Levels Of Cofilin Research Articles

The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module

Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P(2) modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P(2) dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.

Calcineurin regulates endothelial barrier function by interaction with and dephosphorylation of myosin phosphatase

Calcineurin (CN) influences myosin phosphorylation and alters endothelial barrier function; however, the molecular mechanism is still obscure. Here we examine whether CN controls myosin phosphorylation via mediating the phosphorylation state of Thr696 in myosin phosphatase (MP) target subunit 1 (MYPT1), the phosphorylation site inhibitory to the catalytic activity of MP. Exposure of bovine or human pulmonary artery endothelial cells (BPAECs or HPAECs) to the CN inhibitor cyclosporin A (CsA) induces a rise in intracellular Ca(2+) and increases the phosphorylation level of cofilin(Ser3) and MYPT1(Thr696) in a Ca(2+)-and Rho-kinase-dependent manner. An active catalytic fragment of CN overexpressed in tsA201 cells decreases endogenous MYPT-phospho-Thr696 (MYPT1(pThr696)) levels. Purified CN dephosphorylates (32)P-labelled MYPT1, suggesting direct action of CN on this substrate. Interaction of MYPT1 with CN is revealed by MYPT1 pull-down experiments and colocalization in both BPAECs and HPAECs as well as by surface plasmon resonance (SPR)-based binding studies. Stabilization of the MYPT1-CN complex occurs via the MYPT1(300PLIEST305) sequence similar to the CN substrate-docking PxIxIT-motif. Thrombin induces a transient increase of MYPT1(pThr696) in BPAECs, whereas its combination with CsA results in maintained phosphorylation levels of both MYPT1(pThr696) and myosin. These phosphorylation events might correlate with changes in endothelial permeability since CsA slows down the recovery from the thrombin-induced decrease of the transendothelial electrical resistance of the BPAEC monolayer. CN may improve endothelial barrier function via inducing dephosphorylation of cofilin(pSer3) and by interaction with MYPT1 and activating MP through MYPT1(pThr696) dephosphorylation, thereby affecting actin polymerization and decreasing myosin phosphorylation.

Curcumin Attenuates β-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1

Prostate cancer is the most commonly diagnosed cancer affecting 1 in 6 males in the US. Understanding the molecular basis of prostate cancer progression can serve as a tool for early diagnosis and development of novel treatment strategies for this disease. Protein Kinase D1 (PKD1) is a multifunctional kinase that is highly expressed in normal prostate. The decreased expression of PKD1 has been associated with the progression of prostate cancer. Therefore, synthetic or natural products that regulate this signaling pathway can serve as novel therapeutic modalities for prostate cancer prevention and treatment. Curcumin, the active ingredient of turmeric, has shown anti-cancer properties via modulation of a number of different molecular pathways. Herein, we have demonstrated that curcumin activates PKD1, resulting in changes in β-catenin signaling by inhibiting nuclear β-catenin transcription activity and enhancing the levels of membrane β-catenin in prostate cancer cells. Modulation of these cellular events by curcumin correlated with decreased cell proliferation, colony formation and cell motility and enhanced cell-cell aggregation in prostate cancer cells. In addition, we have also revealed that inhibition of cell motility by curcumin is mediated by decreasing the levels of active cofilin, a downstream target of PKD1. The potent anti-cancer effects of curcumin in vitro were also reflected in a prostate cancer xenograft mouse model. The in vivo inhibition of tumor growth also correlated with enhanced membrane localization of β-catenin. Overall, our findings herein have revealed a novel molecular mechanism of curcumin action via the activation of PKD1 in prostate cancer cells.

