Rac is a dominant regulator of cadherin-directed actin assembly that is activated by adhesive ligation independently of Tiam1

2007 ◽  
Vol 292 (3) ◽  
pp. C1061-C1069 ◽  
Author(s):  
Astrid Kraemer ◽  
Marita Goodwin ◽  
Suzie Verma ◽  
Alpha S. Yap ◽  
Radiya G. Ali
Keyword(s):  
Signaling Pathway ◽  
Dominant Negative ◽  
Signaling Cascades ◽  
Potential Contribution ◽  
Actin Assembly ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Signaling Receptors ◽  
The Impact ◽  
E Cadherin

Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp)2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1.

Regulation of epithelial tubule formation by Rho family GTPases

2006 ◽  
Vol 290 (5) ◽  
pp. C1297-C1309 ◽  
Author(s):  
Randi Eisen ◽  
Shereaf Walid ◽  
Don R. Ratcliffe ◽  
George K. Ojakian
Keyword(s):  
Signaling Pathway ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Rho Kinase ◽  
Collagen Gel ◽  
Dominant Negative ◽  
Tubule Formation ◽  
Rho Family Gtpases ◽  
Epithelial Remodeling ◽  
Rho Family

Previous work has established that the integrin signal transduction pathway plays an important role in the regulation of epithelial tubule formation. Furthermore, it has been demonstrated that Rho-kinase, an effector of the Rho signaling pathway, is an important downstream modulator of collagen-mediated renal and mammary epithelial tubule morphogenesis. In the present study, MDCK cells that expressed mutant dominant-negative, constitutively active Rho family GTPases were used to provide further insight into Rho-GTPase signaling and the regulation of epithelial tubule formation. Using collagen gel overlays on MDCK cells as a model system, we observed phosphorylated myosin light chain (pMLC) at the leading edge of migrating lamellipodia. This epithelial remodeling led to the formation of multicellular branching epithelial tubular structures with extensive tight junctions. However, in cells expressing dominant-negative RhoN19, MLC phosphorylation, epithelial remodeling, and tubule formation were inhibited. Instead, only small apical lumens with a solitary tight junctional ring were observed, providing further evidence that Rho signaling through Rho-kinase is important in the regulation of epithelial tubule formation. Because the present model for the Rho signaling pathway proposes that Rac plays a prominent but reciprocal role in cell regulation, experiments were conducted using cells that expressed constitutively active RacV12. When incubated with collagen gels, RacV12-expressing cells formed small apical lumens with simple tight junctions, suggesting that Rac1 signaling also has a prominent role in the regulation of epithelial morphogenesis. Complementary collagen gel overlay experiments with wild-type MDCK cells demonstrated that endogenous Rac1 activation levels decreased over a time course consistent with lamellipodia and tubule formation. Under these conditions, Rac1 was initially localized to the basolateral membrane. However, after epithelial remodeling, activated Rac1 was observed primarily in lamellipodia. These studies support a model in which Rac1 and RhoA are important modulators of epithelial tubule formation.

scholarly journals Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid.

1997 ◽  
Vol 17 (3) ◽  
pp. 1201-1211 ◽  
Author(s):  
R Kozma ◽  
S Sarner ◽  
S Ahmed ◽  
L Lim
Keyword(s):  
Lysophosphatidic Acid ◽  
Growth Cones ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Neurite Retraction ◽  
Rho Family Gtpases ◽  
C3 Exoenzyme ◽  
Rho Family ◽  
Neurite Development

Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells. Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites. These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction. The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA. We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient. ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively. Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N). Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.

scholarly journals A mechanosensitive RhoA pathway that protects epithelia against acute tensile stress

2018 ◽  
Author(s):  
Bipul R Acharya ◽  
Alexander Nestor-Bergmann ◽  
Xuan Liang ◽  
Srikanth Budnar ◽  
Oliver E. Jensen ◽  
...  
Keyword(s):  
Tensile Stress ◽  
Signaling Pathway ◽  
Contractile Response ◽  
Tensile Load ◽  
Actin Assembly ◽  
Epithelial Integrity ◽  
Tissue Movement ◽  
Intrinsic Capacity ◽  
Rhoa Signaling ◽  
E Cadherin

AbstractAdherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. In all these circumstances, it is essential that epithelial integrity be preserved despite the application of tensile stress. In this study, we identify junctional RhoA as a mechanosensitive signaling pathway that responds to epithelial stress. The junctional specificity of this response is mediated by the heterotrimeric protein Gα12, which is recruited by E-cadherin and, in turn, recruits p114 RhoGEF to activate RhoA. Further, we identify Myosin VI as a key mechanosensor, based on its intrinsic capacity to anchor E-cadherin to F-actin when exposed to tensile load. Tension-activated RhoA signaling was necessary to preserve epithelial integrity, which otherwise undergoes fracture when monolayer stress is acutely increased by calyculin. Paradoxically, this homeostatic RhoA signaling pathway increases junctional actomyosin, a contractile response that might be expected to itself promote fracture. Simulations of a vertex-based model revealed that the protective effect of RhoA signaling can be explained through increased yield limit at multicellular vertices, where experiments showed p114 RhoGEF was necessary to increase E-cadherin and promote actin assembly and organization.

scholarly journals Distinct Effects of Rac1 on Differentiation of Primary Avian Myoblasts

1999 ◽  
Vol 10 (10) ◽  
pp. 3137-3150 ◽  
Author(s):  
Rita Gallo ◽  
Marco Serafini ◽  
Loriana Castellani ◽  
Germana Falcone ◽  
Stefano Alemà
Keyword(s):  
Myogenic Differentiation ◽  
Terminal Differentiation ◽  
Dominant Negative ◽  
Skeletal Muscle Differentiation ◽  
Rho Family Gtpases ◽  
Specific Proteins ◽  
Rho Family ◽  
Selective Disassembly ◽  
Jnk Activation ◽  
Constitutively Active

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotubes accumulate severely reduced levels of muscle-specific proteins. This inhibition is the consequence of posttranscriptional events and suggests the presence of a novel level of regulation of myogenesis. We also show that myotubes expressing constitutively active Rac1 fail to assemble ordered sarcomeres. Conversely, a dominant-negative Rac1 variant accelerates sarcomere maturation and inhibits v-Src–induced selective disassembly of I-Z-I complexes. Collectively, our findings provide a role for Rac1 during skeletal muscle differentiation and strongly suggest that Rac1 is required downstream of v-Src in the signaling pathways responsible for the dismantling of tissue-specific supramolecular structures.

scholarly journals Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

1998 ◽  
Vol 72 (11) ◽  
pp. 8806-8812 ◽  
Author(s):  
Erguang Li ◽  
Dwayne Stupack ◽  
Gary M. Bokoch ◽  
Glen R. Nemerow
Keyword(s):  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Cortical Actin ◽  
Lipid Kinase ◽  
Ras Protein ◽  
Cytoskeleton Reorganization ◽  
Pathway Activation ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
In Cells

ABSTRACT Adenovirus (Ad) endocytosis via αv integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.

scholarly journals A Conserved GEF for Rho-Family GTPases Acts in an EGF Signaling Pathway to Promote Sleep-like Quiescence in Caenorhabditis elegans

Genetics ◽  
2016 ◽  
Vol 202 (3) ◽  
pp. 1153-1166 ◽  
Author(s):  
A. L. Fry ◽  
J. T. Laboy ◽  
H. Huang ◽  
A. C. Hart ◽  
K. R. Norman
Keyword(s):  
Caenorhabditis Elegans ◽  
Signaling Pathway ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Egf Signaling

scholarly journals β-Adrenergic Receptor Mediated Protection against Doxorubicin-Induced Apoptosis in Cardiomyocytes: The Impact of High Ambient Glucose

