Expression Of LIM Kinase 1 Research Articles

MiR-20a inhibits cutaneous squamous cell carcinoma metastasis and proliferation by directly targeting LIMK1

Background MicroRNA-20a (miR-20a) plays a key role in tumorigenesis and progression. But its function is reverse in different kinds of malignant tumor, and its role and mechanism in cutaneous squamous cell carcinoma (CSCC) remains unclear. Object To determine the miR-20a’s roles in CSCC and confirm whether LIMK1 is a direct target gene of miR-20a. Methods First miR-20a and LIMK1 expression levels were detected in six pairs of CSCC tissues and corresponding normal skin by qRT-PCR. Then MTT assays and colony formation assays were performed to evaluate the impact of miR-20a on cell proliferation. In addition, scratch migration assays and transwell invasion assays were performed to check miR-20a’s effect on cell metastasis. Since LIMK1 (LIM kinase-1) was predicted as a target gene of miR-20a, the changes of LIMK1 protein and mRNA were measured by western blot and qRT-RCR methods after miR-20a overexpression. Moreover the dual reporter gene assay was performed to confirm whether LIMK1 is a direct target gene of miR-20a. Finally LIMK1 mRNA and miR-20a in other 30 cases of CSCC pathological specimens were determined and a correlation analysis was evaluated. Results The miR-20a significantly low-expressed in CSCC tissues compared with that in matched normal tissues while LIMK1 has a relative higher expression. MiR-20a inhibited A431 and SCL-1 proliferation and metastasis. Both of LIMK1 protein and mRNA levels were downregulated after miR-20a overexpression. The dual reporter gene assays revealed that LIMK1 is a direct target gene of miR-20a. Furthermore, qRT-PCR results of LIMK1 mRNA and miR-20a in 30 cases of CSCC pathological specimens showed miR-20a is inversely correlated with LIMK1 expression. Conclusion Our study demonstrated that miR-20a is involved in the tumor inhibition of CSCC by directly targeting LIMK1 gene. This finding provides potential novel strategies for therapeutic interventions of CSCC.

LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission

Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27(Kip1) expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27(Kip1)-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.

MiR-27b targets LIMK1 to inhibit growth and invasion of NSCLC cells

Non-small cell lung cancer (NSCLC), which accounts for ~80 % of lung cancer cases, is one of the most common causes for cancer-related death. microRNAs (miRNAs) have been found to play critical roles in the development and progression of NSCLC. miR-27b has recently been reported as a tumor suppressor in several cancers, but its role in NSCLC remains poorly understood. In this study, we found that miR-27b was remarkably decreased in both NSCLC tissues and cell lines. Moreover, overexpression of miR-27b significantly suppressed NSCLC cells proliferation and invasion. LIM kinase 1 (LIMK1), an essential protein for malignant transformation, was found to be a target of miR-27b. Ectopic expression of LIMK1 dramatically dampened mir-27b action of cancer inhibition. Finally, LIMK1 was found to be negatively correlated with miR-27b in NSCLC patients. Our results demonstrated a tumor-suppressive role of miR-27b in NSCLC, suggesting a potential therapeutic target for NSCLC.

A conserved region in the 3′ untranslated region of the human LIMK1 gene is critical for proper expression of LIMK1 at the post-transcriptional level

