LIM kinase 1 (LIMK1) is expressed in both cytoplasmic and nuclear compartments, and is a key regulator of cytoskeletal organization involved in cell migration and proliferation. LIMK1 levels are increased in several human cancers, with LIMK1 over-expression in prostate and breast cancer cells leading to tumor progression. While it has been presumed that the mechanism by which LIMK1 promotes cancer progression is via its cytoplasmic effects, the role of nuclear vs cytoplasmic LIMK1 in the tumorigenic process has not been examined.
To determine if cytoplasmic or nuclear LIMK1 expression correlated with breast cancer, we performed immunohistochemical (IHC) analysis of breast tissue microarrays (TMAs), The IHC analysis of breast TMAs revealed that 76% of malignant breast tissue samples strongly expressed LIMK1 in the cytoplasm, with 52% of these specimens also expressing nuclear LIMK1. Only 48% of benign breast samples displayed strong cytoplasmic LIMK1 expression and 27% of these expressed nuclear LIMK1. To investigate the respective roles of cytoplamsic and nuclear LIMK1 in breast cancer progression, we targeted GFP-LIMK1 to cytoplasmic and nuclear subcellular compartments by fusing nuclear export signals (NESs) or nuclear localization sequences (NLS), respectively, to the amino-terminus of GFP-LIMK1. Stable pools of MDA-MB-231 cells were generated by retroviral transduction, and fluorescence microscopy revealed that GFP alone (control) and GFP-LIMK1 were each expressed in both the cytoplasm and nucleus of MDA-MB-231 cells, whereas NLS-GFP-LIMK1 was expressed in the nucleus and NES-GFP-LIMK1 was expressed in the cytoplasm. Western blot analyses revealed equal expression of GFP-LIMK1 and NES-GFP-LIMK1, with NLS-GFP-LIMK1 expression being less but equal to endogenous LIMK1. Also, Western blotting revealed increased levels of phospho-cofilin, phospho-FAK, phospho-paxillin, phospho-Src, phospho-AKT, and phospho-Erk1/2 in cells expressing all GFP-LIMK1 fusions, compared to GFP alone. Invasion assays revealed that all GFP-LIMK1 fusions increased MDA-MB-231 cell invasion ~1.5-fold, compared to GFP-only control cells. Tumor xenograft studies in nude mice revealed that MDA-MB-231 cells stably expressing GFP-LIMK, NLS-GFP-LIMK1 and NES-GFP-LIMK1 enhanced tumor growth 2.5-, 1.6- and 4.7-fold, respectively, compared to GFP-alone.
Taken together, these data demonstrate that LIMK1 activity in both the cytoplasmic and nuclear compartments promotes breast cancer progression, underscoring that nuclear LIMK1 contributes to the transforming function of LIMK1.
Neurofibromatosis Type 2 (NF2) is caused by mutations in the neurofibromatosis 2 (NF2) gene that encodes a tumor suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss of function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates cofilin’s actin severing and depolymerizing activity. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2ΔEx2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2ΔEx2 MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2ΔEx2 MSCs. The decreased viability of Nf2ΔEx2 MSCs was not due to caspase-dependent or -independent apoptosis, but rather, to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.
