LIMK2-1, a new isoform of human LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than that of its two homologs, LIMK2a and LIMK2b

2018 ◽  
Vol 475 (23) ◽  
pp. 3745-3761 ◽  
Author(s):  
Béatrice Vallée ◽  
Hélène Cuberos ◽  
Michel Doudeau ◽  
Fabienne Godin ◽  
David Gosset ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Neurological Diseases ◽  
Mrna Level ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
C Terminus ◽  
Targeted Treatments ◽  
Cytoskeleton Remodeling ◽  
Inhibitory Domain ◽  
Cytoskeleton Dynamics

LIMK1 and LIMK2 (LIMKs, LIM kinases) are kinases that play a crucial role in cytoskeleton dynamics by independently regulating both actin filament and microtubule remodeling. LIMK1 and, more recently, LIMK2 have been shown to be involved in cancer development and metastasis, resistance of cancer cells to microtubule-targeted treatments, neurological diseases, and viral infection. LIMKs have thus recently emerged as new therapeutic targets. Databanks describe three isoforms of human LIMK2: LIMK2a, LIMK2b, and LIMK2-1. Evidence suggests that they may not have completely overlapping functions. We biochemically characterized the three isoforms to better delineate their potential roles, focusing on LIMK2-1, which has only been described at the mRNA level in a single study. LIMK2-1 has a protein phosphatase 1 (PP1) inhibitory domain at its C-terminus which its two counterparts do not. We showed that the LIMK2-1 protein is indeed synthesized. LIMK2-1 does not phosphorylate cofilin, the canonical substrate of LIMKs, although it has kinase activity and promotes actin stress fiber formation. Instead, it interacts with PP1 and partially inhibits its activity towards cofilin. Our data suggest that LIMK2-1 regulates actin cytoskeleton dynamics by preventing PP1-mediated cofilin dephosphorylation, rather than by directly phosphorylating cofilin as its two counterparts, LIMK2a and LIMK2b. This specificity may allow for tight regulation of the phospho-cofilin pool, determining the fate of the cell.

scholarly journals GDI-1 Phosphorylation Switch at Serine 96 Induces RhoA Activation and Increased Endothelial Permeability

2007 ◽  
Vol 27 (18) ◽  
pp. 6323-6333 ◽  
Author(s):  
Nebojsa Knezevic ◽  
Arun Roy ◽  
Barbara Timblin ◽  
Maria Konstantoulaki ◽  
Tiffany Sharma ◽  
...  
Keyword(s):  
Endothelial Permeability ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Mutant Form ◽  
Actin Stress Fiber ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Stress Fiber Formation ◽  
C Terminus ◽  
Actin Stress Fiber Formation

ABSTRACT 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 Cα-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.

scholarly journals AMP-activated Protein Kinase Impairs Endothelial Actin Cytoskeleton Assembly by Phosphorylating Vasodilator-stimulated Phosphoprotein

2006 ◽  
Vol 282 (7) ◽  
pp. 4601-4612 ◽  
Author(s):  
Constanze Blume ◽  
Peter M. Benz ◽  
Ulrich Walter ◽  
Joohun Ha ◽  
Bruce E. Kemp ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Energy Homeostasis ◽  
Serum Response Factor ◽  
Regulatory Protein ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Zdf Rat ◽  
Vasp Phosphorylation ◽  
Amp Activated Protein Kinase

Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that links signaling pathways to remodeling of the cytoskeleton. VASP functions are modulated by protein kinases, which phosphorylate the sites Ser-157, Ser-239, and Thr-278. The kinase responsible for Thr-278 phosphorylation, biological functions of the phosphorylation, and association with disease states have remained enigmatic. Using VASP phosphorylation status-specific antibodies, we identified AMP-activated protein kinase (AMPK), a serine-threonine kinase and fundamental sensor of energy homeostasis, in a screen for kinases that phosphorylate the Thr-278 site of VASP in endothelial cells. Pharmacological AMPK inhibitors and activators and AMPK mutants revealed that the kinase specifically targets residue Thr-278 but not Ser-157 or Ser-239. Quantitative fluorescence-activated cell sorter analysis and serum response factor transcriptional reporter assays, which quantify the cellular F-/G-actin equilibrium, indicated that AMPK-mediated VASP phosphorylation impaired actin stress fiber formation and altered cell morphology. In the Zucker Diabetic Fatty (ZDF) rat model for type II diabetes, AMPK activity and Thr-278 phosphorylation were substantially reduced in arterial vessel walls. These findings suggest that VASP is a new AMPK substrate, that VASP Thr-278 phosphorylation translates metabolic signals into actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessels.