LIM kinase 1 (LIMK1), a cytosolic serine/threonine kinase, regulates actin filament dynamics and reorganization and is involved in neuronal development and brain function. Abnormal expression of LIMK1 is associated with several neurological disorders. In this study, we performed a conservation analysis using Vector NTI (8.0) software. The dualluciferase reporter assay and real-time quantitative RT-PCR were used to assess the protein and mRNA levels of the reporter gene, respectively. We found that a region ranging from nt +884 to +966 in the human LIMK1 3' untranslated region (UTR) was highly conserved in the mouse Limk1 3' UTR and formed a structure containing several loops and stems. Luciferase assay showed that the relative luciferase activity of the mutated construct with the conserved region deleted, pGL4-hLIMK1-3U-M, in SH-SY5Y and HEK-293 cells was only ~60% of that of the wild-type construct pGL4-hLIMK1-3U, indicating that the conserved region is critical for the reporter gene expression. Real-time quantitative RT-PCR analysis demonstrated that the relative Luc2 mRNA levels in SH-SY5Y and HEK293 cells transfected with pGL4-hLIMK1-3U-M decreased to ~50% of that in cells transfected with pGL4-hLIMK1-3U, suggesting an important role of the conserved region in maintaining Luc2 mRNA stability. Our study suggests that the conserved region in the LIMK1 3' UTR is involved in regulating LIMK1 expression at the post-transcriptional level, which may help reveal the mechanism underlying the regulation of LIMK1 expression in the central nervous system and explore the relationship between the 3'-UTR mutant and neurological disorders.

Reduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferation

ObjectiveColorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine...

1023 INVOLVEMENT OF RHO-KINASE/LIM-KINASE/COFILIN SIGNALING PATHWAY IN ERECTILE DYSFUNCTION AND CORPORAL FIBROSIS AFTER CAVERNOUS NERVE INJURY IN MALE RATS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research (II)1 Apr 20131023 INVOLVEMENT OF RHO-KINASE/LIM-KINASE/COFILIN SIGNALING PATHWAY IN ERECTILE DYSFUNCTION AND CORPORAL FIBROSIS AFTER CAVERNOUS NERVE INJURY IN MALE RATS Min Chul Cho, Sang Hoon Song, Sung Yong Cho, Kwanjin Park, Soo Woong Kim, and Jae-Seung Paick Min Chul ChoMin Chul Cho Goyang, Korea, Republic of More articles by this author , Sang Hoon SongSang Hoon Song Seoul, Korea, Republic of More articles by this author , Sung Yong ChoSung Yong Cho Seoul, Korea, Republic of More articles by this author , Kwanjin ParkKwanjin Park Seoul, Korea, Republic of More articles by this author , Soo Woong KimSoo Woong Kim Seoul, Korea, Republic of More articles by this author , and Jae-Seung PaickJae-Seung Paick Seoul, Korea, Republic of More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.608AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Molecular mechanism of corporal fibrosis leading to erectile dysfunction (ED) after cavernous nerve (CN) injury is poorly understood. Recently, we demonstrated that sphingosine-1-phosphate/RhoA/ROCK1 pathway could be involved in corporal fibrosis after CN injury. Activation of ROCK1 is followed by activation of downstream target kinases, LIM-kinase2 (LIMK2) and cofilin, resulting in fibroblast-to-myofibroblast differentiation, a pathophysiologic feature of fibrosis. Thus, the aim of this study was to determine whether LIMK2/cofilin pathway, the downstream effector of ROCK1, was involved in ED and corporal fibrosis after CN injury in male rats. METHODS Forty eight 10-week-old male Sprague-Dawley rats were equally divided into 3 groups: sham surgery (S); bilateral CN crush injury (I); and bilateral CN resection (R). Within each of 3 groups, two subgroups were analyzed as a function of time (1 and 4 weeks postoperatively). At 1 and 4 weeks postoperatively, electrostimulation was performed to assess erectile function by maximal intracavernous pressure (ICP)/mean arterial pressure (MAP) ratio and areas under the curve (AUC). Western blot (ROCK1, phospho/total LIMK2, phospho/total cofilin), immunohistochemistry (α-SMA and phospho-LIMK2) and Masson's trichrome staining were performed. RESULTS At each time point, both I and R groups showed significantly lower ICP/MAP ratio and AUC than S group. Mean ICP/MAP and AUC showed a trend toward higher level in I group than R group, which was not significantly different. At all time points, I and R groups had a significantly decreased SM cell/collagen ratio and expression of α-SMA compared to the S group and a trend toward decreasing over the study period. On immunohistochemical staining, the LIMK2 expression in both I and R groups was significantly higher than that in S group at 1 week, but not at 4 weeks. Densitometry revealed a significantly higher expression of ROCK1 in I and R group compared to S group at 1 and 4 weeks. The ratio of phospho/total LIMK2 protein expression in I and R groups was significantly decreased at 1 week, but not significantly different from that in S group at 4 weeks. The ratio of phospho/total cofilin protein expression in R group significantly decreased starting from 1 week, while that in I group significantly decreased as late as 4 weeks. CONCLUSIONS Our data suggest that LIMK2/cofilin pathway can be involved in ED and corporal fibrosis associated with loss of SM after CN injury, and appears to be functional particularly in the early phase after CN injury. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e419-e420 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Min Chul Cho Goyang, Korea, Republic of More articles by this author Sang Hoon Song Seoul, Korea, Republic of More articles by this author Sung Yong Cho Seoul, Korea, Republic of More articles by this author Kwanjin Park Seoul, Korea, Republic of More articles by this author Soo Woong Kim Seoul, Korea, Republic of More articles by this author Jae-Seung Paick Seoul, Korea, Republic of More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