LIMK; cell proliferation; Neurofibromatosis; schwannomas; cytoskeleton dynamics; cell cycle
BACKGROUND: The malignant astrocytoma is a highly proliferative and invasive neoplasm that infiltrates diffusely into regions of normal brain rendering total surgical resection impossible and effective local radiation therapy difficult. For truly significant advances to be made in the treatment of patients with malignant astrocytoma, we must develop a greater understanding of the molecular machinery driving invasion and identify novel treatment targets. METHODS: We recently compared gene-expression data of 400 invasion/migration genes by performing microarray analysis comparing 10 normal brain samples versus 51 mesenchymal GBM, which are highly invasive, and of poor prognosis. RESULTS: We identified over 141 significant genes with a 1.5 fold change (p-value <0.05, and a false discovery rate (q < 0.05) compared to normal brain. Of the 141 genes, the cofilin pathway, which disassembles actin filaments (namely LIMK1, LIMK2, CFL, CAP1) was highly up-regulated compared to normal brain. Furthermore we identified up-regulation of LIM domain kinase 1 and 2 (LIMK1/2) that phosphorylates and inactivates cofilin (CFL) in an additional data set comparing normal brain to GBM. Using a well-characterized tissue microarray of 20 GBM patients for which a central and a matched periphery sample was available we undertook immunohistochemical (IHC) analysis of LIMK1 and pCFL. We observed significant expression of LIMK1 in the periphery of the GBM compared to the center (11/20 in the periphery versus 2/20 in the center). Previously, we established that pCFL plays an integral role in GBM invasion. Consequently we explored how to target this pathway using small molecule inhibitors. We tested the LIMK1 and LIMK2 inhibitors BMS-5 and Cucurbitacin I. Both BMS-5 and Cucurbitacin I inhibited p-CFL at the protein level at 10uM and 100nM respectively in U87 cells. Using the MTT cell viability assay, we observed significant decrease in cell viability in U87 and T98G cells using doses of 10-20uM BMS-5 and 100nM-10uM Cucurbitacin I while no cytotoxic effect was seen in normal human astrocytes that lack LIMK expression. Both BMS-5 (10uM) and Cucurbitacin I (100nM) promoted increased adhesion in U87 and T98G cells. Interestingly, BMS-5 and Cucurbitacin I reduced migration and invasion of both U87 and T98G cells. CONCLUSIONS: Taken together, the use of LIMK inhibitors to target the invasive machinery in GBM are attractive candidates to move forward with in preclinical trials. SECONDARY CATEGORY: Tumor Biology.
LIM kinase 1 (LIMK1), a LIM domain containing serine/threonine kinase, modulates actin dynamics through inactivation of the actin depolymerizing protein cofilin. Recent studies have indicated an important role of LIMK1 in growth and invasion of prostate and breast cancer cells; however, the molecular mechanism whereby LIMK1 induces tumor progression is unknown. In this study, we investigated the effects of ectopic expression of LIMK1 on cellular morphology, cell cycle progression and expression profile of LIMK1 in prostate tumors.
Ectopic expression of LIMK1 in benign prostatic hyperplasia cells (BPH), which naturally express low levels of LIMK1, resulted in appearance of abnormal mitotic spindles, multiple centrosomes and smaller chromosomal masses. Furthermore, a transient G1/S phase arrest and delayed G2/M progression was observed in BPH cells expressing LIMK1. When treated with chemotherapeutic agent Taxol, no metaphase arrest was noted in these cells. We have also noted increased nuclear staining of LIMK1 in tumors with higher Gleason Scores and incidence of metastasis.
Our results show that increased expression of LIMK1 results in chromosomal abnormalities, aberrant cell cycle progression and alteration of normal cellular response to microtubule stabilizing agent Taxol; and that LIMK1 expression may be associated with cancerous phenotype of the prostate.
The purpose of this study was to determine if downregulation of LIM kinase 1 (LIMK1) by genetic deletion or direct application of LIMK1-targeted siRNA could suppress TGF-β mediated ocular inflammation and fibrosis.
LIMK1 specific siRNAs designed from the human sequence were transfected into human corneal fibroblasts in culture. Immunofluorescence and immunoblotting were performed to examine the fibronectin assembly. The effects of LIMK1 downregulation on actin cytoskeleton organization and focal adhesion formation were studied. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. The in vivo effects of LIMK1 genetic deletion or downregulation by mouse siRNA were evaluated in a mouse model of ocular inflammation generated by subconjunctival injection of phosphate buffered saline and latex beads. Cellularity on tissue sections was examined after staining with hematoxylin and eosin. Anti-CD45 antibody was used for the leukocyte detection.
Downregulation of LIMK1 in cultured corneal fibroblasts impaired fibronectin secretion and assembly, diminished actin polymerization and focal adhesion formation, and retarded cell migration. In the mouse model of ocular inflammation, both genetic deletion and downregulation of LIMK1 by siRNA significantly reduced inflammatory response.
