels with maximal protein expression seen after 6 h of stimulation. Furthermore, cofilin, which is a specific substrate of LIMK-1, shows an increased phosphorylation at Ser-3 in mesangial cells exposed to C6-ceramide. Mechanistically, the ceramide-induced LIMK-1 expression is blocked by the Rho kinase inhibitor Y27632, but not by a farnesyl transferase inhibitor, suggesting the involvement of the small G protein Rho, but not Ras and Rac, in the expressional upregulation. Similar to exogenously added ceramide, also interleukin-1β which is an established activator of the neutral sphingomyelinase that leads to endogenous ceramide formation upregulates LIMK-1 protein expression and activity. In summary, these data demonstrate for the first time that LIMK-1 is a ceramide-induced gene, thus suggesting that LIMK-1 may act as a link between stress-induced ceramide formation and reorganization of the actin cytoskeleton.

Stromal cell-derived factor 1alpha 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.

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.

A role for cofilin and LIM kinase in Listeria-induced phagocytosis.

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1−, or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.

Semaphorin Signaling to Actin

Modulation of the actin cytoskeleton underlies the protrusion and retraction responses of migrating neuronal growth cones. Semaphorins (Semas) are transmembrane proteins that elicit growth cone collapse, but the signaling events that affect actin dynamics when Semas engage their cell surface receptors, called plexins, remain unclear. Aizawa et al. report that Sema 3 may alter actin through the action of cofilin, an actin-binding protein that stimulates actin filament depolymerization. When dorsal root ganglion (DRG) neurons were treated with Sema 3A, sequential phoshorylation and then dephosphorylation of cofilin at the growth cone correlated with growth cone collapse. The authors suggest that serine phosphorylation of cofilin stabilizes peripheral actin filaments in the growth cone, but subsequent dephosphorylation by a phosphatase, that has yet to be identified, then reactivates cofilin, resulting in the rapid actin depolymerization that is characteristic of semaphorins. Inhibition of LIM-kinase in DRG neurons indicates that LIM-kinase is necessary, but not sufficient, for this growth cone collapse response. How plexins activate LIM-kinase remains to be determined. Interestingly, LIM-kinase activity is known to be regulated by Rac, the small GTP-binding protein that has also been implicated in gowth cone collapse by semaphorins. Driessens et al. report that activation of plexin-B1 that is overexpressed in fibroblasts results in activation of the small GTP-binding protein Rho in a Rac -dependent manner, causing actin stress fiber formation. The authors determined that activated Rac binds to the cytoplasmic domain of plexin-B1, but the significance of this interaction is not yet clear as plexin that lacks the Rac-binding region is still functional in control of the cytoskeleton. H. Aizawa, S. Wakatsuki, A. Ishii, K. Moriyama, Y. Sasaki, K. Ohashi, Y. Sekine-Aizawa, A. Sehara-Fujisawa, K. Mizuno, Y. Goshima, I. Yahara, Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nature Neurosci. 4 , 367-373 (2001). [Online Journal] M. H. E. Driessens, H. Hu, C. D. Nobes, A. Self, I. Jordens, C. S. Goodman, A. Hall, Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr. Biol. 11 , 339-344 (2001). [Online Journal]

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.

Rho-associated Kinase ROCK Activates LIM-kinase 1 by Phosphorylation at Threonine 508 within the Activation Loop

LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.

The N-terminal LIM domain negatively regulates the kinase activity ofLIM-kinase 1

LIM-kinase 1 (LIMK1, where LIM is an acronym of the three gene products Lin-11, Isl-1 and Mec-3) is a serine/threonine kinase that phosphorylates cofilin and regulates actin cytoskeletal reorganization. LIMK1 contains two LIM domains and a PDZ (an acronym of the three proteins PSD-95, Dlg and ZO-1) domain in the N-terminal half and a kinase domain in the C-terminal half. In this study we examined the role of the extra-catalytic region in the regulation of kinase activity of LIMK1. Limited proteolysis of LIMK1 resulted in the production of the 35-40-kDa kinase core fragments with 3.5-5.5-fold increased kinase activity. The LIMK1 mutants with deleted LIM domains (δLIM) or conserved cysteines in the two LIM domains replaced with glycines (dmLIMK1) had 3-7-fold higher kinase activitiesin vitro, compared with the wild-type LIMK1. The C-terminal kinase fragment of LIMK1 bound to the LIM domain but not to the PDZ domain. Furthermore, the LIM fragment dose-dependently inhibited the kinase catalytic activity of the kinase core fragment of LIMK1. Taken together, these results suggest that the N-terminal LIM domain negatively regulates the kinase activity of LIMK1 by direct interaction with the C-terminal kinase domain. In addition, expression of the δLIM mutant in cultured cells induced punctate accumulation of actin filaments, an event distinct from the pattern of actin organization induced by expression of the wild-type LIMK1, suggesting that the LIM domain plays a role in the function of LIMK1in vivo.