DADS downregulates the Rac1-ROCK1/PAK1-LIMK1-ADF/cofilin signaling pathway, inhibiting cell migration and invasion

The aim of this study was to explore the molecular mechanisms of the diallyl disulfide (DADS)-mediated downregulation of LIM kinase-1 (LIMK1) and the consequent inhibition of the migration and invasion of human colorectal cancer cells. RNA interference technology was used to establish stable LIMK1-miRNA/SW480 cell lines. The effects of DADS and LIMK1 RNA interference on the migration and invasion of SW480 cells were observed by scratch wound healing assay and Transwell migration assay. The effects of DADS on signaling molecules of the Rac1-Rho kinase (ROCK)1/p21-activated kinase (PAK)1-LIM kinase (LIMK)1-actin depolymerizing factor (ADF)/cofilin pathway in SW480 cells were examined by RT-PCR and western blot analysis. The healing and migration rate of the SW480 cells was significantly reduced and the cell penetrating ability was significantly suppressed (P<0.05) following treatment with DADS (45mg/l). The immunohistochemistry and western blot analysis results showed that DADS significantly downregulated LIMK1 protein expression and suppressed LIMK1 protein phosphorylation. Furthermore, the RT-PCR and western blot analysis results revealed that DADS suppressed Rac1, ROCK1, PAK1, LIMK1 and destrin mRNA and protein expression, as well as the protein phosphorylation of LIMK1 and cofilin1. The data demonstrate that LIMK1 expression positively correlates with the SW480 cell migration and invasion ability. DADS downregulates the Rac1-ROCK1/PAK1-LIMK1-ADF/cofilin signaling pathway, suppressing SW480 cell migration and invasion.

Downregulation of LIMK1 Level Inhibits Migration of Lung Cancer Cells and Enhances Sensitivity to Chemotherapy Drugs

LIM kinase 1 (LIMK1) is a member of a novel class of serine-threonine protein kinases, which plays an important role in malignant transformation. High expression of LIM kinase 1 (LIMK1) has been detected in various invasive cancers. Here, we showed that LIMK1 was overexpressed in non-small cell lung cancer tissues (NSCLC) and cell lines. Expression of LIMK1 was detected in 115 of 150 lung cancer tissues, the frequency being more significant than in lung tissues. In addition, overexpression of LIMK1 was also associated with high TNM stage and lymph node metastasis in NSCLC patients. Moreover, RNAi-mediated suppression of LIMK1 expression markedly inhibited migration and invasion of 801D lung cancer cells. Furthermore, silencing of LIMK1 sensitized 801D cells to chemotherapeutic drugs of cisplatin and gemcitabine. These results indicate that the overexpression of LIMK1 is tightly associated with an aggressive phenotype of lung cancer cells, knockdown of LIMK1 suppressed cell migration and invasion, enhanced chemosensitivity, suggesting a potential therapeutic target for lung cancer.

LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization

Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM kinase 1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow, and lamellipodium extension using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin-stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization, and in MCF-7 cells endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.

Abstract 3613: Down regulation of LIM kinase1 enhances taxane sensitivity of cancer cells

Abstract Taxane compounds, such as docetaxel and paclitaxel, are microtubule-targeted tubulin-polymerizing agents and have proven to be the first line of treatment choice for castration-resistant prostate cancer and tamoxifen-resistant breast cancer cells. However resistance to taxane compounds develops commonly due to altered beta tubulin isotypes or development of multidrug resistance (MDR) through activation of p-glycoprotein drug efflux pump. In addition there are serious side effects and systemic toxicity encountered with docetaxel treatment. There is a need to enhance the efficacy of the taxane compounds by identifying novel targets, inhibition of which would induce mitotic arrest and apoptosis. LIM kinase1 (LIMK1) is a serine/threonine kinase, which coordinates microtubule stability and reorganization of actin cytoskleton through inactivating phosphorylation of cofilin, and is involved in regulation of the mitotic process. LIMK1 is overexpressed in breast and prostate tumors and in breast and prostate cancer cells in culture. Ectopic expression of LIMK1 promotes altered cell cycle progression and mitotic defects. Furthermore, LIMK1 expression prevents normal cellular response to paclitaxel in prostate epithelial cells, such as metaphase arrest and retarded cell growth. In this study, we show that ectopic expression of LIMK1 in BPH-1 cells decreased cytotoxic effects of paclitaxel through reduction of the number of apoptotic cells. Cell viability analysis using MTT assay showed that LIMK1 expressing cells maintained a higher viable cell density compared to control cells following paclitaxel treatment. LIMK1 expression in BPH-1 cells also increased BCL2 expression and decreased translocation of BAX to mitochondria compared to control cells. siRNA-induced down regulation of LIMK1 expression increased docetaxel sensitivity of breast and prostate cancer cells. Flow cytometric analysis of LIMK1 siRNA treated cells showed increased percentage of apoptotic cells compared to control RNA treated cells. Western blot analysis showed increased cleavage of apoptotic markers PARP, caspase 3 and caspase 9 in LIMK1 shRNA transfected prostate cancer cells upon docetaxel treatment compared to cells transfected with scrambled shRNA. Our study suggests that targeted inhibition of LIMK1 expression may potentiate the effects of taxane compounds on breast and prostate cancer cells. This study will have a broad impact on management of castration-resistant prostate and tamoxifen-resistant/ER-apha negative breast cancer cells with increased sensitivity of taxane compounds at a lower dose and hence less systemic toxicity, which could be applicable to other types of cancers as well. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2011-3613

Linking LIMK1 deficiency to hyperacusis and progressive hearing loss in individuals with Williams syndrome

Williams syndrome (a.k.a. Williams-Beuren Syndrome) is a multisystem disorder caused by the hemizygous deletion of a 1.6-Mb region at 7q11.23 encompassing about 26 genes, including that encoding LIM kinase 1 (LIMK1). Individuals with Williams Syndrome manifest hyperacusis and progressive hearing loss, and hyperacusis early onset suggests that it could be associated with one of the deleted genes. Based on our results about the critical role of LIM kinases in the regulation of the motile response of cochlear outer hair cells and cochlear amplification, we propose here that a reduced expression of LIMK1 in outer hair cells would be the major underlying cause of the hyperacusis and progressive hearing loss observed in patients with Williams Syndrome. Moreover, we propose a novel model of gain-control for cochlear amplification based on the LIMK-mediated regulation of slow motility of cochlear outer hair cells.