Cofilin is required for organization of sarcomeric actin filaments in chicken skeletal muscle cells

Cofilin is an actin regulatory protein that plays a critical role in actin filament dynamics in a variety of cells. We have previously demonstrated that excess cofilin in skeletal muscle cells leads to disruption of actin filaments, followed by actin-cofilin rod formation in the cytoplasm. In this study, to further clarify the role of cofilin in actin assembly during myofibrillogenesis, cofilin expression was suppressed in cultured chicken skeletal muscle cells. First, we confirmed that turnover of cofilin in myotubes was much higher than that of actin, and that the cofilin level could be decreased drastically within 2 days when cofilin de novo synthesis was suppressed. Next, cofilin expression in individual myotubes was suppressed by introducing antisense morpholino oligonucleotides into the cells by microinjection. Cofilin depletion at the early phase of myofibrillogenesis caused abnormal actin aggregates in myotubes and impaired actin organization into cross-striated myofibril structures. However, when cofilin expression was suppressed in developed myotubes, actin localization in striated myofibrils was scarcely affected. These results indicate that cofilin plays a critical role in the regulation of actin assembly at the early process of myofibrillogenesis.

Restraint Stress in Rats Alters Gene Transcription and Protein Translation in the Hippocampus

Stress is a relatively new and emerging risk factor for Alzheimer's disease (AD). Severe stress can alter brain characteristics such as neuronal plasticity, due to changes in the metabolism of cytoskeletal proteins. In this study, male Wistar rats were exposed to restraint stress (RS) for 5 h daily for different time periods. At the end of the exposure periods, the amounts of β-actin, cofilin, amyloid precursor protein (APP) and mitogen-activated protein kinase 1 (MAPK-1) RNAs and proteins were investigated. The mRNA expressions of β-actin, cofilin and MAPK-1 followed U-shaped time course. Acute (3 days) and chronic (21 days) RS caused a fourfold and tenfold increases, respectively, in hippocampal β-actin mRNA expression. In the case of cofilin mRNA expression, elevations were detected in the hippocampus on days 3, 7 and 21. The APP mRNA level was increased on day 21. On protein level, chronic stress elevated the levels of β-actin, cofilin and APP in the hippocampus. These results suggest that stress causes the induction of some genes and proteins that are also elevated in AD selectively in the hippocampal region of the rat brain.

Rho GTPase controls Drosophila salivary gland lumen size through regulation of the actin cytoskeleton and Moesin

Generation and maintenance of proper lumen size is important for tubular organ function. We report on a novel role for the Drosophila Rho1 GTPase in control of salivary gland lumen size through regulation of cell rearrangement, apical domain elongation and cell shape change. We show that Rho1 controls cell rearrangement and apical domain elongation by promoting actin polymerization and regulating F-actin distribution at the apical and basolateral membranes through Rho kinase. Loss of Rho1 resulted in reduction of F-actin at the basolateral membrane and enrichment of apical F-actin, the latter accompanied by enrichment of apical phosphorylated Moesin. Reducing cofilin levels in Rho1 mutant salivary gland cells restored proper distribution of F-actin and phosphorylated Moesin and rescued the cell rearrangement and apical domain elongation defects of Rho1 mutant glands. In support of a role for Rho1-dependent actin polymerization in regulation of gland lumen size, loss of profilin phenocopied the Rho1 lumen size defects to a large extent. We also show that Ribbon, a BTB domain-containing transcription factor functions with Rho1 in limiting apical phosphorylated Moesin for apical domain elongation. Our studies reveal a novel mechanism for controlling salivary gland lumen size, namely through Rho1-dependent actin polymerization and distribution and downregulation of apical phosphorylated Moesin.

Role of actin depolymerizing factor in the development of methamphetamine-induced place preference in mice

The present study investigated role of actin depolymerizing factor (ADF) in methamphetamine-induced place preference using ADF mutant (ADFm) and wild-type mice (WT). Whereas methamphetamine developed dose-dependently place preference in wild-type mice, methamphetamine-induced place preference was significantly attenuated by ADF mutation. Moreover, the administration of phalloidin, an F-actin stabilizer, dose-dependently inhibited methamphetamine-induced place preference. Methamphetamine-conditioning significantly increased the protein levels of ADF, cofilin, G-actin and F-actin in the limbic forebrain of both WT and ADFm, though the degree of enhancement of G- and F-actins by methamphetamine in ADFm was significantly smaller than in WT. Immunohistochemical study showed the co-localization of synaptophysin and tyrosine hydroxylase in WT and the potentiation of the immunoreactivity of these proteins by methamphetamine-conditioning, whereas such potentiation was suppressed by ADF mutation. In addition, the protein levels of synaptophysin, post-synaptic density 95 (PSD95), and neuroligin1 significantly increased in the limbic forebrain of WT showing methamphetamine-induced place preference, but not in ADFm. These findings indicate that ADF play an important role in the development of methamphetamine-induced place preference via regulation of actin dynamics.