Downregulation of LIMK1 was efficacious to decrease the ocular inflammation. We disclose a possibility that LIMK1 may mediate TGF-β-dependent signaling during ocular inflammation. A direct application of siRNA into eyes to downregulate LIMK1 expression may provide a novel therapy for suppression and prevention of ocular inflammation and fibrosis.
LIMK regulation of actin and microtubule dynamics is required for epithelial regulation of early- and late-stage cleft stabilization and progression. LIMK stimulates focal adhesion assembly and integrin β1 activation in cleft regions, causing fibronectin fibrillogenesis and promoting cleft progression during salivary gland branching morphogenesis.
Coordinated actin microfilament and microtubule dynamics is required for salivary gland development, although the mechanisms by which they contribute to branching morphogenesis are not defined. Because LIM kinase (LIMK) regulates both actin and microtubule organization, we investigated the role of LIMK signaling in mouse embryonic submandibular salivary glands using ex vivo organ cultures. Both LIMK 1 and 2 were necessary for branching morphogenesis and functioned to promote epithelial early- and late-stage cleft progression through regulation of both microfilaments and microtubules. LIMK-dependent regulation of these cytoskeletal systems was required to control focal adhesion protein–dependent fibronectin assembly and integrin β1 activation, involving the LIMK effectors cofilin and TPPP/p25, for assembly of the actin- and tubulin-based cytoskeletal systems, respectively. We demonstrate that LIMK regulates the early stages of cleft formation—cleft initiation, stabilization, and progression—via establishment of actin stability. Further, we reveal a novel role for the microtubule assembly factor p25 in regulating stabilization and elongation of late-stage progressing clefts. This study demonstrates the existence of multiple actin- and microtubule-dependent stabilization steps that are controlled by LIMK and are required in cleft progression during branching morphogenesis.
Precise regulation of the dendritic spine actin cytoskeleton is critical for neurodevelopment and neuronal plasticity, but how neurons spatially control actin dynamics is not well defined. Here, we identify direct palmitoylation of the actin regulator LIM kinase-1 (LIMK1) as a novel mechanism to control spine-specific actin dynamics. A conserved palmitoyl-motif is necessary and sufficient to target LIMK1 to spines and to anchor LIMK1 in spines. ShRNA knockdown/rescue experiments reveal that LIMK1 palmitoylation is essential for normal spine actin polymerization, for spine-specific structural plasticity and for long-term spine stability. Palmitoylation is critical for LIMK1 function because this modification not only controls LIMK1 targeting, but is also essential for LIMK1 activation by its membrane-localized upstream activator PAK. These novel roles for palmitoylation in the spatial control of actin dynamics and kinase signaling provide new insights into structural plasticity mechanisms and strengthen links between dendritic spine impairments and neuropathological conditions.
Neurons transmit information from one cell to the next by passing signals across junctions called synapses. For the neurons that receive these signals, these junctions are found on fine branch-like structures called dendrites that stick out of the cell. Dendrites themselves are decorated with smaller structures called dendritic spines, which typically receive information from one other neuron via a single synapse. Dendritic spines form in response to the signaling activity of the neuron, and problems with forming these spines have been linked to conditions such as autism and schizophrenia.
Dendritic spines are created by the cell's cytoskeleton—a network of proteins that creates a constantly changing internal scaffold that shapes cells. One cytoskeleton protein called actin exists as thin filaments that can be extended or broken up by other proteins. It is not fully understood how actin is regulated in the dendritic spines. However, some researchers thought that the proteins that control the formation of the actin filaments would need to be localized to the dendritic spines to ensure that the spines form correctly.
Some proteins can be made to localize to cell membranes by attaching a molecule called palmitic acid to them. Previous research has suggested that this ‘palmitoylation’ process is particularly important in neurons. Through a combination of experimental techniques, George et al. now show that palmitoylation is required to localize a protein called LIMK1, which regulates the construction of actin filaments, to the tips of dendritic spines. Further experiments showed that blocking the palmitoylation of LIMK1 alters how actin filaments form, makes spines unstable and causes synapses to be lost.