Abstract 5117: PKD1 suppresses filopodia formation and increases LIM Kinase 1 expression in prostate cancer cells

Abstract Introduction: We have previously demonstrated that transfection of PKD1 into highly metastatic C4-2 prostate cancer cells decreased cell motility. Filopodia are thin, actin-rich plasma-membrane protrusion that have an important role in cell motility and wound healing. Moreover, the initiation and elongation of filopodia depend on the precisely regulated polymerization, convergence and cross linking of actin filaments that are dynamically regulated by a variety of actin-binding proteins including Cofilin. However, Cofilin is inactivated by phosphorylation through the LIM kinase1 (LIMK1), a LIM-domain containing serine-threonine-tyrosine kinase, and activated by dephosphorylation through the Slingshot protein. In this study, we explored that PKD1 increased LIMK1 expression and suppressed filopodia formation with consequent effects on cell motility. Methods: Phosphorylation of Cofilin as well as expression of GFP, PKD1-GFP and LIMK1 was confirmed by western blot analysis. Filopodia formation was detected by phalloidine staining. Wound healing assay was performed for evaluating the cell motility. Results: To find out the role of PKD1 in LIMK1 regulation, we overexpressed PKD1 in PC3 cells followed by stimulation with phorbol 12-myristate 13-acetate (PMA) or Bryostatin-1, a PKD1 selective activator, and observed that the expression of LIMK1 was increased in dose dependent fashion compared to control. We also observed the phosphorylation of Cofilin was increased in PKD1 over expressing cells stimulated with PMA and Bryostatin-1 but not in control cells. Additionally, a number of filopodia were present in every GFP over expressing PC3 cells, whereas were completely absent in PKD1 over expressing PC3 cells. In addition, wound healing assay demonstrated that cell motility was suppressed by PKD1 over expression in PC cells compared to control. Conclusions: Our data shows for first time that PKD1 modulates filopodia formation with increased expression of LIMK1 in PC cells. Additionally, we also found that PKD1 suppressed cell motility with regulation of Cofilin function that might play a role in suppression of metastasis. PKD1 suppressed filopodia formation by modulating LIMK1 expression a novel mechanism leading to altered motility in PC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5117.

LIM-kinase is critical for the mesenchymal-to-amoeboid cell morphological transition in 3D matrices

Tumor cells can migrate in 3D matrices in either a mesenchymal-like or amoeboid mode. HT1080 fibrosarcoma cells cultured in 3D collagen gels change their morphology from mesenchymal-like (elongated) to amoeboid (round) following protease inhibitor (PI) treatment or active Rho or ROCK expression. In this study, we examined the role of LIM-kinase 1 (LIMK1) in the PI- or Rho/ROCK-induced cell morphological change. We showed that LIMK1 was activated after PI treatment of HT1080 cells in 3D collagen gels and this activation was blocked by a ROCK inhibitor. While overexpression of LIMK1 induced cell rounding, knockdown of LIMK1 or the expression of kinase-inactive LIMK1 suppressed PI- or Rho/ROCK-induced cell rounding. These results suggest that LIMK1 plays an essential role in the PI- or Rho/ROCK-induced mesenchymal-to-amoeboid cell morphological transition of HT1080 cells cultured in 3D collagen gels. Furthermore, LIMK1 knockdown suppressed the invasive activity of HT1080 cells in collagen gels with or without PIs, indicating that LIMK1 mediates both the mesenchymal and amoeboid modes of invasion of HT1080 cells.

Expression profiling of G2/M phase regulatory proteins in normal, premalignant and malignant uterine cervix and their correlation with survival of patients

