Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production,cell migration and apoptosis

Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis.     

A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site.

In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling.     

Nox1 redox signaling mediates oncogenic Ras-induced disruption of stress fibers and focal adhesions by down-regulating Rho

Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Nox1 small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1-generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes.     

Ouabain elicits human glioblastoma cells apoptosis by generating reactive oxygen species in ERK-p66SHC-dependent pathway

Excessive reactive oxygen species (ROS) generation has been implicated as one of main agents in ouabain-induced anticancer effect. Unfortunately, the signaling pathways under it are not very clarified. In the present study, we investigated the molecular mechanism involved in ouabain-induced ROS generation and cell apoptosis on human U373MG and U87MG glioma cells. Ouabain-induced glioblastoma cells apoptosis and increased ROS generation. Clearance ROS by three different ROS scavenger partly, but not totally, reversed ouabain’s effect on cell apoptosis. Ouabain-induced ROS generation was not regulated by calcium overload, reduced nicotinamide adenine dinucleotide phosphate oxidation, but by p66Shc phosphorylation. Ouabain treatment increased p66Shc Ser36 phosphorylation. Knockdown of p66Shc by siRNA significantly inhibited ROS generations in response to ouabain. Ouabain-induced p66Shc phosphorylation through Src/Ras/extracellular signal-regulated kinase signal pathway. Our results uncovered a novel signaling pathway with p66Shc, ouabain-induced ROS generation, and glioblastoma cell apoptosis.     

Role of the actin cytoskeleton in angiotensin II signaling in human vascular smooth muscle cells

Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signaling cascades that promote vasoconstriction, growth, and inflammation. Subcellular mechanisms coordinating these processes are unclear. In the present study, we questioned the role of the actin cytoskeleton in Ang II mediated signaling through mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS) in VSMCs. Human VSMCs were studied. Cells were exposed to Ang II (10-7 mol/L) in the absence and presence of cytochalasin B (10-6 mol/L, 60 min), which disrupts the actin cytoskeleton. Phosphorylation of p38MAP kinase, JNK, and ERK1/2 was assessed by immuno blotting. ROS generation was measured using the fluoroprobe chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (4 micromol/L). Interaction between the cytoskeleton and NADPH oxidase was determined by evaluating the presence of p47phox in the Triton X-100 insoluble membrane fraction. Ang II significantly increased phosphorylation of p38MAP kinase, JNK, and ERK1/2 (two- to threefold above control, p < 0.05). Cytochalasin B pretreatment attenuated p38MAP kinase and JNK effects (p < 0.05) without altering ERK1/2 phosphorylation. ROS formation, which was increased in Ang II stimulated cells, was significantly reduced by cytochalasin B (p < 0.01). p47phox, critically involved in NADPH oxidase activation, colocalized with the actin cytoskeleton in Ang II stimulated cells. Our data demonstrate that Ang II mediated ROS formation and activation of p38MAP kinase and JNK, but not ERK1/2, involves the actin cytoskeleton in VSMCs. In addition, Ang II promotes interaction between actin and p47phox. These data indicate that the cytoskeleton is involved in differential MAP kinase signaling and ROS generation by Ang II in VSMCs. Together, these studies suggest that the cytoskeleton may be a central point of crosstalk in growth- and redox-signaling pathways by Ang II, which may be important in the regulation of VSMC function.     

TOR complex 2–Ypk1 signaling regulates actin polarization via reactive oxygen species