George et al. also discovered that palmitoylation is necessary for LIMK1 to be activated by another protein that is found at dendritic spine membranes. This ‘dual-control’ mechanism makes it possible to precisely control where actin filaments form within dendritic spines. In addition to LIMK1, several other enzymes are also modified by palmitoylation. It will therefore be interesting to determine whether this dual control mechanism is broadly used by neurons to precisely regulate the structure and function of individual spines and synapses.
DHHC; dendritic spines; protein trafficking; LTP; glutamate; LIMK1; rat
Chemokine binding to cognate receptors induces actin dynamics that are a major driving force for T cell migration and chemotactic motility. HIV-1 binding to the chemokine coreceptor CXCR4 initiates chemotactic signaling, mimicking chemokine-induced actin dynamics to facilitate infection processes such as entry, early DNA synthesis, and nuclear migration. Recently, we identified that HIV-triggered early actin polymerization is mediated through the Rac1-PAK1/2-LIMK1-cofilin pathway. Inhibition of LIMK1 (LIM domain kinase 1), a kinase phosphorylating cofilin, through shRNA knockdown decreases actin polymerization and T cell chemotaxis toward SDF-1. The LIMK1 knockdown T cells also supported lower viral entry, DNA synthesis and nuclear migration, suggesting a critical role of LIMK1-mediated actin dynamics in the initiation of HIV-1 infection. Surprisingly, LIMK1 knockdown in CEM-SS T cells did not lead to an overall change in the ratio of phospho-cofilin to total cofilin although there was a measurable decrease in the amount of actin filaments in cells. The decrease in filamentous actin in LIMK1 knockdown cells was found to mainly occur in polarized cap region rich in F-actin. These results suggest that LIMK1 may be involved in spontaneous actin polarization in transformed T cells. The inhibition of T cell chemotaxis by LIMK1 knockdown likely result from inhibition of localized LIMK1 activation and cofilin phosphorylation that are required for polarized actin polymerization for directional cell migration. The inhibition of HIV-1 infection by LIMK1 knockdown may also result from the decrease of actin-rich membrane protrusions that may be preferred viral entry sites in T cells.
LIMK1; cofilin; chemotaxis; SDF-1; CXCR4; HIV-1; CD4 T cells; Rac1; Pak1; Pak2
Stromal cell-derived factor 1 α (SDF-1α), 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-1α-induced chemotaxis of T lymphocytes. SDF-1α significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1α also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1α-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-1α-induced activation of LIMK1, which means that SDF-1α-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-1α-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-1α-induced chemotactic response of T lymphocytes.
Leading cells require LIMK for matrix degradation and invadopodia formation during collective cell migration.
LIM kinases 1 and 2 (LIMK1/2) are centrally positioned regulators of actin cytoskeleton dynamics. Using siRNA-mediated knockdown or a novel small molecule inhibitor, we show LIMK is required for path generation by leading tumor cells and nontumor stromal cells during collective tumor cell invasion. LIMK inhibition lowers cofilin phosphorylation, F-actin levels, serum response factor transcriptional activity and collagen contraction, and reduces invasion in three-dimensional invasion assays. Although motility was unaffected, LIMK inhibition impairs matrix protein degradation and invadopodia formation associated with significantly faster recovery times in FRAP assays indicative of reduced F-actin stability. When LIMK is knocked down in MDA-MB-231 cells, they lose the ability to lead strands of collectively invading cells. Similarly, when LIMK activity is blocked in cancer-associated fibroblasts, they are unable to lead the collective invasion of squamous carcinoma cells in an organotypic skin model. These results show that LIMK is required for matrix remodeling activities for path generation by leading cells in collective invasion.
In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.
Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells, however, mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor (ADF), cofilin. Here we report that LIMK1 is also involved in the MTs destabilization. In endothelial cells, endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via PDZ domain. MTs destabilization induced by thrombin or nocodazole resulted in decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MTs destabilization whereas kinase-dead mutant of LIMK1 (KD) did not affect MTs stability. Importantly, downregulation of endogenous LIMK1 by siRNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of ROCK2, which phosphorylates and activates LIMK1, decreases dramatically the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD- or siRNA-LIMK1 prevents thrombin induced microtubule destabilization and Factin formation suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.