SPARC mediates Src-induced disruption of actin cytoskeleton via inactivation of small GTPases Rho–Rac–Cdc42

The matricellular glycoprotein Secreted Protein Acidic and Rich in Cysteine (SPARC) plays an important role in the regulation of cell adhesion and proliferation as well as in tumorigenesis and metastasis. Earlier, we reported that, in addition to its potent anti-angiogenic functions, SPARC also induces apoptosis in medulloblastoma cells, mediated by autophagy. We therefore sought to investigate the underlying molecular mechanism through which SPARC inhibits migration and invasion of Daoy medulloblastoma cells, both in vitro and in vivo. For this study, we used SPARC-overexpressing stable Daoy medulloblastoma cells. SPARC overexpression in Daoy medulloblastoma cells inhibited migration and invasion in vitro. Additionally, SPARC overexpression significantly suppressed the activity of Rho, Rac and Cdc42, which all regulate the actin cytoskeleton. This suppression was accompanied by an increase in the phosphorylation of Src at Tyr-416, which led to a loss of actin stress fibers and focal contacts and a decrease in the phosphorylation level of cofilin. The reduced phosphorylation level of cofilin, which is indicative of receding Rho function, in turn led to inhibition of active Rho A. To confirm the role of SPARC in inhibition of migration and invasion of Daoy medulloblastoma cells, we transfected parental and SPARC-overexpressing Daoy cells with a plasmid vector carrying siRNA against SPARC. Transfection with SPARC siRNA reversed Src-mediated disruption of the cytoskeleton organization as well as dephosphorylation of cofilin and activation of Rho A. Taken together, these results establish SPARC as an effector of Src-induced cytoskeleton disruption in Daoy medulloblastoma cells, which subsequently led to decreased migration and invasion.

Rho inhibitor prevents ischemia–reperfusion injury in rat steatotic liver

Hepatic stellate cells are thought to play a role in modulating intrahepatic vascular resistance based on their capacity to contract via Rho signaling. We investigated the effect of a Rho-kinase inhibitor on ischemia-reperfusion injury in the steatotic liver. Steatotic livers, induced by a choline-deficient diet in rats, were subjected to ischemia-reperfusion injury. Hepatic stellate cells isolated from steatotic livers were analyzed for contractility and Rho signaling activity. The portal pressure of the perfused rat liver and the survival rate after ischemia-reperfusion were also investigated. Hepatic stellate cells from steatotic livers showed increased contractility and upregulation of Rho-kinase 2 compared with those from normal livers. Furthermore, endothelin-1 significantly enhanced the contractility and phosphorylation level of myosin light chain and cofilin in hepatic stellate cells isolated from steatotic livers. A specific Rho-kinase inhibitor, fasudil, significantly suppressed the contractility and decreased the phosphorylation levels of myosin light chain and cofilin. Serum levels of endothelin-1 were markedly increased after IR in rats with steatotic livers, whereas fasudil significantly decreased endothelin-1 serum levels. Rats with steatotic livers showed a significant increase in portal perfusion pressure after ischemia-reperfusion and a significant decrease in survival rate; fasudil treatment significantly reduced these effects. Activation of Rho/Rho-kinase signaling in hepatic stellate cells isolated from steatotic livers is associated with an increased susceptibility to ischemia-reperfusion injury. A Rho-kinase inhibitor attenuated the activation of hepatic stellate cells isolated from steatotic livers and improved ischemia-reperfusion injury in steatotic rats.

