LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.     

Nir2, a novel regulator of cell morphogenesis

Cell morphogenesis requires dynamic reorganization of the actin cytoskeleton, a process that is tightly regulated by the Rho family of small GTPases. These GTPases act as molecular switches by shuttling between their inactive GDP-bound and active GTP-bound forms. Here we show that Nir2, a novel protein related to Drosophila retinal degeneration B (RdgB), markedly affects cell morphology through a novel Rho-inhibitory domain (Rid) which resides in its N-terminal region. Rid exhibits sequence homology with the Rho-binding site of formin-homology (FH) proteins and leads to an apparent loss of F-actin staining when ectopically expressed in mammalian cells. We also show that Rid inhibits Rho-mediated stress fiber formation and lysophosphatidic acid-induced RhoA activation. Biochemical studies demonstrated that Nir2, via Rid, preferentially binds to the inactive GDP-bound form of the small GTPase Rho. Microinjection of antibodies against Nir2 into neuronal cells markedly attenuates neurite extension, whereas overexpression of Nir2 in these cells attenuates Rho-mediated neurite retraction. These results implicate Nir2 as a novel regulator of the small GTPase Rho in actin cytoskeleton reorganization and cell morphogenesis.     

Cdc42: an important regulator of neuronal morphology

Regulation of neuronal morphology and activity-dependent synaptic modifications involves reorganization of the actin cytoskeleton. Dynamic changes of the actin cytoskeleton in many cell types are controlled by small GTPases of the Rho family, such as RhoA, Rac1 and Cdc42. As key regulators of both actin and microtubule cytoskeleton, Rho GTPases have also emerged as important regulators of dendrite and spine structural plasticity. Multiple studies suggest that Rac1 and Cdc42 are positive regulators promoting neurite outgrowth and growth cone protrusion, while the activation of RhoA induces stress fiber formation, leading to growth cone collapse and neurite retraction. This review focuses on recent advances in our understanding of the molecular mechanisms underlying physiological and pathological functions of Cdc42 in the nervous system. We also discuss application of different FRET-based biosensors as a powerful approach to examine the dynamics of Cdc42 activity in living cells.     

Rho plays a key role in TGF-beta1-induced cytoskeletal rearrangement in human retinal pigment epithelium

Proliferative vitroretinopathy (PVR) is caused by retinal pigment epithelial (RPE) cell proliferation and transformation into fibrotic cells that produce extracellular matrix (ECM) components. Transforming growth factor beta1 (TGF-beta1) is known to play an important role in PVR pathogenesis. To determine how TGF-beta1 mediates the pathogenic changes in RPE cells, we characterized the effects of TGF-beta1 on the morphology, ECM accumulation, and stress fiber formation of ARPE-19 cells, a human RPE cell line. We then elucidated the signaling pathways that mediate these effects. Serum-starved ARPE-19 cells were incubated with 10 ng/ml TGF-beta1 and their morphological changes were examined by phase-contrast microscopy. Actin reorganization was examined by immunochemistry and confocal microscopy. Protein phosphorylation was analyzed by Western blot analysis. TGF-beta1 treatment induced cytoskeleton reorganization, alpha-SMA expression, increased the phosphorylation of ERK, Smad2/3, and AKT, and activated RhoA and Rac1. Cytoskeletal rearrangement was prevented by pretreatment with a Rho inhibitor and by expression of a dominant negative form of Rho. TGF-beta1 also increased LIM kinase and cofilin phosphorylation and the Rho inhibitor blocked this effect. We propose that TGF-beta1 induces human RPE cells to undergo cytoskeletal actin rearrangement via Rho GTPase-dependent pathways that modulate LIM kinase and cofilin activity. This inhibits actin depolymerization and induces the cytoskeletal rearrangements in RPE cells that result in the characteristic features of PVR.     

IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways

Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to G(i). In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.     