Endocrinology ◽  
2008 ◽  
Vol 149 (12) ◽  
pp. 6449-6461 ◽  
Author(s):  
Naohiro Yano ◽  
Daisuke Suzuki ◽  
Masayuki Endoh ◽  
Andy Tseng ◽  
Joan P. Stabila ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Intracellular Signaling ◽  
Adrenergic Receptor ◽  
Src Kinase ◽  
Dominant Negative ◽  
Akt Inhibitor ◽  
Antiapoptotic Effect ◽  
Induced Apoptosis ◽  
The Impact

Recent studies have demonstrated that the β2-adrenergic receptor (β2AR)-Gαi signaling pathway exerts a cardiac antiapoptotic effect. The goals of this study were to determine the intracellular signaling factors involved in β2AR-mediated protection against doxorubicin-induced apoptosis in H9c2 cardiomyocyte and explore the impact of high ambient glucose on the antiapoptotic effect. Under physiological glucose environment (100 mg/dl), β2AR stimulation prevented doxorubicin-induced apoptosis, which was attenuated by cotreatment with wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, or transfection of a dominant-negative Akt. Inhibition of Src kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine or cSrc small interfering RNA 32 also attenuated the antiapoptotic effect. Inhibition of platelet-derived growth factor receptor (PDGFR) with AG1296 reversed the β2AR-induced antiapoptotic effect. Transfection of an active Src cDNA (Y529F) alone was sufficient to render the cells resistant to apoptosis, and the resistance was blocked by wortmannin. Transfection of an active PI3K minigene (iSH2-p110) alone also induced resistance to apoptosis, and the resistance was reversed by an Akt-inhibitor but not by AG1296. High ambient glucose (450 mg/dl) caused two major effects: 1) it significantly reduced βAR-induced PDGFR phosphorylation, Src kinase activity, and activation of PI3K signaling pathway; and 2) it partially attenuated β2AR-induced antiapoptotic effect. These data provide in vitro evidence supporting a signaling cascade by which β2AR exerts a protective effect against doxorubicin-induced apoptosis via sequential involvement of Gαi, Gβγ, Src, PDGFR, PI3K, and Akt. High ambient glucose significantly attenuates β2AR-mediated cardioprotection by suppressing factors involved in this cascade including PDGFR, Src, and PI3K/Akt.

scholarly journals Activated PAK4 Regulates Cell Adhesion and Anchorage-Independent Growth

2001 ◽  
Vol 21 (10) ◽  
pp. 3523-3533 ◽  
Author(s):  
Jian Qu ◽  
Marta S. Cammarano ◽  
Qing Shi ◽  
Kenneth C. Ha ◽  
Primal de Lanerolle ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Rho Gtpase ◽  
Morphological Changes ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Oncogenic Transformation ◽  
Wild Type ◽  
Focus Formation ◽  
Rho Family Gtpases ◽  
Rho Family

ABSTRACT The serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family in both sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Since previous characterization of PAK4 was carried out only with the wild-type kinase, we have generated a constitutively active mutant of the kinase to determine whether it has other functions. Expression of activated PAK4 in fibroblasts led to a transient induction of filopodia, which is consistent with its role as an effector for Cdc42. In addition, use of the activated mutant revealed a number of other important functions of this kinase that were not revealed by studying the wild-type kinase. For example, activated PAK4 led to the dissolution of stress fibers and loss of focal adhesions. Consequently, cells expressing activated PAK4 had a defect in cell spreading onto fibronectin-coated surfaces. Most importantly, fibroblasts expressing activated PAK4 had a morphology that was characteristic of oncogenic transformation. These cells were anchorage independent and formed colonies in soft agar, similar to what has been observed previously in cells expressing activated Cdc42. Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for Rho family GTPases. These results provide the first demonstration that a PAK family member can transform cells and indicate that PAK4 may play an essential role in oncogenic transformation by the GTPases. We propose that the morphological changes and changes in cell adhesion induced by PAK4 may play a direct role in oncogenic transformation by Rho family GTPases and their exchange factors.

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