Cadmium affects focal adhesion kinase (FAK) in mesangial cells: Involvement of CaMK‐II and the actin cytoskeleton

The toxic metal ion cadmium (Cd²⁺) induces pleiotropic effects on cell death and survival, in part through effects on cell signaling mechanisms and cytoskeletal dynamics. Linking these phenomena appears to be calmodulin‐dependent activation of the Ca²⁺/calmodulin‐dependent protein kinase II (CaMK‐II). Here we show that interference with the dynamics of the filamentous actin cytoskeleton, either by stabilization or destabilization, results in disruption of focal adhesions at the ends of organized actin structures, and in particular the loss of vinculin and focal adhesion kinase (FAK) from the contacts is a result. Low‐level exposure of renal mesangial cells to CdCl₂ disrupts the actin cytoskeleton and recapitulates the effects of manipulation of cytoskeletal dynamics with biological agents. Specifically, Cd²⁺ treatment causes loss of vinculin and FAK from focal contacts, concomitant with cytoskeletal disruption, and preservation of cytoskeletal integrity with either a calmodulin antagonist or a CaMK‐II inhibitor abrogates these effects of Cd²⁺. Notably, inhibition of CaMK‐II decreases the migration of FAK‐phosphoTyr925 to a membrane‐associated compartment where it is otherwise sequestered from focal adhesions in a Cd²⁺‐dependent manner. These results add further insight into the mechanism of the CaMK‐II‐dependent effects of Cd²⁺ on cellular function. J. Cell. Biochem. 114: 1832–1842, 2013. © 2013 Wiley Periodicals, Inc.     

Low‐concentration heparin suppresses ionomycin‐activated CAMK‐II/EGF receptor‐ and ERK‐mediated signaling in mesangial cells

Heparin and endogenous heparinoids inhibit the proliferation of smooth muscle cells, including renal mesangial cells; multiple effects on signaling pathways are well established, including effects on PKC, Erk, and CaMK‐II. Many studies have used heparin at concentrations of 100 µg/ml or higher, whereas endogenous concentrations of heparinoids are much lower. Here we report the effects of low‐concentration (1 µg/ml) heparin on activation of several kinases and subsequent induction of the c‐fos gene in mesangial cells in response to the calcium ionophore, ionomycin, in the absence of serum factors. Ionomycin rapidly increases the phosphorylation of CaMK‐II (by 30 s), and subsequently of the EGF receptor (EGFR), c‐Src, and Erk 1/2. Low‐dose heparin suppresses the ionomycin‐dependent phosphorylation of EGFR, c‐Src, and Erk 1/2, but not of CaMK‐II, whereas inhibition of activated CaMK‐II reduces phosphorylation of EGFR, c‐Src, and Erk. Our data support a mechanism whereby heparin acts at the cell surface to suppress downstream targets of CaMK‐II, including EGFR, leading in turn to a decrease in Erk‐ (but not c‐Src‐) dependent induction of c‐fos. J. Cell. Physiol. 224: 484–490, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of CaMKKβ/AMPKα pathway by 2‐AG in human platelets

The objective of this study was to determine whether AMPK is activated by 2‐arachidonoylglycerol (2‐AG) and participates to the cytoskeleton control in human platelets. We found that 2‐AG stimulates the AMPKα activation through a Ca²⁺/Calmodulin‐dependent pathway as the specific inhibition of the CaMKKβ by STO‐609 inhibits the AMPKα phosphorylation/activation. Moreover, the CaMKKβ/AMPKα pathway activated by 2‐AG is involved in the phosphorylation of cofilin, vasodilator stimulated phosphoprotein (VASP), and myosin light chain (MLCs). These proteins participate to actin cytoskeletal remodelling during aggregation. We found that the phosphorylation/activation inhibition of these proteins is associated with a significant reduction in actin polymerization, aggregation, ATP, and α‐granule secretion. Finally, AMPKα activation, Cofilin, VASP, and MLCs phosphorylation are significantly reduced by SR141716, the specific inhibitor of type 1 cannabinoid (CB1) receptor, suggesting that the CB1 receptor is involved in the 2‐AG effect. In conclusion, we have shown that the CaMKKβ/AMPKα pathway is activated by 2‐AG in human platelets and controls the phosphorylation of key proteins involved in actin polymerization and aggregation.     