The evolutionarily conserved mTOR complex 2 (mTORC2) signaling pathway is an important regulator of actin cytoskeletal architecture and, as such, is a candidate target for preventing cancer cell motility and invasion. Remarkably, the precise mechanism(s) by which mTORC2 regulates the actin cytoskeleton have remained elusive. Here we show that in budding yeast, TORC2 and its downstream kinase Ypk1 regulate actin polarization by controlling reactive oxygen species (ROS) accumulation. Specifically, we find that TORC2-Ypk1 regulates actin polarization both by vacuole-related ROS, controlled by the phospholipid flippase kinase Fpk1 and sphingolipids, and by mitochondria-mediated ROS, controlled by the PKA subunit Tpk3. In addition, we find that the protein kinase C (Pkc1)/MAPK cascade, a well-established regulator of actin, acts downstream of Ypk1 to regulate ROS, in part by promoting degradation of the oxidative stress responsive repressor, cyclin C. Furthermore, we show that Ypk1 regulates Pkc1 activity through proper localization of Rom2 at the plasma membrane, which is also dependent on Fpk1 and sphingolipids. Together these findings demonstrate important links between TORC2/Ypk1 signaling, Fpk1, sphingolipids, Pkc1, and ROS as regulators of actin and suggest that ROS may play an important role in mTORC2-dependent dysregulation of the actin cytoskeleton in cancer cells.     

Galpha13 activation rescues moesin-depletion induced apoptosis in F9 teratocarcinoma cells

Mouse F9 cells differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm in response to RA and dibutyryl (db-) cAMP. G protein signaling either blocks or mimics RA-induced differentiation, the latter signaling through the Wnt-beta-catenin pathway. In our study, we found that a constitutively active Galpha13 mutant induces F9 cells to differentiate into parietal endoderm in the absence of exogenous agents. Galpha13 expression and subsequent differentiation are accompanied by beta-catenin translocation to the nucleus. Differentiation and changes in cell morphology are supported by rearrangements to the F-actin cytoskeleton. ERM (ezrin-radixin-moesin) proteins, known to link F-actin to transmembrane receptors, are also redistributed during differentiation. Furthermore, morpholino antisense and shRNA approaches show that moesin expression is essential since its knockdown leads to altered F-actin distribution and subsequent apoptosis. Moesin-depleted cells, however, remain attached to the substrate when Galpha13 is constitutively expressed, but they do not differentiate into extraembryonic endoderm. Our study demonstrates a link between Galpha13 signaling that regulates differentiation of F9 cells through primitive to parietal endoderm and a moesin requirement for cell survival.     

Methyl β-cyclodextrin reduces accumulation of reactive oxygen species and cell death in yeast

Stabilized F-actin structures have been shown to be detrimental to both mammalian and yeast cells. In yeast, stabilization of actin caused by addition of jasplakinolide, by point mutations in the act1 gene, or by deletion of certain genes that regulate F-actin leads to cell death with hallmarks of apoptosis. In particular, there is an elevation in the levels of reactive oxygen species, and we have shown the importance of the Ras/cAMP pathway for this effect. Here we show that in yeast cells deleted for end3, which functions to regulate actin organization during endocytosis, treatment of cells with methyl β-cyclodextrin reduces levels of reactive oxygen species and inhibits cell death progression. Methyl β-cyclodextrin is widely used to disrupt lipid rafts that contain cholesterol. The mechanism through which the rescue is achieved was investigated and we demonstrate that methyl β-cyclodextrin reduces accumulation of Ras2 at the plasma membrane in Δend3 cells. We use FRAP and live cell imaging to determine the possible mechanism through which methyl β-cyclodextrin functions to elicit this effect on Ras2 localization. Finally, we demonstrate that addition of methyl β-cyclodextrin to wild-type cells can act to protect cells from acute oxidative stress caused by addition of hydrogen peroxide.     

Desensitization of the PDGFbeta receptor by modulation of the cytoskeleton: the role of p21(Ras) and Rho family GTPases

Ligand-induced PDGF-type beta receptor (PDGFbeta-R) autophosphorylation is profoundly suppressed in cells transformed by activated p21(Ras). We report here that the integrity of the actin cytoskeleton is a critical regulator of PDGFbeta-R function in the presence of p21(Ras). Morphological reversion of Balb cells expressing a constitutively activated p21(Ras), with re-formation of actin stress fibers and cytoskeletal architecture, rendering them phenotypically similar to untransformed fibroblasts, allowed recovery of ligand-dependent PDGFbeta-R autophosphorylation. Conversely, disruption of the actin cytoskeleton in Balb/c-3T3 cells obliterated the normal ligand-induced phosphorylation of the PDGFbeta-R. The Rho family GTPases Rac and Rho are activated by p21(Ras) and are critical mediators of cell motility and morphology via their influence on the actin cytoskeleton. Transient expression of wild-type or constitutively active mutant forms of RhoA suppressed ligand-dependent PDGFbeta-R autophosphorylation and downstream signal transduction. These studies demonstrate the necessary role of Rho in the inhibition of PDGFbeta-R autophosphorylation in cells containing activated p21(Ras) and also demonstrate the importance of cell context and the integrity of the actin cytoskeleton in the regulation of PDGFbeta-R ligand-induced autophosphorylation.     