The N-terminal LIM domain negatively regulates the kinase activity ofLIM-kinase 1

LIM-kinase 1 (LIMK1, where LIM is an acronym of the three gene products Lin-11, Isl-1 and Mec-3) is a serine/threonine kinase that phosphorylates cofilin and regulates actin cytoskeletal reorganization. LIMK1 contains two LIM domains and a PDZ (an acronym of the three proteins PSD-95, Dlg and ZO-1) domain in the N-terminal half and a kinase domain in the C-terminal half. In this study we examined the role of the extra-catalytic region in the regulation of kinase activity of LIMK1. Limited proteolysis of LIMK1 resulted in the production of the 35-40-kDa kinase core fragments with 3.5-5. 5-fold increased kinase activity. The LIMK1 mutants with deleted LIM domains (DeltaLIM) or conserved cysteines in the two LIM domains replaced with glycines (dmLIMK1) had 3-7-fold higher kinase activities in vitro, compared with the wild-type LIMK1. The C-terminal kinase fragment of LIMK1 bound to the LIM domain but not to the PDZ domain. Furthermore, the LIM fragment dose-dependently inhibited the kinase catalytic activity of the kinase core fragment of LIMK1. Taken together, these results suggest that the N-terminal LIM domain negatively regulates the kinase activity of LIMK1 by direct interaction with the C-terminal kinase domain. In addition, expression of the DeltaLIM mutant in cultured cells induced punctate accumulation of actin filaments, an event distinct from the pattern of actin organization induced by expression of the wild-type LIMK1, suggesting that the LIM domain plays a role in the function of LIMK1 in vivo.

Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics.

Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. Here we show that p21-activated kinase (Pak1) phosphorylates LIM-kinase at threonine residue 508 within LIM-kinase's activation loop, and increases LIM-kinase-mediated phosphorylation of the actin-regulatory protein cofilin tenfold in vitro. In vivo, activated Rac or Cdc42 increases association of Pak1 with LIM-kinase; this association requires structural determinants in both the amino-terminal regulatory and the carboxy-terminal catalytic domains of Pak1. A catalytically inactive LIM-kinase interferes with Rac-, Cdc42- and Pak1-dependent cytoskeletal changes. A Pak1-specific inhibitor, corresponding to the Pak1 autoinhibitory domain, blocks LIM-kinase-induced cytoskeletal changes. Activated GTPases can thus regulate actin depolymerization through Pak1 and LIM-kinase.

Signal-Regulated Activation of Serum Response Factor Is Mediated by Changes in Actin Dynamics

Serum response factor (SRF) regulates transcription of many serum-inducible and muscle-specific genes. Using a functional screen, we identified LIM kinase-1 as a potent activator of SRF. We show that SRF activation by LIM kinase-1 is dependent on its ability to regulate actin treadmilling. LIM kinase activity is not essential for SRF activation by serum, but signals depend on alterations in actin dynamics. Studies with actin-binding drugs, the actin-specific C2 toxin, and actin overexpression demonstrate that G-actin level controls SRF. Regulation of actin dynamics is necessary for serum induction of a subset of SRF target genes, including vinculin, cytoskeletal actin, and srf itself, and also suffices for their activation. Actin treadmilling provides a convergence point for both serum- and LIM kinase-1-induced signaling to SRF.

Structural Features of LIM Kinase That Control Effects on the Actin Cytoskeleton

LIM kinase phosphorylates and inactivates the actin binding/depolymerizing factor cofilin and induces actin cytoskeletal changes. Several unique structural features within LIM kinase were investigated for their roles in regulation of LIM kinase activity. Disruption of the second LIM domain or the PDZ domain or deletion of the entire amino terminus increased activity in vivo measured as increasing aggregation of the actin cytoskeleton. A kinase-deleted alternate splice product was identified and characterized. This alternate splice product and a kinase inactive mutant inhibited LIM kinase in vivo, indicating that the amino terminus suppresses activity of the kinase domain. Mutation of threonine 508 in the activation loop to valine abolished activity whereas replacement with 2 glutamic acid residues resulted in a fully active enzyme. Dephosphorylation of LIM kinase inhibited cofilin phosphorylation. Mutation of the basic insert in the activation loop inhibited activity in vivo, but not in vitro. These results indicate phosphorylation is an essential regulatory feature of LIM kinase and indicate that threonine 508 and the adjacent basic insert sequences of the activation loop are required for this process. A combination of structural features are thus involved in receiving upstream signals that regulate LIM kinase-induced actin cytoskeletal reorganization.

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.