Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells

The pathways by which activation of the small GTP-binding protein Rac causes cytoskeletal changes are not fully understood but are likely to involve both assembly of new actin filaments and reorganization of actin filaments driven by the actin-dependent ATPase activity of myosin II. Here we show that expression of active RacQ61 in growing HeLa cells, in addition to inducing ruffling, substantially enhances the level of phosphorylation of serine-19 of the myosin II regulatory light chain (MLC), which would increase actomyosin II ATPase and motor activities. Phosphorylated myosin was localized to RacQ61-induced ruffles and stress fibers. RacQ61-induced phosphorylation of MLC was reduced by a maximum of about 38% by an inhibitor (Tat-PAK) of p21-activated kinase (PAK), about 35% by an inhibitor (Y-27632) of Rho kinase, 51% by Tat-PAK plus Y-27632, and 10% by an inhibitor (ML7) of myosin light chain kinase. Staurosporine, a non-specific inhibitor of serine/threonine kinases, reduced RacQ61-induced phosphorylation of MLC by about 58%, at the maximum concentration that did not kill cells. Since Rac activates PAK and PAK can phosphorylate MLC, these data strongly suggest that PAK is responsible for a significant fraction of RacQ61-induced MLC phosphorylation. To our knowledge, this is the first evidence that active Rac causes phosphorylation of MLC in cells, thus implicating activation of the ATPase activity of actomyosin II as one of the ways by which Rac may induce cytoskeletal changes.     

Involvement of Local Lamellipodia in Endothelial Barrier Function

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.     

Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling

During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism. In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction. All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632. These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC. Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation. cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase. Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP.     

Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.     

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.     

GDI-1 phosphorylation switch at serine 96 induces RhoA activation and increased endothelial permeability

We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Calpha-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 --> Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.     

Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

The retinal pigment epithelium (RPE) forms the outer blood–retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial–mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR‐1 induces the time‐ and dose‐dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho‐MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y‐27632, whereas MLCK inhibitor ML‐7 and PLC‐β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC‐β‐mediated calcium increase. Additionally, thrombin‐induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP₃/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin‐induced RPE cell transformation subsequent to BRB dysfunction. J. Cell. Physiol. 226: 414–423, 2011. © 2010 Wiley‐Liss, Inc.     

Stromal Cell-Derived Factor 1α Activates LIM Kinase 1 and Induces Cofilin Phosphorylation for T-Cell Chemotaxis

Stromal cell-derived factor 1 alpha (SDF-1alpha), the ligand for G-protein-coupled receptor CXCR4, is a chemotactic factor for T lymphocytes. LIM kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing and -severing protein, at Ser-3 and regulates actin reorganization. We investigated the role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes. SDF-1alpha significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1alpha-induced LIMK1 activation, thus indicating that Gi protein is involved in LIMK1 activation. Expression of dominant negative Rac (DN-Rac), but not DN-Rho or DN-Cdc42, blocked SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. We used a cell-permeable peptide (S3 peptide) that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibited the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibited the SDF-1alpha-induced cofilin phosphorylation, actin reorganization, and chemotactic response of Jurkat cells. These results suggest that the phosphorylation of cofilin by LIMK1 plays a critical role in the SDF-1alpha-induced chemotactic response of T lymphocytes.     

Redox-dependent downregulation of Rho by Rac

Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases.     

Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction

Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling.     

Rho kinase signaling pathways during stretch in primary alveolar epithelia

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.     

S1P induces FA remodeling in human pulmonary endothelial cells: role of Rac, GIT1, FAK, and paxillin.

Sphingosine 1-phosphate (S1P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest 108: 689-701, 2001). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S1P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S1P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S1P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S1P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S1P induced a transient association of GIT1 with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S1P-mediated barrier enhancement via Rac- and GIT-mediated processes.     

Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activation of Rho/Rho kinase and inhibition of myosin light chain phosphatase.

