Identification of Lim-kinase

A new brain serine/threonine protein kinase may be a target for the p21ras-related proteins Cdc42 and Rac1. The kinase sequence is related to that of the yeast protein STE20, implicated in pheromone-response pathways. The kinase complexes specifically with activated (GTP-bound) p21, thereby inhibiting p21 GTPase activity and leading to kinase autophosphorylation and activation. Autophosphorylated kinase has a decreased affinity for Cdc42/Rac, freeing the p21 for further stimulatory activities or downregulation by GTPase-activating proteins. This bimolecular interaction provides a model for studying p21 regulation of mammalian phosphorylation signalling pathways (Manseur, 1994).

By low-stringency screening of a human hepatoma HepG2 cell cDNA library, using the genomic fragment of chick c-sea receptor tyrosine kinase as a probe, overlapping cDNAs were isolated encoding a novel protein kinase, which was termed LIM-kinase (LIMK). The predicted open reading frame encodes a 647-amino-acid polypeptide containing a putative protein kinase structure in the C-terminal half. In addition, LIMK has two repeats of cysteine-rich LIM/double zinc finger motif at the most N-terminus. This is the first protein kinase seen to contain the LIM motif(s) in the molecule. Although the protein kinase domain of LIMK has highly conserved sequence elements of protein kinases, phylogenetic analysis revealed that LIMK cannot be classified into any subfamily of known protein kinases. Northern blot analysis revealed that the single species of LIMK mRNA of 3.3 kb is expressed in various human epithelial and hematopoietic cell lines. In rat tissues, LIMK mRNA is expressed in the brain, at the highest level. LIM is suggested to be involved in protein-protein interactions by binding to another LIM motif. Since the LIM domain is frequently present in the homeodomain-containing transcriptional regulators and oncogenic nuclear proteins, LIMK may be involved in developmental or oncogenic processes through interactions with these LIM-containing proteins (Mizuno, 1994).

Expression of Lim-kinase

The murine Limk1 gene is a single copy gene located at the distal end of mouse chromosome 5. Limk1 exhibits a 95% homology to the human homologue, LIMK, which contains two LIM domains and a putative protein kinase domain. Although Limk1 and LIMK contain all motifs found in catalytic kinase domains, amino acids previously described to be diagnostic of either serine/threonine- or tyrosine-kinases are not present. It is demonstrated that GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro and that mutation of residue D460 within the IHRDL motif abolishes kinase activity. Northern blot showed preferential expression of a 3.5 kb message in adult spinal cord and brain. In situ hybridisation confirmed high expression levels in the nervous system, particularly in the spinal cord and the cranial nerve and dorsal root ganglia. Limk1 also contains two tandem LIM-domains. These zinc-finger like domains can mediate protein-protein interactions and have been described in nuclear and cytoskeletal proteins. The combination of LIM- and kinase domains may provide a novel route by which intracellular signalling can be integrated (Proschel, 1995).

Throughout vertebrate embryogenesis, membrane bound and intracellular protein kinases govern the fundamental decisions necessary for coordinated cell growth and differentiation. Limk, a novel protein kinase with serine threonine substrate specificity, also contains two LIM domains. Northern blot and in situ hybridization techniques were used to determine its pattern of expression in early mouse development. Between 7.5 and 8.5 d.p.c., limk is expressed in three broad domains within the embryo, the neuroectodermal of the prospective forebrain and mid-brain regions, the cardiac mesoderm, and the newly formed definitive endodermal derivatives the foregut and hindgut. By 10.0 d.p.c. limk remains prominently expressed in the ventromedial regions of the developing forebrain and midbrain, with continued expression in the hindgut. In adults limk is expressed most prominently in the brain. Additionally, limk is most abundantly expressed in the trophoblast giant cells, from 4.5 d.p.c. onwards. Moreover, high levels of limk expression are associated with the overt formation of giant cells from diploid progenitors, suggesting an involvement for limk in the differentiation of this highly specialized extra-embryonic cell type (Cheng, 1995).