A Temporal Model of Cofilin Regulation and the Early Peak of Actin Barbed Ends in Invasive Tumor Cells

Cofilin is an important regulator of actin polymerization, cell migration, and chemotaxis. Recent experimental data on mammary carcinoma cells reveal that stimulation by epidermal growth factor (EGF) generates a pool of active cofilin that results in a peak of actin filament barbed ends on the timescale of 1 min. Here, we present results of a mathematical model for the dynamics of cofilin and its transition between several pools in response to EGF stimulation. We describe the interactions of phospholipase C, membrane lipids (PIP 2), and cofilin bound to PIP 2 and to F-actin, as well as diffusible cofilin in active G-actin-monomer-bound or phosphorylated states. We consider a simplified representation in which the thin cell edge (lamellipod) and the cell interior are represented by two compartments that are linked by diffusion. We demonstrate that a high basal level of active cofilin stored by binding to PIP 2, as well as the highly enriched local milieu of F-actin at the cell edge, is essential to capture the EGF-induced barbed-end amplification observed experimentally.

Citron kinase regulates axon growth through a pathway that converges on cofilin downstream of RhoA

Axon regeneration in the adult central nervous system (CNS) is prevented by inhibitory molecules present in myelin, which bind to a receptor complex that leads to downstream RhoGTP activation and axon growth cone collapse. Here, we compared expression of Citron kinase (Citron-K), a target molecule of RhoGTP in non-regenerating dorsal root ganglion neurons (DRGN) after dorsal column (DC) injury, and in regenerating DRGN after either sciatic nerve (SN) injury or preconditioning SN + DC lesion models. We show by microarray that Citron-K mRNA levels in DRGN of a non-regenerating DC injury model were elevated 2-fold compared to those of intact control DRGN. Conversely, Citron-K levels were reduced by 2 and 2.4-fold at 10 days post lesion in the regenerating SN and preconditioning SN + DC lesion models, respectively, compared to levels in control intact DRGN. Western blotting and immunohistochemistry confirmed these observations and localised Citron-K immunostaining to both DRGN and satellite glia. In dissociated, adult rat DRG cell cultures, 80% knockdown of Citron-K, in the presence of inhibitory concentrations of CNS myelin extract (CME), promoted significant disinhibited DRGN neurite outgrowth, only when cells were stimulated with neurotrophic factors. The levels of RhoGTP remained unchanged after Citron-K knockdown in the presence of CME while enhanced cofilin levels correlated with disinhibited DRGN neurite outgrowth. This observation suggests that Citron-K plays a role in axon growth downstream of Rho activation. We conclude that Citron-K regulates actin polymerisation downstream of RhoA and may offer a potentially novel therapeutic approach for promoting CNS axon regeneration.

A study of the expression and interaction of Destrin, cofilin, and LIMK in Debakey I type thoracic aortic dissection tissue

Objective. Our previous proteomic research has indicated that some cytoskeleton proteins show differential expression between thoracic aortic dissection and normal control groups, which suggests a possible mechanism involved in the pathogenesis of the vascular remodeling of this disease. This study was to investigate the expression of these cytoskeleton proteins and their possible molecular pathway in the remodeling process of thoracic aortic dissection. Methods. Ascending aortic segments were obtained from thoracic aortic dissection patients (Debakey type I, n = 13) and age-matched normal donors (n = 8). Quantitative differences of Destrin, cofilin, and LIM protein kinases (LIMK) were investigated using RT-PCR and Western blot analysis. The relationships between the expression of these proteins and clinical parameters such as age, hypertension and maximal aortic diameter of the patients were analysed statistically. Results. Western blotting showed that the protein expression of cofilin and LIMK was significantly decreased in thoracic aortic dissection tissue compared with normal control, (p = 0.004 for cofilin, p < 0.001 for LIMK). The mRNA levels of cofilin and LIMK were lower in thoracic aortic dissection than normal control and were coincident with the protein expression (p = 0.0039 for cofilin, p = 0.017 for LIMK). A significant correlation (Spearman's rho = −0.521, p = 0.019) was found between LIMK protein expression and maximal aortic diameter; lower levels of LIMK expression were associated with larger aortic diameters. Conclusions. Changes in the expression of cytoskeletal regulatory proteins such as LIMK and cofilin may play a role in weakening thoracic aortic medial tissue, as a precondition to thoracic aortic dissection.