Mildly oxidized low density lipoprotein (mox-LDL) is critically involved in the early atherogenic responses of the endothelium and increases endothelial permeability through an unknown signal pathway. Here we show that (i) exposure of confluent human endothelial cells (HUVEC) to mox-LDL but not to native LDL induces the formation of actin stress fibers and intercellular gaps within minutes, leading to an increase in endothelial permeability; (ii) mox-LDL induces a transient decrease in myosin light chain (MLC) phosphatase that is paralleled by an increase in MLC phosphorylation; (iii) phosphorylated MLC stimulated by mox-LDL is incorporated into stress fibers; (iv) cytoskeletal rearrangements and MLC phosphorylation are inhibited by C3 transferase from Clostridium botulinum, a specific Rho inhibitor, and Y-27632, an inhibitor of Rho kinase; and (v) mox-LDL does not increase intracellular Ca(2+) concentration. Our data indicate that mox-LDL induces endothelial cell contraction through activation of Rho and its effector Rho kinase which inhibits MLC phosphatase and phosphorylates MLC. We suggest that inhibition of this novel cell signaling pathway of mox-LDL could be relevant for the prevention of atherosclerosis.     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)‐elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP‐mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre‐contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine‐induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine‐induced RhoA activation, measured by both stress fibre formation and pull‐down assay whereas the same Epac activation prevented methacholine‐induced Rac1 inhibition measured by pull‐down assay. Epac‐driven inhibition of both methacholine‐induced muscle contraction by Toxin B‐1470, and MLC phosphorylation by the Rac1‐inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac‐mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre‐contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.     

betaPak-interacting exchange factor-mediated Rac1 activation requires smgGDS guanine nucleotide exchange factor in basic fibroblast growth factor-induced neurite outgrowth

Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that betaPak-interacting exchange factor (betaPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that betaPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of betaPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, betaPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of betaPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby betaPIX can regulate Rac1 activity.     

Actin-depolymerizing factor mediates Rac/Rop GTPase-regulated pollen tube growth

Pollen tube elongation is a rapid tip growth process that is driven by a dynamic actin cytoskeleton. A ubiquitous family of actin binding proteins, actin-depolymerizing factors (ADFs)/cofilins, bind to actin filaments, induce severing, enhance depolymerization from their slow-growing end, and are important for maintaining actin dynamics in vivo. ADFs/cofilins are regulated by multiple mechanisms, among which Rho small GTPase-activated phosphorylation at a terminal region Ser residue plays an important role in regulating their actin binding and depolymerizing activity, affecting actin reorganization. We have shown previously that a tobacco pollen-specific ADF, NtADF1, is important for maintaining normal pollen tube actin cytoskeleton organization and growth. Here, we show that tobacco pollen grains accumulate phosphorylated and nonphosphorylated forms of ADFs, suggesting that phosphorylation could be a regulatory mechanism for their activity. In plants, Rho-related Rac/Rop GTPases have been shown to be important regulators for pollen tube growth. Overexpression of Rac/Rop GTPases converts polar growth into isotropic growth, resulting in pollen tubes with ballooned tips and a disrupted actin cytoskeleton. Using the Rac/Rop GTPase-induced defective pollen tube phenotype as a functional assay, we show that overexpression of NtADF1 suppresses the ability of NtRac1, a tobacco Rac/Rop GTPase, to convert pollen tube tip growth to isotropic growth. This finding suggests that NtADF1 acts in a common pathway with NtRac1 to regulate pollen tube growth. A mutant form of NtADF1 with a nonphosphorylatable Ala substitution at its Ser-6 position [NtADF1(S6A)] shows increased activity, whereas the mutant NtADF1(S6D), which has a phospho-mimicking Asp substitution at the same position, shows reduced ability to counteract the effect of NtRac1. These observations suggest that phosphorylation at Ser-6 of NtADF1 could be important for its integration into the NtRac1 signaling pathway. Moreover, overexpression of NtRac1 diminishes the actin binding activity of green fluorescent protein (GFP)-NtADF1 but has little effect on the association of GFP-NtADF1(S6A) with actin cables in pollen tubes. Together, these observations suggest that NtRac1-activated activity regulates the actin binding and depolymerizing activity of NtADF1, probably via phosphorylation at Ser-6. This notion is further supported by the observation that overexpressing a constitutively active NtRac1 in transformed pollen grains significantly increases the ratio of phosphorylated to nonphosphorylated ADFs. Together, the observations reported here strongly support the idea that NtRac1 modulates NtADF1 activity through phosphorylation at Ser-6 to regulate actin dynamics.     

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.     

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.     

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).