SUPPRESSION OF AcMNPV REPLICATION BY ADF AND THYMOSIN PROTEIN UP‐REGULATION IN A NEW TESTIS CELL LINE,Ha‐shl‐t

Host cytoskeletons facilitate the entry, replication, and egress of viruses because cytoskeletons are essential for viral survival. One mechanism of resisting viral infections involves regulating cytoskeletal polymerization/depolymerization. However, the molecular mechanisms of regulating these changes in cytoskeleton to suppress viral replication remain unclear. We established a cell line (named Ha‐shl‐t) from the pupal testis of Helicoverpa armigera (Lepidoptera: Noctuidae). The new testis cell line suppresses Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) replication via disassembly of cytoskeleton. Up‐regulation of thymosin (actin disassembling factor) and adf (actin depolymerizing factor) reduces F‐actin. Silencing thymosin or adf or treating cells with the F‐actin stabilizer phalloidin led to increased AcMNPV replication, while treating cells with an F‐actin assembly inhibitor cytochalasin B decreased viral replication. We infer that Ha‐shl‐t cells utilize F‐actin depolymerization to suppress AcMNPV replication by up‐regulating thymosin and adf. We propose Ha‐shl‐t as a model system for investigating cytoskeletal regulation in antiviral action and testicular biology generally.     

Calmodulin-dependent regulation of hypotonicity-induced translocation of ENaC in renal epithelial A6 cells

Hypotonicity stimulates translocation of epithelial Na(+) channel (ENaC) to the apical membrane from the intracellular store site of ENaC by activating protein tyrosine kinase (PTK) in renal epithelial A6 cells. Based upon the fact that calmodulin shows its action on other enzymes through PTK caused phosphorylation of tyrosine residues of calmodulin itself, we studied whether a calmodulin-dependent pathway is involved in the action of hypotonicity on ENaC. W7, an antagonist of calmodulin, diminished the stimulatory action of hypotonicity on ENaC, irrespective of W7 treatment before or after application of hypotonicity. Calmodulin is known to regulate three pathways: (1) protein phosphatase 2B (PP2B), (2) Ca(2+)/calmodulin-dependent protein kinase II (CaMK II), and (3) myosin light chain kinase (MLCK). Pretreatment with cyclosporin A, an inhibitor of PP2B, did not influence the hypotonicity action on ENaC. The hypotonicity action on ENaC was partially inhibited by pretreatment with KN93, an inhibitor of CaMK II, but not by addition of KN93 after hypotonic stimulation had been applied. ML-7, an inhibitor of MLCK, showed the action similar to KN93. These observations indicate that: (1) the hypotonicity-induced translocation of ENaC depends on CaMK II and MLCK and (2) ENaC translocated to the apical membrane by hypotonicity is maintained in its activity and/or stability at the apical membrane through a calmodulin-dependent pathway.     

Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein

Receptor-mediated stimulation induces massive actin polymerization and cyto-skeletal reorganization. The activity of a potent actin-modulating protein, gelsolin, is regulated both by Ca²⁺ and polyphos-phoinositides, and it may have a pivotal role in restructuring the actin cytoskeleton in response to agonist stimulation. Structure-function analysis of gelsolin has (1) indicated that its NH₂-terminal half is primarily responsible for modulating actin filament length and polymerization; and (2) elucidated mechanisms by which Ca²⁺ and phospholipids may regulate such functions. Gelsolin is functionally and structurally similar to villin, another Ca²⁺-activated actin-severing protein found in microvilli, suggesting that gelsolin may be a prototype of this family of actin-modulating proteins. A molecular variant of gelsolin is secreted and may be involved in the clearance of actin filaments released during tissue damage. The two forms of gelsolin are encoded by a single gene, and distinct messages are derived by alternative message splicing.     

CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on the Glut4 gene

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 x 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased approximately 2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene.     

