scholarly journals Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

2009 ◽  
Vol 296 (3) ◽  
pp. C463-C475 ◽  
Author(s):  
Ana C. P. Thirone ◽  
Pam Speight ◽  
Matthew Zulys ◽  
Ori D. Rotstein ◽  
Katalin Szászi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Rho Kinase ◽  
Small Gtpases ◽  
Hyperosmotic Stress ◽  
Dominant Negative ◽  
Lim Kinase ◽  
Tubular Cells ◽  
Cofilin Phosphorylation ◽  
Underlying Mechanisms

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.

scholarly journals Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation

2012 ◽  
Vol 302 (3) ◽  
pp. C605-C614 ◽  
Author(s):  
Kathleen L. O'Connor ◽  
Min Chen ◽  
L. Nicole Towers
Keyword(s):  
Kinase Inhibitor ◽  
De Novo ◽  
Rho Kinase ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Rhoa Activity ◽  
Nucleotide Exchange Factor ◽  
Integrin Α6β4 ◽  
Dependent Cell

The α6β4 integrin promotes carcinoma invasion through its ability to promote directed migration and polarization of carcinoma cells. In this study, we explore how the α6β4 integrin cooperates with lysophosphatidic acid (LPA) to activate Rho and Rac small GTPases. Through the use of dominant negative Rho constructs, C3 exotransferase, and Rho kinase inhibitor, we find that Rho is critical for LPA-dependent chemotaxis and lamellae formation. However, utilization of specific Rho isoforms depends on integrin α6β4 expression status. Integrin α6β4-negative MDA-MB-435 cells utilize only RhoC for motility, whereas integrin α6β4-expressing cells utilize RhoC but additionally activate and utilize RhoA for LPA-dependent cell motility and lamellae formation. Notably, the activation of RhoA by cooperative LPA and integrin α6β4 signaling requires the Rho guanine nucleotide exchange factor AKAP-Lbc. We also determine that integrin α6β4 cannot activate Rac1 directly but promotes LPA-mediated Rac1 activation that is dependent on RhoA activity and de novo β1 integrin ligation. Finally, we find that the regulation of Rac1 and RhoA in response to LPA is differentially regulated by phosphodiesterases, PKA, and phosphatidylinositol 3-kinase, thus supporting their spatially distinct compartmentalization. In summary, signaling from integrin α6β4 facilitates LPA-stimulated chemotaxis through preferential activation of RhoA, which, in turn, facilitates activation of Rac1.

scholarly journals Hyperosmotic stress induces Rho‐Rho kinase‐LIM kinase‐mediated cofilin phosphorylation

2007 ◽  
Vol 21 (6) ◽  
Author(s):  
Andras Kapus ◽  
Matthew Zulys ◽  
Stine F. Pedersen ◽  
Ana C. P. Thirone
Keyword(s):  
Rho Kinase ◽  
Hyperosmotic Stress ◽  
Lim Kinase ◽  
Cofilin Phosphorylation

Osmotic stress-induced remodeling of the cortical cytoskeleton

2002 ◽  
Vol 283 (3) ◽  
pp. C850-C865 ◽  
Author(s):  
Caterina Di Ciano ◽  
Zilin Nie ◽  
Katalin Szászi ◽  
Alison Lewis ◽  
Takehito Uruno ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
De Novo ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Signaling Mechanism ◽  
Actin Binding Domain ◽  
Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

scholarly journals A Cdc42 Activation Cycle Coordinated by PI 3-Kinase during Fc Receptor-mediated Phagocytosis

2010 ◽  
Vol 21 (3) ◽  
pp. 470-480 ◽  
Author(s):  
Peter Beemiller ◽  
Youxin Zhang ◽  
Suresh Mohan ◽  
Erik Levinsohn ◽  
Isabella Gaeta ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Plasma Membranes ◽  
Dominant Negative ◽  
Subsequent Increase ◽  
Pi3k Activation ◽  
Concentration Changes ◽  
Rho Family ◽  
Phagocytic Cups ◽  
Underlying Mechanisms ◽  
Activation Cycle

