scholarly journals Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.

1996 ◽  
Vol 7 (9) ◽  
pp. 1419-1427 ◽  
Author(s):  
B C Tilly ◽  
M J Edixhoven ◽  
L G Tertoolen ◽  
N Morii ◽  
Y Saitoh ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Chloride Channels ◽  
Kinase Activity ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Cell Swelling ◽  
Phosphatidylinositol 3 Kinase ◽  
C3 Exoenzyme ◽  
Original Cell ◽  
Adp Ribosyltransferase

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

scholarly journals Phosphatidylinositol 3-Kinase, Cdc42, and Rac1 Act Downstream of Ras in Integrin-Dependent Neurite Outgrowth in N1E-115 Neuroblastoma Cells

2000 ◽  
Vol 20 (1) ◽  
pp. 158-172 ◽  
Author(s):  
Shula Sarner ◽  
Robert Kozma ◽  
Sohail Ahmed ◽  
Louis Lim
Keyword(s):  
Neurite Outgrowth ◽  
Kinase Activity ◽  
Neuroblastoma Cells ◽  
Dominant Negative ◽  
Serum Starvation ◽  
Phosphatidylinositol 3 Kinase ◽  
Serum Factors ◽  
Rac1 Activity ◽  
Neurite Formation ◽  
Phosphatidylinositol 3

ABSTRACT Ras and Rho family GTPases have been ascribed important roles in signalling pathways determining cellular morphology and growth. Here we investigated the roles of the GTPases Ras, Cdc42, Rac1, and Rho and that of phosphatidylinositol 3-kinase (PI 3-kinase) in the pathway leading from serum starvation to neurite outgrowth in N1E-115 neuroblastoma cells. Serum-starved cells grown on a laminin matrix exhibited integrin-dependent neurite outgrowth. Expression of dominant negative mutants of Ras, PI 3-kinase, Cdc42, or Rac1 all blocked this neurite outgrowth, while constitutively activated mutants of Ras, PI 3-kinase, or Cdc42 were each sufficient to promote outgrowth even in the presence of serum. A RasH40C;G12V double mutant which binds preferentially to PI 3-kinase also promoted neurite formation. Activated RasG12V-induced outgrowth required PI 3-kinase activity, but activated PI 3-kinase-induced outgrowth did not require Ras activity. Although activated Rac1 by itself did not induce neurites, neurite outgrowth induced by activated Cdc42G12Vwas Rac1 dependent. Cdc42G12V-induced neurites appeared to lose their normal polarization, almost doubling the average number of neurites produced by a single cell. Outgrowth induced by activated Ras or PI 3-kinase required both Cdc42 and Rac1 activity, but Cdc42G12V-induced outgrowth did not need Ras or PI 3-kinase activity. Active RhoG14V reduced outgrowth promoted by RasG12V. Finally, expression of dominant negative Jun N-terminal kinase or extracellular signal-regulated kinase did not inhibit outgrowth, suggesting these pathways are not essential for this process. Our results suggest a hierarchy of signalling where Ras signals through PI 3-kinase to Cdc42 and Rac1 activation (and Rho inactivation), culminating in neurite outgrowth. Thus, in the absence of serum factors, Ras may initiate cell cycle arrest and terminal differentiation in N1E-115 neuroblastoma cells.

Polyamines regulate Rho-kinase and myosin phosphorylation during intestinal epithelial restitution

2003 ◽  
Vol 284 (4) ◽  
pp. C848-C859 ◽  
Author(s):  
Jaladanki N. Rao ◽  
Xin Guo ◽  
Lan Liu ◽  
Tongtong Zou ◽  
Karnam S. Murthy ◽  
...  
Keyword(s):  
Cell Migration ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intestinal Epithelial ◽  
Epithelial Restitution ◽  
Mlc Phosphorylation

Polyamines are required for the early phase of mucosal restitution that occurs as a consequence of epithelial cell migration. Our previous studies have shown that polyamines increase RhoA activity by elevating cytosolic free Ca2+ concentration ([Ca2+]cyt) through controlling voltage-gated K+ channel expression and membrane potential ( E m) during intestinal epithelial restitution. The current study went further to determine whether increased RhoA following elevated [Ca2+]cyt activates Rho-kinase (ROK/ROCK) resulting in myosin light chain (MLC) phosphorylation. Studies were conducted in stable Cdx2-transfected intestinal epithelial cells (IEC-Cdx2L1), which were associated with a highly differentiated phenotype. Reduced [Ca2+]cyt, by either polyamine depletion or exposure to the Ca2+-free medium, decreased RhoA protein expression, which was paralleled by significant decreases in GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. The reduction of [Ca2+]cyt also inhibited cell migration after wounding. Elevation of [Ca2+]cyt induced by the Ca2+ ionophore ionomycin increased GTP-bound RhoA, ROCK-1, and ROKα proteins, Rho-kinase activity, and MLC phosphorylation. Inhibition of RhoA function by a dominant negative mutant RhoA decreased the Rho-kinase activity and resulted in cytoskeletal reorganization. Inhibition of ROK/ROCK activity by the specific inhibitor Y-27632 not only decreased MLC phosphorylation but also suppressed cell migration. These results indicate that increase in GTP-bound RhoA by polyamines via [Ca2+]cytcan interact with and activate Rho-kinase during intestinal epithelial restitution. Activation of Rho-kinase results in increased MLC phosphorylation, leading to the stimulation of myosin stress fiber formation and cell migration.

Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts

2003 ◽  
Vol 285 (4) ◽  
pp. C935-C944 ◽  
Author(s):  
Iris Carton ◽  
Diane Hermans ◽  
Jan Eggermont
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Cell Types ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cell Swelling ◽  
Toxin B ◽  
Rhodamine Phalloidin ◽  
Actin Reorganization ◽  
Membrane Protrusions

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way.

scholarly journals Role of RHOB in the antiproliferative effect of glucocorticoid receptor on macrophage RAW264.7 cells

2008 ◽  
Vol 200 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Xiaohui Wang ◽  
Yuxia Chen ◽  
Yan Wang ◽  
Xiaoyan Zhu ◽  
Yuanyuan Ma ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Rho Gtpases ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Antiproliferative Effect ◽  
Raw264.7 Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Anti Cancer ◽  
Macrophage Killing

Although glucocorticoid (GC) has been reported to inhibit macrophage killing activity and cytokine production in response to proinflammatory stimuli, the effect of GC on macrophage proliferation is controversial. In our previous study, we found that inhibition of glucocorticoid receptor (GR) expression in murine macrophage cell line RAW264.7 cells (RAW-GR(−) cells) by RNAi significantly promoted cell proliferation. In the present study, we provide the evidence that the expression ofRhob, a member of Rho GTPases with anti-cancer character, remarkably decreased in RAW-GR(−) and RAW264.7 cells transiently transfected with GR-RNAi vector. Overexpression or constitutive activation ofRhobin RAW-GR(−) and RAW264.7 cells by transfection with wild-typeRhobexpression vector (Rhob-wt) or constitutively activatedRhobplasmid (Rhob-V14) resulted in decreased proliferation of the two cell lines. Oppositely, the proliferation of RAW264.7 cells was significantly increased when the expression ofRhobby RNA interference technique or the activity ofRhobby transfection with dominant negativeRhobmutant that is defective in nucleotide binding (Rhob-N19) was inhibited. In addition, enhanced activity of Akt, but not MAPK3/1 or MAPK14, was found in RAW-GR(−) cells. Blocking the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt with the specific inhibitor LY294002 decreased the proliferation and elevated RHOB protein level, indicating that PI3K/Akt signal plays its role of proliferation modulation upstream of RHOB protein. In conclusion, these results demonstrate thatRhobplays an important role in the antiproliferative effect of GR on RAW264.7 cells by GR→Akt→Rhobsignaling andRhobnegatively regulates the proliferation of RAW264.7 cells.

scholarly journals Amino acid availability regulates p70 S6 kinase and multiple translation factors

1998 ◽  
Vol 334 (1) ◽  
pp. 261-267 ◽  
Author(s):  
Xuemin WANG ◽  
Linda E. CAMPBELL ◽  
Christa M. MILLER ◽  
Christopher G. PROUD
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Kinase Activity ◽  
Cell Swelling ◽  
Phosphatidylinositol 3 Kinase ◽  
S6 Kinase ◽  
P70 S6 Kinase ◽  
Amino Acid Availability ◽  
Translation Factors ◽  
Phosphatidylinositol 3

Incubation of Chinese hamster ovary cells without amino acids for up to 60 min caused a rapid marked decrease in p70 S6 kinase activity and increased binding of initiation factor eIF4E to its inhibitory regulator protein 4E-BP1. This was associated with dephosphorylation of 4E-BP1 and eIF4E and dissociation of eIF4E from eIF4G. All these effects were rapidly reversed by resupplying a mixture of amino acids and this was blocked by rapamycin and by inhibitors of phosphatidylinositol 3-kinase, implying a role for phosphatidylinositol 3-kinase in the signalling pathway linking amino acids with the control of p70 S6 kinase activity and the phosphorylation of these translation factors. Amino acid withdrawal also led to changes in the phosphorylation of other translation factors; phosphorylation of eIF4E decreased whereas elongation factor eEF2 became more heavily phosphorylated, each of these changes being associated with decreased activity of the factor in question. Earlier studies have suggested that protein kinase B (PKB) may act upstream of p70 S6 kinase. However, amino acids did not affect the activity of PKB, indicating that amino acids activate p70 S6 kinase through a pathway independent of this enzyme. Studies with individual amino acids suggested that the effects on p70 S6 kinase activity and translation-factor phosphorylation were independent of cell swelling. The data show that amino acid supply regulates multiple translation factors in mammalian cells.

scholarly journals Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function

2004 ◽  
Vol 166 (3) ◽  
pp. 317-323 ◽  
Author(s):  
Rebecca L. Berdeaux ◽  
Begoña Díaz ◽  
Lomi Kim ◽  
G. Steven Martin
Keyword(s):  
Specific Inhibitor ◽  
Stress Fiber ◽  
Small Gtpase ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Stress Fiber Formation ◽  
Transformed Fibroblasts ◽  
C3 Exoenzyme ◽  
And Function

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.

scholarly journals Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

2011 ◽  
Vol 208 (5) ◽  
pp. 1055-1068 ◽  
Author(s):  
Bebhinn Treanor ◽  
David Depoil ◽  
Andreas Bruckbauer ◽  
Facundo D. Batista
Keyword(s):  
Actin Cytoskeleton ◽  
B Cell ◽  
Protein Function ◽  
Actin Polymerization ◽  
Lymphocyte Activation ◽  
Dominant Negative ◽  
Temporary Increase ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Diffusion Dynamics

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

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