Differential protein and mRNA expression of CaMKs during osteoclastogenesis and its functional implications

The calmodulin-dependent kinase (CaMK) family has been recently recognized to participate in the regulation of osteoclastogenesis. However, there are some controversial reports regarding the mRNA expression patterns of CaMKs during osteoclastogenesis, although the protein expression pattern of most CaMKs during osteoclastogenesis have not been studied. In the present study, we attempted to address this issue by using a mouse bone marrow monocyte model and parallel Western blotting and quantitative real-time PCR. Our results revealed some interesting expression patterns of CaMKs during the process. Among all CaMKs examined, only CaMKIIδ exhibited consistent expression patterns between its mRNA and protein with both rising remarkably during osteoclastogenesis. CaMKIV protein was not detectable during the first three days of cell culture, but it rose on Day 5. The CaMK inhibitor, KN93, subdued osteoclastogenesis during the first three days of cell culture, a time when CaMKIV was absent while other KN93-sensitive CaMKs presented. In addition, KN93 was found to inhibit the expression of some early receptor activator of NF-κB (RANK) signaling intermediates (extracellular signal-regulated kinase (ERK) and Akt) in the non-differentiated mouse bone marrow monocytes. Collectively, these data reveal differential expression patterns of KN93-sensitive CaMK proteins and their mRNAs during osteoclastogenesis, supporting a CaMKII-RANK signaling interaction in the regulation of early osteoclastogenesis.     

Stimulus-Induced Selective Association of Actin-Associated Proteins (α-Actinin) and Protein Kinase C Isoforms with the Cytoskeleton of Human Neutrophils

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein α-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal α-actinin and actin. Increased association of PKCβI and βII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, α-actinin, and PKCβII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal α-actinin and PKCβII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 μM) completely blocked PMA-induced increases in cytoskeletal α-actinin but reduced cytoskeletal recruitment of PKCβII only by 16%. Higher concentrations of latrunculin A (4 μM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCβII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

Biphasic Pattern of Gelsolin Expression and Variations in Gelsolin-Actin Interactions during Myogenesis

During myogenesis in vitro, the amount of gelsolin in myogenic cells increased by a factor of 3 from about 200 ng to a maximum of 750 ng per milligram of total protein. Gelsolin increased steadily from the myoblast state to terminally differentiated myotubes containing abundant cross-striated myofibrils. At the same time, the amount of total actin varied by only about 30%, the molar ratio of gelsolin:actin increased from 1:500 to approximately 1:150. This modulation of gelsolin expression was observed both in avian and mammalian myocultures. Once the state of terminal differentiation in myocultures was attained, the amount of gelsolin decreased again. On the other hand, gelsolin decreased continuously in the postnatal mouse muscle by a factor of 5 between Day 1 and Day 12 after birth. When myogenic cells from various stages of differentiation were extracted with Triton X-100, the majority of gelsolin was soluble, whereas a minor fraction was tightly associated with the cytoskeleton. The actual amount of insoluble gelsolin depended on both the Ca²⁺ concentration during extraction and the degree of differentiation. Whereas at [Ca²¹] > 10⁵M about one-third of the total gelsolin was associated with the cytoskeleton at all stages of differentiation, the amount of insoluble gelsolin after Triton extraction in the presence of EGTA increased from 3 to 17% during differentiation. The amount of soluble actin decreased from 40 to 25% during the same period, independent of the Ca²⁺ concentration. We calculated that the amount of gelsolin associated with the cytoskeletal or myofibrillar system is approximately 20-fold higher in differentiated myotubes than in early myotubes, indicating a functional role of gelsolin for myofibrillar assembly.     

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‐Iβ 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.     