Inhibition of GGTase‐I and FTase disrupts cytoskeletal organization of human PC‐3 prostate cancer cells

The mevalonate synthesis pathway produces intermediates for isoprenylation of small GTPases, which are involved in the regulation of actin cytoskeleton and cell motility. Here, we investigated the role of the prenylation transferases in the regulation of the cytoskeletal organization and motility of PC-3 prostate cancer cells. This was done by using FTI-277, GGTI-298 or NE-10790, the specific inhibitors of FTase (farnesyltransferase), GGTase (geranylgeranyltransferase)-I and -II, respectively. Treatment of PC-3 cells with GGTI-298 and FTI-277 inhibited migration and invasion in a time- and dose-dependent manner. This was associated with disruption of F-actin organization and decreased recovery of GFP-actin. Immunoblot analysis of various cytoskeleton-associated proteins showed that the most striking change in GGTI-298- and FTI-277-treated cells was a markedly decreased level of total and phosphorylated cofilin, whereas the level of cofilin mRNA was not decreased. The treatment of PC-3 cells with GGTI-298 also affected the dynamics of GFP-paxillin and decreased the levels of total and phosphorylated paxillin. The levels of phosphorylated FAK (focal adhesion kinase) and PAK (p-21-associated kinase)-2 were also lowered by GGTI-298, but levels of paxillin or FAK mRNAs were not affected. In addition, GGTI-298 had a minor effect on the activity of MMP-9. RNAi knockdown of GGTase-Ibeta inhibited invasion, disrupted F-actin organization and decreased the level of cofilin in PC-3 cells. NE-10790 did not have any effect on PC-3 prostate cancer cell motility or on the organization of the cytoskeleton. In conclusion, our results demonstrate the involvement of GGTase-I- and FTase-catalysed prenylation reactions in the regulation of cytoskeletal integrity and motility of prostate cancer cells and suggest them as interesting drug targets for development of inhibitors of prostate cancer metastasis.

Subcellular distribution and expression of cofilin and ezrin in human colon adenocarcinoma cell lines with different metastatic potential.

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells.In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.

Skeletal muscle apoptosis following 7 days of denervation‐induced muscle disuse

Chronic decreases in skeletal muscle contractile activity result in muscle atrophy, reduced force production and increased fatigue. However, the mechanisms of disuse‐induced activation of apoptotic signaling cascades which lead to atrophy are not fully established. To study these pathways C57BL/6 (WT) and Bax/Bak knockout (KO) animals were denervated for 7 days. This model of muscle disuse resulted in a decrease in mitochondrial respiration and a 2‐fold increase in the production of reactive oxygen species. Activity of the anti‐oxidant enzyme catalase was elevated by 2.5‐fold in subsarcolemmal (SS) mitochondria from WT and KO animals. Denervation resulted in higher cofilin levels within SS mitochondria, suggesting an increased rate of cofilin translocation from the cytosol. This translocation of cofilin may have contributed to the approximate 2‐fold increase in cleaved Poly‐ADP ribose polymerase (Parp) following disuse. Denervation also resulted in a 1.7‐fold increase in DNA fragmentation in WT animals, but no fragmentation was evident in the KO group. These data suggest that: 1) Bax and Bak contribute to the degree of DNA fragmentation following denervation and 2) chronic muscle disuse increases oxidative stress which stimulates the translocation of pro‐apoptotic proteins, such as cofilin, to promote protein release, and contribute to the loss of muscle mass. This work was supported by NSERC.

Proteomic change of peripheral lymphocytes from scald injury and Pseudomonas aeruginosa sepsis in rabbits

Background: Increased susceptibility to infection has been related to impairment of lymphocyte-regulated immune responses after severe burn. The aim of this study is to identify the differential expression of proteins in circulating lymphocytes from scald injury and Pseudomonas aeruginosa sepsis in rabbits to provide a basis for pathogenesis of burns and sepsis. Methods: Rabbits were subjected to sham burn (A), 30% scald (B), A + bacterial challenge (C) or B + bacterial challenge (D). Bacterial challenge was inflicted by an injection of 2.0 × 10 8 CFU P. aeruginosa (ATCC27853) in the auricular vein 22 h after the burn procedure. The animals were sacrificed 24 h later. Lymphocytes were isolated, and the differential proteins in the lymphocytes from the experimental and control animals were identified by two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), two of which were confirmed by Western blotting. Results: Nineteen differential protein spots were found by 2-DE and 12 spots (11 proteins) were identified. Differential expression of peroxiredoxin and annexin I was validated by Western blotting. Among the identified proteins, the expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate kinase, glutamate dehydrogenase and annexin I were down-regulated in group B, excessively down-regulated in group D, but mildly in group C, and peroxiredoxin was up-regulated in groups B and D. Conclusions: Proteome changes in lymphocytes from P. aeruginosa sepsis in the scalded rabbits were revealed, which are related to immune suppression and the pathogenesis of sepsis after scald injury.