Fcγ Receptor (FcR)-mediated phagocytosis by macrophages requires phosphatidylinositol 3-kinase (PI3K) and activation of the Rho-family GTPases Cdc42 and Rac1. Cdc42 is activated at the advancing edge of the phagocytic cup, where actin is concentrated, and is deactivated at the base of the cup. The timing of 3′ phosphoinositide (3′PI) concentration changes in cup membranes suggests a role for 3′PIs in deactivation of Cdc42. This study examined the relationships between PI3K and the patterns of Rho-family GTPase signaling during phagosome formation. Inhibition of PI3K resulted in persistently active Cdc42 and Rac1, but not Rac2, in stalled phagocytic cups. Patterns of 3′PIs and Rho-family GTPase activities during phagocytosis of 5- and 2-μm-diameter microspheres indicated similar underlying mechanisms despite particle size–dependent sensitivities to PI3K inhibition. Expression of constitutively active Cdc42(G12V) increased 3′PI concentrations in plasma membranes and small phagosomes, indicating a role for Cdc42 in PI3K activation. Cdc42(G12V) inhibited phagocytosis at a later stage than inhibition by dominant negative Cdc42(N17). Together, these studies identified a Cdc42 activation cycle organized by PI3K, in which FcR-activated Cdc42 stimulates PI3K and actin polymerization, and the subsequent increase of 3′PIs in cup membranes inactivates Cdc42 to allow actin recycling necessary for phagosome formation.

scholarly journals Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets

Blood ◽  
2006 ◽  
Vol 107 (2) ◽  
pp. 575-583 ◽  
Author(s):  
Dharmendra Pandey ◽  
Pankaj Goyal ◽  
James R. Bamburg ◽  
Wolfgang Siess
Keyword(s):  
Shape Change ◽  
Rho Kinase ◽  
Lim Kinase ◽  
Kinase Activation ◽  
Integrin Αiibβ3 ◽  
Lim Kinase 1 ◽  
Filament Dynamics ◽  
Cofilin Phosphorylation ◽  
High Concentrations ◽  
Platelet Shape

Abstract Cofilin is a regulator of actin filament dynamics. We studied whether during platelet activation Rho kinase stimulates LIM kinase (LIMK) leading to subsequent phosphorylation and inactivation of cofilin. Platelet shape change and aggregation/secretion were induced by low and high concentrations of thrombin, respectively. We found that during these platelet responses Rho kinase activation was responsible for mediating rapid Thr508 phosphorylation and activation of LIMK-1 and for the F-actin increase during shape change and, in part, during secretion. Surprisingly, during shape change cofilin phosphorylation was unaltered, and during aggregation/secretion cofilin was first rapidly dephosphorylated by an okadaic acid–insensitive phosphatase and then slowly rephosphorylated by LIMK-1. LIMK-1 phosphorylation and cofilin dephosphorylation and rephosphorylation during aggregation were independent of integrin αIIbβ3 engagement. Cofilin phosphorylation did not regulate cofilin association with F-actin and was unrelated to the F-actin increase in thrombin-activated platelets. Our study identifies LIMK-1 as being activated by Rho kinase in thrombin-stimulated platelets. Two counteracting pathways, a cofilin phosphatase and LIMK-1, are activated during platelet aggregation/secretion regulating cofilin phosphorylation sequentially and independently of integrin αIIbβ3 engagement. Rho kinase–mediated F-actin increase during platelet shape change and secretion involves a mechanism other than LIMK-1–mediated cofilin phosphorylation, raising the possibility of another LIMK substrate regulating platelet actin assembly.

scholarly journals Baculovirus induces host cell aggregation via a Rho/Rok-dependent mechanism

2014 ◽  
Vol 95 (10) ◽  
pp. 2310-2320 ◽  
Author(s):  
Zihao Deng ◽  
Zhihong Huang ◽  
Meijin Yuan ◽  
Kai Yang ◽  
Yi Pang
Keyword(s):  
Rna Interference ◽  
Host Cell ◽  
Actin Polymerization ◽  
Cell Aggregation ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cell Contact ◽  
Amoeboid Movement ◽  
Pathway Inhibition