LIMK1, LIMK-domain-containing kinase 1; ROCK2, Rho kinase 2; ADF, actin depolymerization factor ; PAK, p21 activated kinases; HUVEC, human umbilical vein endothelial cells; MAPs, microtubule-associated proteins; GEF, guanine nucleotide exchange factor; HBSS, Hank’s balanced salt solution; PBS, phosphate buffered saline; BSA, bovine serum albumin; GST, glutathione S-transferase
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.
cytoskeleton; actin depolymerization; LIM-kinase; Rho family GTPases; stress fibers
Prostate cancer (PC) affects a large proportion of the male population, and is primarily driven by androgen receptor (AR) activity. First-line treatment typically consists of reducing AR signaling by hormone-depletion, but resistance inevitably develops over time. One way to overcome this issue is to block AR function via alternative means, preferably by inhibiting protein targets that are more active in tumors than in normal tissue. By staining PC tumor sections, elevated LIM kinase 1 (LIMK1) expression and increased phosphorylation of its substrate cofilin were found to be associated with poor outcome and reduced survival in patients with non-metastatic PC. A LIMK selective small molecule inhibitor (LIMKi) was used to determine whether targeted LIMK inhibition was a potential PC therapy. LIMKi reduced PC cell motility, as well as inhibiting proliferation and increasing apoptosis in androgen-dependent PC cells more effectively than in androgen-independent PC cells. LIMK inhibition blocked ligand-induced AR nuclear translocation, reduced AR protein stability and transcriptional activity, consistent with its effects on proliferation and survival acting via inhibition of AR activity. Furthermore, inhibition of LIMK activity increased αTubulin acetylation and decreased AR interactions with αTubulin, indicating that the role of LIMK in regulating microtubule dynamics contributes to AR function. These results indicate that LIMK inhibitors could be beneficial for the treatment of PC both by reducing nuclear AR translocation, leading to reduced proliferation and survival, and by inhibiting PC cell dissemination.
LIM kinase 1 (LIMK1) is an actin and microtubule cytoskeleton modulatory protein that is overexpressed in a number of cancerous tissues and cells and also promotes invasion and metastasis of prostate and breast cancer cells. Membrane type matrix metalloproteinase 1 (MT1-MMP) is a critical modulator of extracellular matrix (ECM) turnover through pericellular proteolysis and thus plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and its substrates pro-MMP-2 and pro-MMP-9 are often overexpressed in a variety of cancers including prostate cancer and the expression levels correlate with the grade of malignancy in prostate cancer cells. The purpose of this study is to determine any functional relation between LIMK1 and MT1-MMP and its implication in cell invasion.
Our results showed that treatment with the hydroxamate inhibitor of MT1-MMP, MMP-2 and MMP-9 ilomastat inhibited LIMK1-induced invasion of benign prostate epithelial cells. Over expression of LIMK1 resulted in increased collagenolytic activity of MMP-2, and secretion of pro-MMP2 and pro-MMP-9. Cells over expressing LIMK1 also exhibited increased expression of MT1-MMP, transcriptional activation and its localization to the plasma membrane. LIMK1 physically associates with MT1-MMP and is colocalized with it to the Golgi vesicles. We also noted increased expression of both MT1-MMP and LIMK1 in prostate tumor tissues.
Our results provide new information on regulation of MT1-MMP function by LIMK1 and showed for the first time, involvement of MMPs in LIMK1 induced cell invasion.
The polarity protein Par-3 plays critical roles in axon specification and the establishment of epithelial apico-basal polarity. Par-3 associates with Par-6 and atypical protein kinase C and is required for the proper assembly of tight junctions, but the molecular basis for its functions is poorly understood. We now report that depletion of Par-3 elevates the phosphorylated pool of cofilin, a key regulator of actin dynamics. Expression of a nonphosphorylatable mutant of cofilin partially rescues tight junction assembly in cells lacking Par-3, as does the depletion of LIM kinase 2 (LIMK2), an upstream kinase for cofilin. Par-3 binds to LIMK2 but not to the related kinase LIMK1. Par-3 inhibits LIMK2 activity in vitro, and overexpressed Par-3 suppresses cofilin phosphorylation that is induced by lysophosphatidic acid. Our findings identify LIMK2 as a novel target of Par-3 and uncover a molecular mechanism by which Par-3 could regulate actin dynamics during cell polarization.