Two novel serine/threonine kinase genes containing the LIM motif (LIMK-1 and LIMK-2) were isolated from a rat cDNA library. To examine the functions of these genes, in situ hybridization was performed in the developing rat nervous system. LIMK-1 and LIMK-2 mRNAs mostly co-localize during development and are expressed preferentially in the central nervous system during mid-to-late gestation but the signals decrease during the post-natal period. However, differential gene expression was observed in some nuclei in the CNS; LIMK-1 mRNA is intensely expressed in the facial motor nucleus, the hypoglossal nucleus, deep nuclei of the cerebellum and the layers 3, 5 and 6 of the adult cerebral cortex while only LIMK-2 mRNA is preferentially expressed in the some parts of the epithelium. In the nasal cavity, LIMK-1 and LIMK-2 mRNAs are expressed complementarily. These results suggest that LIMK-1 and LIMK-2 may have different functions in these regions during development (Mori, 1997).

LIM kinase 1 (LIMK1) is a cytoplasmic protein kinase that is highly expressed in neurons. In transfected cells, LIMK1 binds to the cytoplasmic tail of neuregulins and regulates the breakdown of actin filaments. To identify potential functions of LIMK1 in vivo, the subcellular distribution of LIMK1 protein was determined within neurons of the rat by using immunomicroscopy. At neuromuscular synapses in the adult hindlimb, LIMK1 is concentrated in the presynaptic terminal. However, little LIMK1 immunoreactivity is detected at neuromuscular synapses before the 2nd week after birth, and most motoneuron terminals are not strongly LIMK1 immunoreactive until the 3rd week after birth. Thus, LIMK1 accumulation at neuromuscular synapses coincides with their maturation. In contrast, SV2, like many other presynaptic terminal proteins, can be readily detected at neuromuscular synapses in the embryo. Similar to its late accumulation at developing synapses, LIMK1 accumulation at regenerating neuromuscular synapses occurs long after these synapses first form. In the adult ventral spinal cord, LIMK1 is concentrated in a subset of presynaptic terminals. LIMK1 gradually accumulates at spinal cord synapses postnatally, reaching adult levels only after P14. This study is the first to implicate LIMK1 in the function of presynaptic terminals. The concentration of LIMK1 in adult, but not nascent, presynaptic terminals suggests a role for this kinase in regulating the structural or functional characteristics of mature synapses (Wang, 2000).

The olfactory epithelium is the only neuronal tissue capable of generating new neurons during adult life and hence must express genes responsible for this phenomenon. Therefore, mRNA from immortalized olfactory epithelial cells was used to search for novel protein tyrosine kinases by polymerase chain reaction, using as primers conserved sequences from the catalytic domain of known kinase genes. A full-length complementary DNA clone corresponding to one such polymerase chain reaction product was isolated and sequenced. This complementary DNA, designated Kiz-1 (LIMK1), encodes a protein containing two prominent domains; the NH2-terminal region contains a cysteine/histidine-rich moiety previously identified as a zinc-finger domain in proteins of the LIM family, while the COOH-terminus contains a kinase domain. Kiz-1 is expressed mainly in the brain of adult mice but also in a range of cultured cell lines, regardless of their tissue of origin. Immunohistochemical studies on adult mouse brain demonstrated that Kiz-1 is expressed exclusively in neurons, not in astrocytes or oligodendrocytes. In the developing embryo, however, Kiz-1 is expressed in all tissues. In COS cells transfected with Kiz-1 complementary DNA and in the immortalized olfactory epithelial cells, Kiz-1 was found mainly in the cytoplasm, but in neurons of the adult brain, it resided also in the nucleus. Two Kiz-1 mRNA species are expressed in cell lines as well as in the murine and human brain. One transcript lacks a region of 60 nucleotides. This region lies within the catalytic domain of the kinase and is encoded by a separate exon. These results suggest that Kiz-1 may play distinct roles in dividing cells and in differentiated neurons (Bernard, 2004).

Signaling upstream of LIM-kinase

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) are respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylates cofilin and induces formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation is stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induces stress fibers and filopodia, whereas both Rho-induced stress fibers and Cdc42-induce filopodia are abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac does 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 (Sumi, 1999).

Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. 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 (Edwards, 1999).

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 (Lawler, 1999) as it phosphorylates and inactivates cofilin, an actin depolymerizing factor. During a search for LIMK1-interacting proteins, clones encompassing the tail region of BMPR-II were isolated. 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 (Foletta, 2003).