Several baculoviruses can induce host cell aggregation during infection; however, the molecular basis remains unknown. The Rho family of small GTPases, including Rho1, Racs and Cdc42, plays important roles in cell migration and cell–cell contact. Activated GTPases target actin polymerization to discrete sites on the plasma membrane, thereby inducing membrane protrusions. In this study, we demonstrated that Spodoptera litura nucleopolyhedrovirus (SpltNPV) infection induced the amoeboid movement and aggregation of SpLi-221 cells in vitro. The amount of Rho1-GTP increased in the infected cells, which suggested that Rho1 was activated upon infection. RNA interference and superinfection of dominant-negative recombinants revealed that the SpltNPV-induced SpLi-221 cell aggregation was dependent on the Rho1, but not Racs or Cdc42, signalling pathway. Inhibition of Rho-associated protein kinase (Rok) activity by the inhibitor Y-27632 significantly reduced SpLi-221 cell aggregation. Silencing Rho1 expression with RNA interference decreased SpltNPV propagation by approximately 40 % in vitro, when SpLi-221 cells were infected at a low, but not high, m.o.i., suggesting that the SpltNPV-induced cell aggregation may benefit SpltNPV spread.

Phosphatidyl Inositol (4,5)P2 Marks Megakaryocyte Internal Membranes and Is Associated with Megakaryocyte Maturation and Platelet Release.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 732-732
Author(s):  
Harald Schulze ◽  
Manav Korpal ◽  
Jonathan Hurov ◽  
Sang-We Kim ◽  
Jinghang Zhang ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Blood Platelets ◽  
Membrane Surface ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Pleckstrin Homology Domain ◽  
Acceptor Site ◽  
Cell Periphery ◽  
Filamentous Actin ◽  
Fluorescence Protein

Abstract To produce blood platelets, the megakaryocyte (MK) cytoplasm elaborates proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. Invaginated demarcation membranes (DMS) are thought to be the source for the proplatelet and platelet membranes, however, they have THUS far BEEN INSUFFICIENTLY characterized. We first used a mouse model where the cDNA encoding enhanced yellow fluorescence protein (EYFP) with a C-terminally introduced myristoyl acceptor site has been introduced into the GPIIb locus. Heterozygous knock-in mice reveal yellow fluroescent MKs with an internal staining pattern that resembles the reticiulated pattern of the DMS as found in micrographs. Proplatelet-forming MKs reveal contiguous membrane connection between the internally stained membranes and the outlines of the proplatelet shaft resulting in production of fluorescent platelets. We next sought to characterize the internal membranes biochemically and retrovirally infected MKs to express the green fluorescence protein (EGFP) tagged with the pleckstrin homology domain of phospholipase Cδ1 (PLCδ1) which binds with high specificity to phosphatidylinositol(4,5)P2 (PIP2). Young MKs stain the cell periphery as described for most other cell types. Mature MKs, however, stain the internal membranes, whereas the plasma membrane becomes PIP2-negative as shown by co-staining with CD41. Proplatelet membranes emanate from these internal PIP2-positive membranes, proving that the DMS is indeed the membrane reservoir during platelet biogenesis. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinaseα (PI4Kα), a protein highly expressed in MKs and platelets, as shown by overexpressing EGFP-tagged kinase in primary MKs. In addition, shRNA-mediated loss of PIP4Kα or depletion of its presumptive substrate block DMS development and expansion of MK size. Thus, PI-4,5-P2 is a marker and essential component of internal membranes and is most likely introduced about the non-canonical pathway using PI5P as the substrate. PI-4,5-P2 promotes actin polymerization by activating small GTPases from the Rac/Rho superfamily as well as Wiskott-Aldrich Syndrome (WASp) family proteins. Indeed, PIP2 is associated with filamentous actin when MKs are co-stained with phalloidin. Expression of a dominant-negative N-WASp C-terminal fragment (CA-domain) that inactivats all WASp/WAVE family members leads to Arp3 binding without assembling the complete Arp2/3 complex, thus inhibiting actin filament nucleation. F-Actin staining in the infected MKs reveals a pattern similar to that of MKs treated with pharmacologic dosage of actin polymerization-antagonists like cytochalasin D, which disrupts actin filaments and inhibits proplatelet formation when administered early in MK culture. Dominant-negative WASp impairs proplatelet elaboration similarly, acting at a step past expansion of the cell volume. These observations implicate a signaling pathway wherein PI-4,5-P2 facilitates DMS development and suggests a pathway that links a DMS lipid marker with local assembly of actin fibers as a requirement for platelet biogenesis.

scholarly journals LIMK (LIM Kinase) Inhibition Prevents Vasoconstriction- and Hypertension-Induced Arterial Stiffening and Remodeling