DGCR6L, a novel PAK4 interaction protein, regulates PAK4-mediated migration of human gastric cancer cell via LIMK1

Overexpression, genetic amplification and mutations of p21-activated kinase 4 (PAK4) were found in a variety of human cancers. PAK4 regulated actin cytoskeleton reorganization by phosphorylating LIMK1 and promoted cancer cells migration. Using yeast two-hybrid screen, we identified a novel PAK4 binding protein, DGCR6L, which was associated with cancer cell metastasis. We confirmed PAK4 binding to the DGCR6L specifically by GST pull-down assay, and found an association between endogenous PAK4 and DGCR6L by immunoprecipitation in mammalian cells. Furthermore, L115 of DGCR6L was the critical amino acid to bind 466–572aa in the very C-terminus of PAK4. Importantly, DGCR6L was required for the formation of PAK4-DGCR6L-β-actin complex. Overexpressed DGCR6L promoted migration of AGS cells mediated by PAK4, whereas knock-down of DGCR6L markedly inhibited the migration of those cells. Moreover, DGCR6L (L115V), which did not bind to PAK4, lost the ability to promote AGS cells migration. DGCR6L colocalized with PAK4 or F-actin and enhanced the phosphorylation level of LIMK1 and cofilin in a dose dependent manner. Taken together, our results demonstrated that DGCR6L, a novel PAK4 interacting protein, regulated PAK4-mediated migration of human gastric cancer cells via LIMK1.

Regulated expression of cofilin and the consequent regulation of p27kip1 are essential for G1 phase progression

Cofilin, a ubiquitously expressed actin binding protein, is responsible for the formation of the actin cytoskeleton and is indispensable for cell cycle control. However, the association between cofilin expression and the cell cycle remains to be elucidated. In this study, we found that the expression level of cofilin up-regulated in G1 phase-arrested confluent cells, while knockdown of cofilin expression by small interference RNA (siRNA) in these cells led to a reduction in the population of G1 cells. To investigate the role of cofilin in the control of G1 phase progression, a tet-on gene expression system was introduced to over-express different concentrations of cofilin in cells. The results showed that G1 phase progression was blocked following induction of exogenous cofilin. A survey of the cell cycle proteins controlling the G1 phase progression revealed that the cyclin-dependent kinase inhibitor (CKI) p27kip1 was the primary molecule induced by over-expressed cofilin in a time and dose dependent manner. Up-regulated p27kip1 repressed phosphorylation of the retinoblastoma protein (Rb) mediated by cyclin D1/CDK4 activity. Conversely, siRNA against p27kip1 expression in the cofilin over-expressing cells released the G1 phase arrest. Furthermore, we found that over-expression of cofilin led to induction of p27kip1 gene promoter transactivation using luciferase reporter gene assay. This effect was associated with increase of p27kip1 mRNA transiently. In addition, inhibition of threonine-187 phosphorylation of p27kip1 protein for ubiquitinyl-proteasomal mediated degradation was also involved in up-regulation of p27 kip1. These data suggest that cofilin expression and its regulation of p27kip1 expression is important for the control of G1 phase progression.

14-3-3 ζ/τ heterodimers regulate Slingshot activity in migrating keratinocytes

Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an α6β4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3 ζ, we demonstrate that in keratinocytes 14-3-3 ζ/ τ heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3 ζ acts as a dominant negative and inhibits the interaction between 14-3-3 τ and SSH1. These results implicate 14-3-3 ζ/ τ heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.

Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.