Bone morphogenetic proteins (BMPs) regulate multiple cellular processes, including cell differentiation and migration. Their signals are transduced by the kinase receptors BMPR-I and BMPR-II, leading to Smad transcription factor activation via BMPR-I. LIM kinase (LIMK) 1 is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin depolymerizing factor. During a search for LIMK1-interacting proteins, we isolated clones encompassing the tail region of BMPR-II. Although the BMPR-II tail is not involved in BMP signaling via Smad proteins, mutations truncating this domain are present in patients with primary pulmonary hypertension (PPH). Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4. A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1. This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.
LIM kinase 1; BMPR-II; cytoskeleton; F-actin; cofilin
The cytoskeleton determines cell shape and is involved in cell motility. It also plays a role in differentiation and in modulating specialized cellular functions. LIM kinase 1 (LIMK1) participates in cytoskeletal remodeling by phosphorylating and inactivating the actin-severing protein, cofilin. Severing F-actin to release G-actin monomers is required for actin cytoskeletal remodeling. Although less well established, LIMK1 may also influence the cell cycle and modulate metalloproteinase activity. Since the role of LIMK1 in bone cell biology has not been reported, the skeletal phenotype of LIMK1−/− mice was examined. LIMK1−/− mice had significantly reduced trabecular bone mass when analyzed by micoCT (p <0.01). Histomorphometric analyses demonstrated a 31% reduction in the number osteoblasts (p=0.0003) and a 23% reduction in osteoid surface (p=0.0005). The number of osteoclasts was no different in control and knock out animals. Consistent with the in vivo findings in osteoblasts, the number of osteoblast colony forming units in LIMK1−/− bone marrow was reduced by nearly 50%. Further, osteoblasts isolated from LIMK1−/− mice showed significantly reduced rates of mineralization in vitro. Osteoclasts from LIMK1−/− mice evidenced more rapid cytoskeletal remodeling in response to treatment with CSF1. In keeping with this latter finding, basal levels of phospho-cofilin were reduced in LIMK1−/− osteoclasts. LIMK1−/− osteoclasts also resorbed dentine slices to a greater extent in vitro and were more active in a pit assay. These data support the hypothesis that LIMK1 is required for normal osteoblast differentiation. In addition, its absence leads to increased cytoskeletal remodeling and bone resorption in osteoclasts.
osteopenia; LIM kinase 1; osteoblasts; osteoclasts; CSF1
Nischarin is a novel protein that regulates cell migration by inhibiting p21-activated kinase (PAK). LIM kinase (LIMK) is a downstream effector of PAK, and it is known to play an important role in cell invasion. Here we show that nischarin also associates with LIMK to inhibit LIMK activation, cofilin phosphorylation, and LIMK-mediated invasion of breast cancer cells, suggesting that nischarin regulates cell invasion by negative modulation of the LIMK/cofilin pathway. The amino terminus of nischarin binds to the PDZ and kinase domains of LIMK. Although LIMK activation enhances the interaction with nischarin, only phosphorylation of threonine 508 of LIMK is crucial for the interaction. Inhibition of endogenous nischarin expression by RNA interference stimulates breast cancer cell invasion. Also, nischarin small interfering RNA (siRNA) enhances cofilin phosphorylation. In addition, knock-down of nischarin showed branched projection actin structures. Collectively these data indicate that nischarin siRNA may enhance random migration, resulting in stimulation of invasion.