Hypertension ◽  
2020 ◽  
Vol 76 (2) ◽  
pp. 393-403
Author(s):  
Mariana Morales-Quinones ◽  
Francisco I. Ramirez-Perez ◽  
Christopher A. Foote ◽  
Thaysa Ghiarone ◽  
Larissa Ferreira-Santos ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Arterial Stiffness ◽  
Vascular Smooth Muscle ◽  
Actin Polymerization ◽  
Fiber Formation ◽  
Cell Cortex ◽  
Lim Kinase ◽  
Arterial Stiffening ◽  
Cofilin Phosphorylation ◽  
Visceral Arteries

Increased arterial stiffness and vascular remodeling precede and are consequences of hypertension. They also contribute to the development and progression of life-threatening cardiovascular diseases. Yet, there are currently no agents specifically aimed at preventing or treating arterial stiffening and remodeling. Previous research indicates that vascular smooth muscle actin polymerization participates in the initial stages of arterial stiffening and remodeling and that LIMK (LIM kinase) promotes F-actin formation and stabilization via cofilin phosphorylation and consequent inactivation. Herein, we hypothesize that LIMK inhibition is able to prevent vasoconstriction- and hypertension-associated arterial stiffening and inward remodeling. We found that small visceral arteries isolated from hypertensive subjects are stiffer and have greater cofilin phosphorylation than those from nonhypertensives. We also show that LIMK inhibition prevents arterial stiffening and inward remodeling in isolated human small visceral arteries exposed to prolonged vasoconstriction. Using cultured vascular smooth muscle cells, we determined that LIMK inhibition prevents vasoconstrictor agonists from increasing cofilin phosphorylation, F-actin volume, and cell cortex stiffness. We further show that localized LIMK inhibition prevents arteriolar inward remodeling in hypertensive mice. This indicates that hypertension is associated with increased vascular smooth muscle cofilin phosphorylation, cytoskeletal stress fiber formation, and heightened arterial stiffness. Our data further suggest that pharmacological inhibition of LIMK prevents vasoconstriction-induced arterial stiffening, in part, via reductions in vascular smooth muscle F-actin content and cellular stiffness. Accordingly, LIMK inhibition should represent a promising therapeutic means to stop the progression of arterial stiffening and remodeling in hypertension.

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

2002 ◽  
Vol 22 (3) ◽  
pp. 774-783 ◽  
Author(s):  
Michiru Nishita ◽  
Hiroyuki Aizawa ◽  
Kensaku Mizuno
Keyword(s):  
T Lymphocytes ◽  
Stromal Cell ◽  
Small Gtpases ◽  
Chemotactic Response ◽  
Jurkat Cells ◽  
Lim Kinase ◽  
Actin Reorganization ◽  
Lim Kinase 1 ◽  
Cofilin Phosphorylation ◽  
Stromal Cell Derived Factor

ABSTRACT Stromal cell-derived factor 1 α (SDF-1α), 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-1α-induced chemotaxis of T lymphocytes. SDF-1α significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1α also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1α-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-1α-induced activation of LIMK1, which means that SDF-1α-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-1α-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-1α-induced chemotactic response of T lymphocytes.

Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors

2007 ◽  
Vol 292 (3) ◽  
pp. G806-G817 ◽  
Author(s):  
Ann M. Hopkins ◽  
A’Drian A. Pineda ◽  
L. Matthew Winfree ◽  
G. Thomas Brown ◽  
Mike G. Laukoetter ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Binding ◽  
Rock Inhibitor ◽  
Cell Sheets ◽  
Lim Kinase ◽  
Rock Inhibition

Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector Rho-associated coil-coil-forming kinase (ROCK) influences coordinated epithelial cell motility. Although cells exposed to a pharmacological ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated: the formation of aberrant protrusions at the migrating front and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent insolubility of the actin-binding protein ezrin and enhanced coassociation of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain (MLC) phosphorylation with the MLC kinase inhibitor ML-7, the latter did not influence protrusiveness and, in fact, significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflects an influence of ROCK on F-actin stability via LIM kinase 1 (LIMK-1), which phosphorylates and inactivates cofilin. Y-27632 reduced the levels of both active LIMK-1 and inactive cofilin (phospho forms), and expression of a dominant negative LIMK-1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by overexpression of LIMK-1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organized epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.

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