Cell regulatory G2/M phase proteins are the key regulators of mitosis and have been reported with abnormal expressions in various malignancies. To determine the expressions of these proteins in neoplastic uterine cervix tissue. This study evaluates the G2/M phase regulatory protein expression of Cyclin B1, Aurora-B, Pololike kinase 1 (PLK1) and LIM kinase1 (LIMK1) in tissues of 25 normal (control), 16 dysplastic (dysplasia) and 34 neoplastic (cancer) patients of uterine cervix. The expressions of different proteins were obtained by using Western Blot technique. One way analysis of variance (ANOVA), Pearson correlation, Kaplan-Meier and other tests are used for analysis. The level of expression of LIMK1 in cervical cancer patients was found to be significantly higher (P < 0.01) than both the controls and dysplasia. The expression of Aurora B and PLK1 in cervical cancer patients was also found to be significantly higher ( P < 0.05) than controls but it did not differ with dysplasia. However, the expression of Cyclin B1 was similar among cervical cancer patients, dysplasia and controls ( P> 0.05). The expression of all the above proteins showed significant ( P < 0.01) and inverse relation with the survival of cancer patients. Among the selected candidate proteins, it was LIMK1 that showed the most positive correlation with the aggressiveness of the disease and negative correlation (r= -0.64; P < 0.01) with the survival of patients.

LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs

LIM-kinase 2 (LIMK2) belongs to the LIMK family of proteins, which comprises LIMK1 and LIMK2. Both proteins regulate actin polymerization through phosphorylation and inactivation of the actin depolymerizing factor cofilin. In this study, we show that the level of LIMK2 protein is increased in neuroblastoma, BE(2)-C cells, selected for resistance to microtubule-destabilizing agents, vincristine and colchicine. However, the level of phosphorylated LIMK1 and LIMK2 was similar in the resistant and parental BE(2)-C cells. In contrast, the level of phospho-cofilin was greatly increased in the drug-resistant cells. Downregulation of LIMK2 expression increases sensitivity of neuroblastoma SH-EP cells to vincristine and vinblastine but not to microtubule-stabilizing agents, while it's overexpression increased its resistance to vincristine. Its vincristine-induced mitotic arrest was moderately inhibited in the LIMK2 knockdown cells, suggesting that the increased drug sensitivity is through an alternative mechanism other then mitotic arrest and apoptosis. Moreover, downregulation of LIMK2 expression induces formation of abnormal mitotic spindles, an effect enhanced in the presence of microtubule-destabilizing agents. LIMK2 is important for normal mitotic spindle formation and altered LIMK2 expression mediates sensitivity to microtubule destabilizing agents. These findings suggest that inhibition of LIMK2 activity may be used for the treatment of tumors resistant to microtubule-destabilizing drugs.

Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth

Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaMKs), and STO-609, an inhibitor of CaMK kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation.

The activity status of cofilin is directly related to invasion, intravasation, and metastasis of mammary tumors

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.

LIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase‐type plasminogen activator system

Mammalian LIM kinase 1 (LIMK1) phosphorylates and inactivates the actin-binding and -depolymerizing factor cofilin and induces actin cytoskeletal changes. LIMK1 is reported to play an important role in cell motility, but the mechanism of induction of cell motility and the role of LIMK1 in tumor growth, angiogenesis and invasion are poorly understood. Here we show that expression of LIMK1 in MDA-MB-435 human breast cancer cells enhanced cell proliferation and cell invasiveness and promoted in vitro angiogenesis. Since tumor metastasis requires degradation of the extracellular matrix by the serine protease urokinase type plasminogen activator (uPA), we examined the role of LIMK1 in the regulation of uPA/uPAR system. LIMK1 overexpression in breast cancer cells upregulated the uPA system, increased uPA promoter activity, induced uPA and uPAR mRNA and protein expression and induced uPA secretion. In contrast, cells transfected with the catalytically inactive LIMK mutant D460N-LIMK1 did not exhibit these phenotypic changes. Blocking antibodies against uPA and uPAR suppressed LIMK1-induced cell invasiveness. In addition, LIMK1 overexpression increased tumor growth in female athymic nude mice, promoted tumor angiogenesis and induced metastasis to livers and lungs, possibly by increasing uPA expression in the tumors. Finally, LIMK1 and uPAR were coordinately overexpressed in human breast tumors. These results suggested an important role for LIMK1 signaling in breast cancer tumor growth, angiogenesis and invasion and a regulatory connection between LIMK1 and the uPA system.

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

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. In this study, we demonstrate that 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 induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed 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.

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. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels 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, we 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, as 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.