Vibration activates the actin/NF‐κB axis and upregulates IL‐6 and IL‐8 expression in human periodontal ligament cells

We previously reported that mechanical vibration‐induced proinflammatory cytokines, interleukin‐6 (IL‐6) and IL‐8, expression in human periodontal ligament (hPDL) cells, however, the underlying mechanism remained unclear. Mechanical stimuli are able to activate cellular responses by inducing the activation of several signaling pathways including cytoskeletal changes and inflammation. The actin cytoskeleton is a highly dynamic network and plays many important roles in intracellular events. Here, we aimed to investigate the involvement of a pivotal mediator of inflammatory responses, nuclear factor‐κB (NF‐κB), and actin polymerization in vibration‐induced upregulation of IL‐6 and IL‐8 expression in hPDL cells. hPDL cells were pretreated with the NF‐κB inhibitor BAY 11‐7082 or cytochalasin D, respectively, before exposure to vibration. IL‐6 and IL‐8 messenger RNA (mRNA) and protein expression were quantified by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assays, respectively. Subcellular localization of the NF‐κB p65 subunit was visualized by immunofluorescent staining. We found an increase in NF‐κB nuclear translocation in vibrated cells compared with control cells. Pretreatment with BAY 11‐7082 significantly inhibited vibration‐induced IL‐6 and IL‐8 mRNA and protein expression in hPDL cells. Moreover, pretreatment with cytochalasin D inhibited NF‐κB nuclear translocation and attenuated upregulation of IL‐6 and IL‐8 mRNA and protein in vibrated cells. Therefore, modulation of actin cytoskeletal polymerization in response to vibration may activate the NF‐κB signaling pathway and subsequently upregulate IL‐6 and IL‐8 expression in hPDL cells.     

Actin ADP‐ribosylation at Threonine148 by Photorhabdus luminescens toxin TccC3 induces aggregation of intracellular F‐actin

Intoxication of eukaryotic cells by Photorhabdus luminescens toxin TccC3 induces cell rounding and detachment from the substratum within a few hours and compromises a number of cell functions like phagocytosis. Here, we used morphological and biochemical procedures to analyse the mechanism of TccC3 intoxication. Life imaging of TccC3‐intoxicated HeLa cells transfected with AcGFP‐actin shows condensation of F‐actin into large aggregates. Life cell total internal reflection fluorescence (TIRF) microscopy of identically treated HeLa cells confirmed the formation of actin aggregates but also disassembly of F‐actin stress fibres. Recombinant TccC3 toxin ADP‐ribosylates purified skeletal and non‐muscle actin at threonine148 leading to a strong propensity to polymerize and F‐actin bundle formation as shown by TIRF and electron microscopy. Native gel electrophoresis shows strongly reduced binding of Thr148‐ADP‐ribosylated actin to the severing proteins gelsolin and its fragments G1 and G1–3, and to ADF/cofilin. Complexation of actin with these proteins inhibits its ADP‐ribosylation. TIRF microscopy demonstrates rapid polymerization of Thr148‐ADP‐ribosylated actin to curled F‐actin bundles even in the presence of thymosin β4, gelsolin or G1–3. Thr148‐ADP‐ribosylated F‐actin cannot be depolymerized by gelsolin or G1–3 as verified by TIRF, co‐sedimentation and electron microscopy and shows reduced treadmilling as indicated by a lack of stimulation of its ATPase activity after addition of cofilin‐1.     

Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion-activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion-targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2-gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.     

β‐PIX controls intracellular viscoelasticity to regulate lung cancer cell migration

Cancer metastasis occurs via a progress involving abnormal cell migration. Cell migration, a dynamic physical process, is controlled by the cytoskeletal system, which includes the dynamics of actin organization and cellular adhesive organelles, focal adhesions (FAs). However, it is not known whether the organization of actin cytoskeletal system has a regulatory role in the physiologically relevant aspects of cancer metastasis. In the present studies, it was found that lung adenocarcinoma cells isolated from the secondary lung cancer of the lymph nodes, H1299 cells, show specific dynamics in terms of the actin cytoskeleton and FAs. This results in a higher level of mobility and this is regulated by an immature FA component, β‐PIX (PAK‐interacting exchange factor‐β). In H1299 cells, β‐PIX's activity was found not to be down‐regulated by sequestration onto stress fibres, as the cells did not bundle actin filaments into stress fibres. Thus, β‐PIX mainly remained localized at FAs, which allowed maturation of nascent adhesions into focal complexes; this resulted in actin polymerization, increased actin network integrity, changes in the intracellular microrheology at the peripheral of the cell, and cell polarity, which in turn regulated cell migration. Perturbation of β‐PIX caused an inhibition of cell migration, including migration velocity, accumulated distance and directional persistence. Our results demonstrate the importance of β‐PIX to the regulation of high mobility of lung adenocarcinoma cell line H1299 and that this occurs via regulation of FA dynamics, changes in actin cytoskeleton organization and cell polarity.     