The growth and morphological differentiation of dendrites are critical events in the establishment of proper neuronal connectivity and neural function. One extrinsic factor, BMP7, has been shown to specifically affect dendritic morphogenesis; however, the underlying mechanism by which this occurs is unknown. This study shows that LIM kinase 1 (LIMK1), a key downstream effector of Rho GTPases, colocalizes with the BMP receptor, BMPRII, in the tips of neurites and binds to BMPRII. This interaction is required for BMP-dependent induction of the dendritic arbor in cortical neurons. Furthermore, the physical interaction of LIMK1 with BMPRII synergizes with the Rho GTPase, Cdc42, to activate LIMK1 catalytic activity. These studies thus define a Smad-independent pathway that directly links the BMP receptor to regulation of actin dynamics and provides insights into how extracellular signals modulate LIMK1 activity to permit fine spatial control over cytoskeletal remodelling during dendritogenesis (Lee-Hoeflich, 2004).

LATS (large tumour suppressor) is a family of conserved tumour suppressors identified in Drosophila and mammals. Human LATS1 binds to LIMK1 in vitro and in vivo and colocalizes with LIMK1 at the actomyosin contractile ring during cytokinesis. LATS1 inhibits both the phosphorylation of cofilin by LIMK1 and LIMK1-induced cytokinesis defects. Inactivation of LATS1 by antibody microinjection or RNA-mediated interference in cells, or gene knockout in mice, abrogates cytokinesis and increases the percentage of multinucleate cells. These findings indicate that LATS1 is a novel cytoskeleton regulator that affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1 (X. Yang, 2004).

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, it has been demonstrated that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predict two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulates LIMK2 phosphorylation at Ser-283 and inhibits nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicates that LIMK2 phosphorylation at Ser-283 but not Thr-494 is functional. Serum stimulation of endothelial cells also inhibits nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. This study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhances cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, it is suggestd that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2 (Goyal, 2005).

Myelin-associated inhibitors (MAIs) signal through a tripartate receptor complex on neurons to limit axon regeneration in the CNS. Inhibitory influences ultimately converge on the cytoskeleton to mediate growth cone collapse and neurite outgrowth inhibition. Rho GTPase and its downstream effector Rho kinase are key signaling intermediates in response to MAIs; however, the links between Rho and the actin cytoskeleton have not been fully defined. This study found that Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM kinase and Slingshot (SSH) phosphatase to regulate the phosphorylation profile of the actin depolymerization factor cofilin. Blockade of LIMK1 activation and subsequent cofilin phosphorylation circumvents myelin-dependent inhibition in chick dorsal root ganglion neurons, suggesting that phosphorylation and inactivation of cofilin is critical for neuronal inhibitory responses. Subsequent activation of SSH1 phosphatase mediates cofilin dephosphorylation and reactivation. Overexpression of SSH1 does not mimic the neurite outgrowth inhibitory effects of myelin, suggesting an alternative role in MAI inhibition. It is speculated that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors (Hsieh, 2006).

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. Activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induces LIMK1 activation and cofilin phosphorylation, and this is inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylates LIMK1 at Ser-310, it fails to activate LIMK1 directly; however, MK2 activates LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppresses VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhances these processes. Knockdown of MK2 by siRNA suppresses VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppresses VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells (Kobayashi, 2006).

LIM-kinase acts upstream of cofilin

Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. A pathway has been identified for the regulation of cofilin (see Drosophila Twinstar), a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinase 1 inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augments cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibits these processes. These results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis (Arber, 1998).

Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at position 3; however, the kinases responsible for this phosphorylation have not been identified. LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, these results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin (Yang, 1998).

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. The role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse was investigated. Semaphorin 3A induces phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibits LIM-kinase in vitro and in vivo, and essentially suppresses semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which can not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A (Aizawa, 2001).

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. The role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes was investigated. SDF-1alpha significantly induces the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induces cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibits 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, blocks SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. A cell-permeable peptide (S3 peptide) was used that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibits the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibits 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 (Nishita, 2002)

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, ADF/cofilin's activity was inhibited in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). Expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation (Zebda, 2000).