Centaureidin promotes dendrite retraction of melanocytes by activating Rho

Melanosomes synthesized within melanocytes are transferred to keratinocytes through dendrites, resulting in a constant supply of melanin to the epidermis, and this process determines skin pigmentation. During screening for inhibitors of melanosome transfer, we found a novel reagent, centaureidin, that induces significant morphological changes in normal human epidermal melanocytes and inhibits melanocyte dendrite elongation, resulting in a reduction of melanosome transfer in an in vitro melanocyte-keratinocyte co-culture system. Since members of the Rho family of small GTP-binding proteins act as master regulators of dendrite formation, and activated Rho promotes dendrite retraction, we studied the effects of centaureidin on the small GTPases. In in vitro binding assay, centaureidin activated Rho and furthermore, a Rho inhibitor (C. botulinum C3 exoenzyme), a Rho kinase inhibitor (Y27632) and a small GTPase inhibitor (Toxin B) blocked dendrite retraction induced by centaureidin. These results suggest centaureidin could act via the Rho signaling pathway, and it may directly or indirectly activate Rho. Thus, centaureidin appears to inhibit dendrite outgrowth from melanocytes by activating Rho, resulting in the inhibition of melanosome transfer from melanocytes to keratinocytes.     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.     

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.     

Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase

LIM-kinase 1 (LIMK1) and LIM-kinase 2 (LIMK2) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases. We report our findings that ROCK, a downstream protein kinase of Rho, specifically activates LIMK2 but not LIMK1 downstream of RhoA. LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK (ROCK-Delta3), whereas full-length ROCK selectively activates LIMK2 but not LIMK1. Activation of LIMK2 by RhoA was inhibited by Y-27632, a specific inhibitor of ROCK, but Rac1-mediated activation of LIMK1 was not. ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity. A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes, whereas replacement by glutamate enhanced the protein kinase activity and stress fiber formation by LIMK2. These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization. Together with the finding that LIMK1 is regulated by Pak1, LIMK1 and LIMK2 are regulated by different protein kinases downstream of distinct Rho family GTPases.     

Phosphorylation of critical serine residues in Gem separates cytoskeletal reorganization from down-regulation of calcium channel activity

Gem is a small GTP-binding protein that has a ras-like core and extended chains at each terminus. The primary structure of Gem and other RGK family members (Rad, Rem, and Rem2) predicts a GTPase deficiency, leading to the question of how Gem functional activity is regulated. Two functions for Gem have been demonstrated, including inhibition of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. These functions for Gem have been ascribed to its interaction with the calcium channel beta subunit and Rho kinase beta, respectively. We show here that these functions are separable and regulated by distinct structural modifications to Gem. Phosphorylation of serines 261 and 289, located in the C-terminal extension, is required for Gem-mediated cytoskeletal reorganization, while GTP and possibly calmodulin binding are required for calcium channel inhibition. In addition to regulating cytoskeletal reorganization, phosphorylation of serine 289 in conjunction with serine 23 results in bidentate 14-3-3 binding, leading to increased Gem protein half-life. Evidence presented shows that phosphorylation of serine 261 is mediated via a cdc42/protein kinase Czeta-dependent pathway. These data demonstrate that phosphorylation of serines 261 and 289, outside the GTP-binding region of Gem, controls its inhibition of Rho kinase beta and associated changes in the cytoskeleton.     

Tenascin-C suppresses Rho activation

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.     

Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms

Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.     

Tetraspanin CD82 Inhibits Protrusion and Retraction in Cell Movement by Attenuating the Plasma Membrane-Dependent Actin Organization

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.     

Increased expression of Gem after rat sciatic nerve injury

Gem belongs to the Rad/Gem/Kir subfamily of Ras-related GTPases, whose expression is induced in several cell types upon activation by extracellular stimuli. Two functions of Gem have been demonstrated, including regulation of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. Because of the essential relationship between actin reorganization and peripheral nerve regeneration, we investigated the spatiotemporal expression of Gem in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Gem had a significant up-regulation from 1 day, peaked at day 5 and then gradually decreased to the normal level. At its peak expression, Gem expressed mainly in Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, the peak expression of Gem was in parallel with PCNA, and numerous SCs expressing Gem were PCNA positive. Thus, all of our findings suggested that Gem may be involved in the pathophysiology of sciatic nerve after SNC.     

Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization

Most animal cells use a combination of actin-myosin–based contraction and actin polymerization– based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389–399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53–62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401–410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.     

Functions of a rho-specific guanine nucleotide exchange factor in neurite retraction. Possible role of a proline-rich motif of KIAA0380 in localization

The Rho/Rho kinase signaling pathway plays an essential role in neurite retraction and cell rounding in response to G(12/13)-coupled receptor activation in neuronal cells. The Rho guanine nucleotide exchange factor involved in these processes has not been identified. To monitor the activation state of Rho kinase, we developed a vimentin head/Rho kinase chimera, which is intramolecularly phosphorylated in a Rho-dependent manner at Ser(71) of the fused vimentin head. Using this system, we identified a clone termed KIAA0380, which contains the G alpha(12/13)-binding domain as well as a tandem of the Dbl homology/pleckstrin homology (DH/PH) domain, as an activator of Rho/Rho kinase signaling. Molecular dissection analyses revealed that a proline-rich motif C-terminally adjacent to DH/PH domain is essential for plasma membrane localization of KIAA0380 and cortical actin reorganization followed by cell rounding. In contrast, the DH/PH domain of KIAA0380 is localized in the cytoplasm, where it activates Rho/Rho kinase and induces stress fiber formation, consistent with results using p115 Rho guanine nucleotide exchange factor, which has a similar structure to KIAA0380 but lacks a proline-rich motif. These results suggest that upon stimulation, KIAA0380 translocates to the plasma membrane via the proline-rich motif and there activates Rho/Rho kinase signaling. In neuroblastoma Neuro2a cells, KIAA0380 was observed in the tips of neurites, a location where cortical actin reorganization is induced upon stimulation with lysophosphatidic acid. Ectopic expression of the N-terminal fragment inhibited lysophosphatidic acid-induced neurite retraction of Neuro2a cells. These results suggest that KIAA0380 plays an important role in neurite retraction through Rho-dependent signaling.     

Tumor Necrosis Factor-α Induces Stress Fiber Formation through Ceramide Production: Role of Sphingosine Kinase

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.     

Rho-A is critical for osteoclast podosome organization, motility, and bone resorption

Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in alpha(v)beta(3)/gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho(Val-14) (constitutively active) and Rho(Asn-19) (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90-100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho(Val-14) transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/alpha(v)beta(3.) The effects of Rho(Val-14) transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of Rho(Val-14) and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho(Asn-19) produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.     

Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction

Cell repulsion responses to Eph receptor activation are linked to rapid actin cytoskeletal reorganizations, which in turn are partially mediated by Rho-ROCK (Rho kinase) signalling, driving actomyosin contractility. In the present study, we show that Rho alone is not sufficient for this repulsion response. Rather, Cdc42 (cell division cycle 42) and its effector MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) are also critical for ephrinB-induced cell retraction. Stimulation of endothelial cells with ephrinB2 triggers rapid, but transient, cell retraction. We show that, although membrane retraction is fully blocked by blebbistatin (a myosin-II ATPase inhibitor), it is only partially blocked by inhibiting Rho-ROCK signalling, suggesting that there is ROCK-independent signalling to actomyosin contractility downstream of EphBs. We find that a combination of either Cdc42 or MRCK inhibition with ROCK inhibition completely abolishes the repulsion response. Additionally, endocytosis of ephrin-Eph complexes is not required for initial cell retraction, but is essential for subsequent Rac-mediated re-spreading of cells. Our data reveal a complex interplay of Rho, Rac and Cdc42 in the process of EphB-mediated cell retraction-recovery responses.