Cytoskeletal mechanics during Shigella invasion and dissemination in epithelial cells

The actin cytoskeleton is key to the barrier function of epithelial cells, by permitting the establishment and maintenance of cell–cell junctions and cell adhesion to the basal matrix. Actin exists under monomeric and polymerized filamentous form and its polymerization following activation of nucleation promoting factors generates pushing forces, required to propel intracellular microorganisms in the host cell cytosol or for the formation of cell extensions that engulf bacteria. Actin filaments can associate with adhesion receptors at the plasma membrane via cytoskeletal linkers. Membrane anchored to actin filaments are then subjected to the retrograde flow that may pull membrane‐bound bacteria inside the cell. To induce its internalization by normally non‐phagocytic cells, bacteria need to establish adhesive contacts and trick the cell into apply pulling forces, and/or to generate protrusive forces that deform the membrane surrounding its contact site. In this review, we will focus on recent findings on actin cytoskeleton reorganization within epithelial cells during invasion and cell‐to‐cell spreading by the enteroinvasive pathogen Shigella, the causative agent of bacillary dysentery.     

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

AimsFormation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.Main methodsColon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.Key findingsIncreased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.SignificanceOur experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.     

Binding of Protein Kinase C Isoforms to Actin in Aplysia

Abstract: Recently, two of the 10 vertebrate protein kinase C (PKC) isoforms, PKCβII and PKCε, have been shown to bind specifically to actin filaments, suggesting that these kinases may regulate cytoskeletal dynamics. Here, we present evidence that two PKCs from the marine mollusk Aplysia californica , PKC Apl I, a Ca²⁺-activated PKC, and PKC Apl II, a Ca²⁺-independent PKC most similar to PKCε and η, also bind F-actin. First, they both cosedimented with purified actin filaments in a phorbol ester-dependent manner. Second, they both translocated to the Triton-insoluble fraction of the nervous system after phorbol ester treatment. PKC Apl II could also partially translocate to actin filaments and associate with the Triton-insoluble fraction in the absence of phorbol esters. Translocation to purified actin filaments was increased in the presence of a PKC inhibitor, suggesting that PKC phosphorylation reduces PKC bound to actin. Although both kinases bound F-actin, actin was not sufficient to activate the kinases. In support of a physiological role for actin-PKC interactions, immunochemical localization of PKC Apl II in neuronal growth cones revealed a striking colocalization with F-actin. Our results are consistent with a role for actin-PKC interactions in regulating cytoskeletal dynamics in Aplysia .     

Gelsolin overexpression enhances neurite outgrowth in PC12 cells.

The rational design of therapies for treating nerve injuries requires an understanding of the mechanisms underlying neurite extension. Neurite motility is driven by actin polymerization; however, the mechanisms are not clearly understood. One actin accessory protein, gelsolin, is involved with remodeling the cytoskeleton, although its role in cell motility is unclear. We report a two-fold upregulation of gelsolin upon differentiation with nerve growth factor. Cells that were genetically modified to overexpress gelsolin have longer neurites and a greater neurite motility rate compared to controls. These data suggest that gelsolin plays an important role in neurite outgrowth.     

LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro‐inflammatory mediators. We examined mechanisms of LPS‐stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage‐like J774 cells. A wound‐healing assay revealed that LPS also activated directed cell movement that was followed by TNF‐α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5‐bisphosphate. Fractionation of Triton X‐100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin‐regulatory proteins, paxillin on tyrosine 118 by 80% and N‐WASP on serine 484/485 by 20%, and these events preceded activation of NF‐κB. LPS‐induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N‐WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS‐stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF‐α production suggesting that LPS‐induced cell motility is not required for TNF‐α release. J. Cell. Biochem. 113: 80–92, 2012. © 2011 Wiley Periodicals, Inc.