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 induces rapid actin polymerization into stress fibers and concomitantly activates RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants block TGF-beta1-induced actin reorganization. Since Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), it was found that TGF-beta1 induces LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitate and cofilin becomes phosphorylated in response to TGF-beta1, while RNA interference against LIMK2 blocks formation of new stress fibers by TGF-beta1. Since the kinase ROCK1 links Rho GTPases to LIMK2, it was found that inhibiting ROCK1 activity completely blocks TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. Whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization was tested. Adenoviruses expressing constitutively activated TGF-beta type I receptor leads to robust actin reorganization and Rho activation, while the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) does not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibits TGF-beta1-induced Rho activation and cytoskeletal reorganization. These data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2 (Vardouli, 2005).

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. 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. These 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 (Chen, 2006).

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 reached 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. This paradox has been resolved 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 (Wang, 2006).

Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, this study established that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. It is proposed that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals (Delorme, 2007).

A multi-protein complex consisting of Slingshot, Lim kinases, actin, 14-3-3 and cofilin regulate ADF/cofilin activity

Slingshot (SSH) phosphatases (see Drosophila Slingshot) and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. 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. 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. A novel ADF/cofilin phosphoregulatory complex is described, and a new mechanism is suggested for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton (Soosairajah, 2005).

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. Cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppresses chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produces and retaines multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. These results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. It is proposed that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement (Nishita, 2005).

LIM kinase 1 activates cAMP-responsive element-binding protein during the neuronal differentiation of immortalized hippocampal progenitor cells

LIM kinase 1 (LIMK1), a novel member of a subclass of the protein-serine/threonine kinases, is known to play a role in the development and maintenance of neuronal circuits that mediate cognitive function. Genetic studies have implicated a mutation of LIMK1 as a causative factor in the impairment of visuospatial cognition in a neurodevelopmental disorder, Williams syndrome. A transcriptional factor, cAMP-responsive element-binding protein (CREB), is thought to be involved in the formation of many types of synaptic plasticity involving learning and memory. LIMK1 activity is markedly induced during the differentiation of immortalized hippocampal progenitor (H19-7) cells. The addition of neurogenic growth factor to H19-7 cells induces specific binding between LIMK1 and active CREB, LIMK1 directly phosphorylates CREB, and this leads to the stimulation of subsequent cAMP-responsive element-mediated gene transcription during H19-7 cell neuronal differentiation. In addition, LIMK1 activation occurs through Rac/Cdc42- and p21-activated kinase-mediated signaling pathways. Moreover, when the plasmid encoding kinase-inactive LIMK1 is transfected to block the activation of endogenous LIMK1, the neuronal differentiation of H19-7 cells is significantly suppressed. These findings suggest that LIMK1 activation and subsequent CREB phosphorylation are important in the neuronal differentiation of central nervous system hippocampal progenitor cells (E. J. Yang, 2004)

Phosphorylation of actin-depolymerizing factor/cofilin by LIM-kinase mediates amyloid beta-induced degeneration: a potential mechanism of neuronal dystrophy in Alzheimer's disease

Deposition of fibrillar amyloid beta (fAbeta) plays a critical role in Alzheimer's disease (AD). fAbeta-induced dystrophy requires the activation of focal adhesion proteins and the formation of aberrant focal adhesion structures, suggesting the activation of a mechanism of maladaptative plasticity in AD. Focal adhesions are actin-based structures that provide a structural link between the extracellular matrix and the cytoskeleton. To gain additional insight in the molecular mechanism of neuronal degeneration in AD, this study explored the involvement of LIM kinase 1 (LIMK1), actin-depolymerizing factor (ADF), and cofilin in Abeta-induced dystrophy. ADF/cofilin are actin-binding proteins that play a central role in actin filament dynamics, and LIMK1 is the kinase that phosphorylates and thereby inhibits ADF/cofilin. The data indicate that treatment of hippocampal neurons with fAbeta increases the level of Ser3-phosphorylated ADF/cofilin and Thr508-phosphorylated LIMK1 (P-LIMK1), accompanied by a dramatic remodeling of actin filaments, neuritic dystrophy, and neuronal cell death. A synthetic peptide, S3 peptide, which acts as a specific competitor for ADF/cofilin phosphorylation by LIMK1, inhibits fAbeta-induced ADF/cofilin phosphorylation, preventing actin filament remodeling and neuronal degeneration, indicating the involvement of LIMK1 in Abeta-induced neuronal degeneration in vitro. Immunofluorescence analysis of AD brain showed a significant increase in the number of P-LIMK1-positive neurons in areas affected with AD pathology. P-LIMK1-positive neurons also show early signs of AD pathology, such as intracellular Abeta and pretangle phosphorylated tau. Thus, LIMK1 activation may play a key role in AD pathology (Heredia, 2006).

LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the 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, cofilin. LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole results in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 results in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) does not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA results in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. These findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton (Gorovoy, 2005).

Control of growth cone motility and morphology by LIM kinase and Slingshot via phosphorylation and dephosphorylation of cofilin

Growth cone motility and morphology are based on actin-filament dynamics. Cofilin plays an essential role for the rapid turnover of actin filaments by severing and depolymerizing them. The activity of cofilin is repressed by phosphorylation at Ser3 by LIM kinase (LIMK, in which LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) and is reactivated by dephosphorylation by phosphatases, termed Slingshot (SSH). The roles of cofilin, LIMK, and SSH in the growth cone motility and morphology and neurite extension were investigated by expressing fluorescence protein-labeled cofilin, LIMK1, SSH1, or their mutants in chick dorsal root ganglion (DRG) neurons and then monitoring live images of growth cones by time-lapse video fluorescence microscopy. The expression of LIMK1 remarkably represses growth cone motility and neurite extension, whereas the expression of SSH1 or a nonphosphorylatable S3A mutant of cofilin enhances these events. The fan-like shape of growth cones was disorganized by the expression of any of these proteins. The repressive effects on growth cone behavior by LIMK1 expression were significantly rescued by the coexpression of S3A-cofilin or SSH1. These findings suggest that LIMK1 and SSH1 play critical roles in controlling growth cone motility and morphology and neurite extension by regulating the activity of cofilin and may be involved in signaling pathways that regulate stimulus-induced growth cone guidance. Using various mutants of cofilin, evidence was obtained that the actin-filament-severing activity of cofilin is critical for growth cone motility and neurite extension (Endo, 2003).

BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin

Bone morphogenic proteins (BMPs) are involved in axon pathfinding, but how they guide growth cones remains elusive. This study reports that a BMP7 gradient elicits bidirectional turning responses from nerve growth cones by acting through LIM kinase (LIMK) and Slingshot (SSH) phosphatase to regulate actin-depolymerizing factor (ADF)/cofilin-mediated actin dynamics. Xenopus laevis growth cones from 4-8-h cultured neurons are attracted to BMP7 gradients but become repelled by BMP7 after overnight culture. The attraction and repulsion are mediated by LIMK and SSH, respectively, which oppositely regulate the phosphorylation-dependent asymmetric activity of ADF/cofilin to control the actin dynamics and growth cone steering. The attraction to repulsion switching requires the expression of a transient receptor potential (TRP) channel TRPC1 and involves Ca2+ signaling through calcineurin phosphatase for SSH activation and growth cone repulsion. Together, this study shows that spatial regulation of ADF/cofilin activity controls the directional responses of the growth cone to BMP7, and Ca2+ influx through TRPC tilts the LIMK-SSH balance toward SSH-mediated repulsion (Wen, 2007).

Role of LIM kinases in normal and psoriatic human epidermis

Evidence is presented that LIM kinases can control cell adhesion and compaction in human epidermis. LIMK2 is expressed in the epidermal basal layer and signals downstream of the GTPase Rac1 to promote extracellular matrix adhesion and inhibit terminal differentiation. Conversely, LIMK1 is expressed in the upper granular layers and phosphorylates and inhibits cofilin. Expression of LIMK1 is lost in psoriatic lesions and other skin disorders characterized by lack of cell compaction in the differentiating cell layers. In psoriatic lesions down-regulation of LIMK1 correlates with up-regulation of Myc. Expression of constitutively active cofilin or Myc in reconstituted human epidermis blocks cell compaction. Overexpression of LIMK1 leads to down-regulation of Myc, whereas inhibition of Rho kinase, an upstream activator of LIMK1, stimulates Myc expression. Inhibition of Myc by LIMK1 is via inhibition of Stat3 phosphorylation, because constitutively active cofilin or inhibition of Rho kinase results in Stat3 phosphorylation and increased Myc levels, whereas dominant negative Stat3 abolishes the effect. In conclusion, a novel antagonistic relationship has been uncovered between the LIMK1/phosphocofilin and Myc/Stat3 pathways in the differentiating layers of human epidermis and it is proposed that down-regulation of LIMK1 contributes to one of the pathological features of psoriatic epidermal lesions (Honma, 2006).

Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylating and inactivating cofilin, an actin-depolymerizing factor of actin filaments. A detailed analysis is described of the cell-cycle-dependent activity of LIMK2, and a subcellular localization of LIMK1 and LIMK2. The activity of LIMK2, distinct from LIMK1, toward cofilin phosphorylation does not change in the normal cell division cycle. In contrast, LIMK2 is hyperphosphorylated and its activity is markedly increased when HeLa cells are synchronized at mitosis with nocodazole treatment. Immunofluorescence analysis showed that LIMK1 is localized at cell-cell adhesion sites in interphase and prophase, redistributes to the spindle poles during prometaphase to anaphase, and accumulates at the cleavage furrow in telophase. In contrast, LIMK2 is diffusely localized in the cytoplasm during interphase, redistributed to the mitotic spindle, and finally to the spindle midzone during anaphase to telophase. These findings suggest that LIMK2 is activated in response to microtubule disruption, and that LIMK1 and LIMK2 may play different roles in regulating for the mitotic spindle organization, chromosome segregation, and cytokinesis during the cell division cycle (Sumi, 2006).

Mutation of Lim-kinase

To identify genes important for human cognitive development, Williams syndrome (WS), a developmental disorder that includes poor visuospatial constructive cognition, was studied. Two families with a partial WS phenotype are described; affected members have the specific WS cognitive profile and vascular disease, but lack other WS features. Submicroscopic chromosome 7q11.23 deletions cosegregate with this phenotype in both families. DNA sequence analyses of the region affected by the smallest deletion (83.6 kb) have revealed two genes, elastin (ELN) and LIM-kinase1 (LIMK1). The latter encodes a novel protein kinase with LIM domains and is strongly expressed in the brain. Because ELN mutations cause vascular disease but not cognitive abnormalities, these data implicate LIMK1 hemizygosity in imparied visuospatial constructive cognition (Frangiskakis, 1996).

In vitro studies indicate a role for the LIM kinase family in the regulation of cofilin phosphorylation and actin dynamics. In addition, abnormal expression of LIMK-1 is associated with Williams syndrome, a mental disorder with profound deficits in visuospatial cognition. However, the in vivo function of this family of kinases remains elusive. Using LIMK-1 knockout mice, a significant role has been demonstrated for LIMK-1 in vivo in regulating cofilin and the actin cytoskeleton. Furthermore, the knockout mice exhibit significant abnormalities in spine morphology and in synaptic function, including enhanced hippocampal long-term potentiation. The knockout mice also show altered fear responses and spatial learning. These results indicate that LIMK-1 plays a critical role in dendritic spine morphogenesis and brain function (Meng, 2002).

The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. The role of the RING finger protein Rnf6 during neuronal development has been investigated and high Rnf6 protein levels were detected in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, it was found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons; the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics (Tursun, 2005).

A brain-specific microRNA regulates dendritic spine development acting through Limk1

MicroRNAs are small, non-coding RNAs that control the translation of target messenger RNAs, thereby regulating critical aspects of plant and animal development. In the mammalian nervous system, the spatiotemporal control of mRNA translation has an important role in synaptic development and plasticity. Although a number of microRNAs have been isolated from the mammalian brain, neither the specific microRNAs that regulate synapse function nor their target mRNAs have been identified. This study shows that a brain-specific microRNA, miR-134, is localized to the synapto-dendritic compartment of rat hippocampal neurons and negatively regulates the size of dendritic spines -- postsynaptic sites of excitatory synaptic transmission. This effect is mediated by miR-134 inhibition of the translation of an mRNA encoding a protein kinase, Limk1, that controls spine development. Exposure of neurons to extracellular stimuli such as brain-derived neurotrophic factor relieves miR-134 inhibition of Limk1 translation and in this way may contribute to synaptic development, maturation and/or plasticity (Schratt, 2006).

LIM-kinase1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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