Ras Regulates the Polarity of the Yeast Actin Cytoskeleton through the Stress Response Pathway

Polarized growth in yeast requires cooperation between the polarized actin cytoskeleton and delivery of post-Golgi secretory vesicles. We have previously reported that loss of the major tropomyosin isoform, Tpm1p, results in cells sensitive to perturbations in cell polarity. To identify components that bridge these processes, we sought mutations with both a conditional defect in secretion and a partial defect in polarity. Thus, we set up a genetic screen for mutations that conferred a conditional growth defect, showed synthetic lethality with tpm1Delta, and simultaneously became denser at the restrictive temperature, a hallmark of secretion-defective cells. Of the 10 complementation groups recovered, the group with the largest number of independent isolates was functionally null alleles of RAS2. Consistent with this, ras2Delta and tpm1Delta are synthetically lethal at 35 degrees C. We show that ras2Delta confers temperature-sensitive growth and temperature-dependent depolarization of the actin cytoskeleton. Furthermore, we show that at elevated temperatures ras2Delta cells are partially defective in endocytosis and show a delocalization of two key polarity markers, Myo2p and Cdc42p. However, the conditional enhanced density phenotype of ras2Delta cells is not a defect in secretion. All the phenotypes of ras2Delta cells can be fully suppressed by expression of yeast RAS1 or RAS2 genes, human Ha-ras, or the double disruption of the stress response genes msn2Deltamsn4Delta. Although the best characterized pathway of Ras function in yeast involves activation of the cAMP-dependent protein kinase A pathway, activation of the protein kinase A pathway does not fully suppress the actin polarity defects, suggesting that there is an additional pathway from Ras2p to Msn2/4p. Thus, Ras2p regulates cytoskeletal polarity in yeast under conditions of mild temperature stress through the stress response pathway.     

Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function,the Actin Cytoskeleton,and Cell Adhesion

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.     

Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C-dependent p38 mitogen-activated protein kinase signaling

Disruption of the actin cytoskeleton in subconfluent mesenchymal cells induces chondrogenic differentiation via protein kinase C (PKC) alpha signaling. In this study, we investigated the role of p38 mitogen-activated protein (MAP) kinase in the chondrogenic differentiation of mesenchymal cells that is induced by depolymerization of the actin cytoskeleton. Treatment of mesenchymal cells derived from chick embryonic limb buds with cytochalasin D (CD) disrupted the actin cytoskeleton with concomitant chondrogenic differentiation. The chondrogenesis was accompanied by an increase in p38 MAP kinase activity and inhibition of p38 MAP kinase with SB203580 blocked chondrogenesis. Together these results suggest an essential role for p38 MAP kinase in chondrogenesis. In addition, inhibition of p38 MAP kinase did not alter CD-induced increased expression and activity of PKC alpha, whereas down-regulation of PKC by prolonged exposure of cells to phorbol ester inhibited CD-induced p38 MAP kinase activation. Our results therefore suggest that PKC is involved in the regulation of chondrogenesis induced by disruption of the actin cytoskeleton via a p38 MAP kinase signaling pathway.     