The assembly and release of retroviruses from the host cells require dynamic interactions between viral structural proteins and a variety of cellular factors. It has been long speculated that the actin cytoskeleton is involved in retrovirus production, and actin and actin-related proteins are enriched in HIV-1 virions. However, the specific role of actin in retrovirus assembly and release remains unknown. Here we identified LIM kinase 1 (LIMK1) as a cellular factor regulating HIV-1 and Mason-Pfizer monkey virus (M-PMV) particle release. Depletion of LIMK1 reduced not only particle output but also virus cell-cell transmission and was rescued by LIMK1 replenishment. Depletion of the upstream LIMK1 regulator ROCK1 inhibited particle release, as did a competitive peptide inhibitor of LIMK1 activity that prevented cofilin phosphorylation. Disruption of either ROCK1 or LIMK1 led to enhanced particle accumulation on the plasma membrane as revealed by total internal reflection fluorescence microscopy (TIRFM). Electron microscopy demonstrated a block to particle release, with clusters of fully mature particles on the surface of the cells. Our studies support a model in which ROCK1- and LIMK1-regulated phosphorylation of cofilin and subsequent local disruption of dynamic actin turnover play a role in retrovirus release from host cells and in cell-cell transmission events.
IMPORTANCE Viruses often interact with the cellular cytoskeletal machinery in order to deliver their components to the site of assembly and budding. This study indicates that a key regulator of actin dynamics at the plasma membrane, LIM kinase, is important for the release of viral particles for HIV as well as for particle release by a distantly related retrovirus, Mason-Pfizer monkey virus. Moreover, disruption of LIM kinase greatly diminished the spread of HIV from cell to cell. These findings suggest that LIM kinase and its dynamic modulation of the actin cytoskeleton in the cell may be an important host factor for the production, release, and transmission of retroviruses.
Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine–threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have made opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression (Zebda, N., O. Bernard, M. Bailly, S. Welti, D.S. Lawrence, and J.S. Condeelis. 2000. J. Cell Biol. 151:1119–1128; Davila, M., A.R. Frost, W.E. Grizzle, and R. Chakrabarti. 2003. J. Biol. Chem. 278:36868–36875; Yoshioka, K., V. Foletta, O. Bernard, and K. Itoh. 2003. Proc. Natl. Acad. Sci. USA. 100:7247–7252; Nishita, M., C. Tomizawa, M. Yamamoto, Y. Horita, K. Ohashi, and K. Mizuno. 2005. J. Cell Biol. 171:349–359). We resolve this paradox by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells.
In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.
It is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation.
Agents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1.
We have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.
Damnacanthal is identified as an effective inhibitor of LIM-kinase. It inhibits chemotaxis of T-cells and migration and invasion of breast carcinoma cells in culture and hapten-induced migration of epidermal Langerhans cells in mouse ears. Damnacanthal is a useful tool for investigating the cellular and physiological functions of LIM-kinase.
LIM-kinases (LIMKs) play crucial roles in various cell activities, including migration, division, and morphogenesis, by phosphorylating and inactivating cofilin. Using a bimolecular fluorescence complementation assay to detect the actin–cofilin interaction, we screened LIMK1 inhibitors and identified two effective inhibitors, damnacanthal (Dam) and MO-26 (a pyrazolopyrimidine derivative). These compounds have already been shown to inhibit Lck, a Src family tyrosine kinase. However, in vitro kinase assays revealed that Dam inhibited LIMK1 more effectively than Lck. Dam suppressed LIMK1-induced cofilin phosphorylation and deceleration of actin retrograde flow in lamellipodia in N1E-115 cells. Dam impaired CXCL12-induced chemotactic migration of Jurkat T lymphocytes and Jurkat-derived, Lck-deficient JCaM1.6 cells and also inhibited serum-induced migration and invasion of MDA-MB-231 breast carcinoma cells. These results suggest that Dam has the potential to suppress cell migration and invasion primarily through the inhibition of LIMK kinase activity. Topical application of Dam also suppressed hapten-induced migration of epidermal Langerhans cells in mouse ears. Dam provides a useful tool for investigating cellular and physiological functions of LIMKs and holds promise for the development of agents against LIMK-related diseases. The bimolecular fluorescence complementation assay system used in this study will provide a useful method to screen for inhibitors of various protein kinases.
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.
Listeria monocytogenes; cytoskeleton; Met; InlB; actin-based motility