Cyclic AMP inhibits JNK activation by CREB-mediated induction of c-FLIP(L) and MKP-1, thereby antagonizing UV-induced apoptosis

The cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress apoptosis, in a cell context-dependent manner. Our previous study has shown that cAMP, by protein kinase A (PKA)-cAMP response element-binding protein (CREB)-dynein light chain (DLC) pathway, negatively regulates mitogen-activated protein kinase p38 activation, thereby contributing to tumor necrosis factor (TNF)-alpha-induced apoptosis in certain types of cells. However, it remains largely unknown how cAMP suppresses apoptosis. Here we report that cAMP antagonized UV-induced apoptosis in Rat-1 and NIH 3T3 cells. Despite that cAMP significantly suppressed UV-induced p38 activation, inhibition of p38 activity showed no significant effect on UV-induced cell death in both cell lines. Further studies revealed that cAMP antagonized UV-induced apoptosis by inhibition of c-Jun N-terminal protein kinase (JNK) activation. The induction of the long form of cellular FLICE-inhibitory protein (c-FLIP(L)) and mitogen-activated protein kinase phosphatase-1 (MKP-1), but not DLC and p21(WAF1) by CREB was required for cAMP-mediated inhibition of JNK activation. The suppression by cAMP of UV-induced apoptosis was reversed by c-FLIP(L) small-interfering RNA (siRNA) or MKP-1 siRNA, which released the inhibition of JNK activation by cAMP. Thus, our results provide a molecular mechanism by which cAMP suppresses JNK activation and antagonizes apoptosis.     

Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H₂O₂ treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H₂O₂-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47^phox. Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.     

Transduction of the chemotactic signal to the actin cytoskeleton of Dictyostelium discoideum

Dictyostelium discoideum amebae chemotax toward folate during vegetative growth and toward extracellular cAMP during the aggregation phase that follows starvation. Stimulation of starving amebae with extracellular cAMP leads to both actin polymerization and pseudopod extension (Hall et al., 1988, J. Cell. Biochem. 37, 285-299). We have identified an actin nucleation activity (NA) from starving amebae that is regulated by cAMP receptors and controls actin polymerization (Hall et al., 1989, J. Cell Biol., in press). We show here that NA from vegetative cells is also regulated by chemotactic receptors for folate. Our studies indicate that NA is an essential effector in control of the actin cytoskeleton by chemotactic receptors. Guided by a recently proposed model for signal transduction from the cAMP receptor (Snaar-Jagalska et al., 1988, Dev. Genet. 9, 215-225), we investigated which of three signaling pathways activates the NA effector. Treatment of whole cells with a commercial pertussis toxin preparation (PT) inhibited cAMP-stimulated NA. However, endotoxin contamination of the PT appears to account for this effect. The synag7 mutation and caffeine treatment do not inhibit activation of NA by cAMP. Thus, neither activation of adenylate cyclase nor a G protein sensitive to PT treatment of whole cells is necessary for the NA response. Actin nucleation activity stimulated with folate is normal in vegetative fgdA cells. However, cAMP suppresses rather than activates NA in starving fgdA cells. This indicates that the components of the actin nucleation effector are present and that a pathway regulating the inhibitor(s) of nucleation remains functional in starving fgdA cells. The locus of the fgdA defect, a G protein implicated in phospholipase C activation, is directly or indirectly responsible for transduction of the stimulatory chemotactic signal from cAMP receptors to the nucleation effector in Dictyostelium.     

Cyclic AMP inhibits p38 activation via CREB-induced dynein light chain

The mitogen-activated protein kinase p38 plays a critical role in inflammation, cell cycle progression, differentiation, and apoptosis. The activity of p38 is stimulated by a variety of extracellular stimuli, such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), and subjected to regulation by other intracellular signaling pathways, including the cyclic AMP (cAMP) pathway. Yet the underlying mechanism by which cAMP inhibits p38 activation is unknown. Here we show that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of p38 activation. cAMP inhibits p38 activation via the protein kinase A-CREB pathway. The inhibition is mediated by the CREB target gene Dlc, whose protein product, DLC, interferes with the formation of the MKK3/6-p38 complex, thereby suppressing p38 phosphorylation activation by MKK3/6. The inhibition of p38 activation by cAMP leads to suppression of NF-kappaB activity and promotion of apoptosis in response to TNF-alpha. Thus, our results identify DLC as a novel inhibitor of the p38 pathway and provide a molecular mechanism by which cAMP suppresses p38 activation and promotes apoptosis.