scholarly journals Nir2, a Novel Regulator of Cell Morphogenesis

2002 ◽  
Vol 22 (8) ◽  
pp. 2650-2662 ◽  
Author(s):  
Donghua Tian ◽  
Vladimir Litvak ◽  
Maria Toledo-Rodriguez ◽  
Shari Carmon ◽  
Sima Lev
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Fiber Formation ◽  
Cell Morphogenesis ◽  
Neurite Retraction ◽  
Cytoskeleton Reorganization ◽  
Apparent Loss ◽  
Inhibitory Domain

ABSTRACT 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.

scholarly journals B-RAF Regulation of Rnd3 Participates in Actin Cytoskeletal and Focal Adhesion Organization

2008 ◽  
Vol 19 (2) ◽  
pp. 498-508 ◽  
Author(s):  
R. Matthew Klein ◽  
Laurie S. Spofford ◽  
Ethan V. Abel ◽  
Arisa Ortiz ◽  
Andrew E. Aplin
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Constitutive Expression ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Mitogen Activated Protein Kinase ◽  
Actin Stress Fiber ◽  
Lim Kinase ◽  
Stress Fiber Formation

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.

scholarly journals Identification of Filamin as a Novel Ligand for Caveolin-1: Evidence for the Organization of Caveolin-1–associated Membrane Domains by the Actin Cytoskeleton

2000 ◽  
Vol 11 (1) ◽  
pp. 325-337 ◽  
Author(s):  
Martin Stahlhut ◽  
Bo van Deurs
Keyword(s):  
Actin Cytoskeleton ◽  
Spatial Organization ◽  
Hybrid Approach ◽  
Fiber Formation ◽  
Membrane Domains ◽  
Dimerization Domain ◽  
Double Labeling ◽  
Caveolin 1 ◽  
C Terminus

Reports on the ultrastructure of cells as well as biochemical data have, for several years, been indicating a connection between caveolae and the actin cytoskeleton. Here, using a yeast two-hybrid approach, we have identified the F-actin cross-linking protein filamin as a ligand for the caveolae-associated protein caveolin-1. Binding of caveolin-1 to filamin involved the N-terminal region of caveolin-1 and the C terminus of filamin close to the filamin-dimerization domain. In in vitro binding assays, recombinant caveolin-1 bound to both nonmuscle and muscle filamin, indicating that the interaction might not be cell type specific. With the use of confocal microscopy, colocalization of caveolin-1 and filamin was observed in elongated patches at the plasma membrane. Remarkably, when stress fiber formation was induced with Rho-stimulating Escherichia coli cytotoxic necrotizing factor 1, the caveolin-1–positive structures became coaligned with stress fibers, indicating that there was a physical link connecting them. Immunogold double-labeling electron microscopy confirmed that caveolin-1–labeled racemose caveolae clusters were positive for filamin. The actin network, therefore, seems to be directly involved in the spatial organization of caveolin-1–associated membrane domains.

scholarly journals An Emerging Role of PRRT2 in Regulating Growth Cone Morphology

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2666
Author(s):  
Elisa Savino ◽  
Fabrizia Guarnieri ◽  
Jin-Wu Tsai ◽  
Anna Corradi ◽  
Fabio Benfenati ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Growth Cone ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Neurological Diseases ◽  
Growth Cones ◽  
Cone Morphology ◽  
Infantile Seizures ◽  
Cytoskeleton Dynamics

Mutations in the PRRT2 gene are the main cause for a group of paroxysmal neurological diseases including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. In the mature central nervous system, the protein has both a functional and a structural role at the synapse. Indeed, PRRT2 participates in the regulation of neurotransmitter release, as well as of actin cytoskeleton dynamics during synaptogenesis. Here, we show a role of the protein also during early stages of neuronal development. We found that PRRT2 accumulates at the growth cone in cultured hippocampal neurons. Overexpression of the protein causes an increase in the size and the morphological complexity of growth cones. In contrast, the growth cones of neurons derived from PRRT2 KO mice are smaller and less elaborated. Finally, we demonstrated that the aberrant shape of PRRT2 KO growth cones is associated with a selective alteration of the growth cone actin cytoskeleton. Our data support a key role of PRRT2 in the regulation of growth cone morphology during neuronal development.

scholarly journals The blood flow-klf6a-tagln2 axis drives vessel pruning in zebrafish by regulating endothelial cell rearrangement and actin cytoskeleton dynamics

PLoS Genetics ◽  
2021 ◽  
Vol 17 (7) ◽  
pp. e1009690
Author(s):  
Lin Wen ◽  
Tao Zhang ◽  
Jinxuan Wang ◽  
Xuepu Jin ◽  
Muhammad Abdul Rouf ◽  
...  
Keyword(s):  
Blood Flow ◽  
Endothelial Cell ◽  
Actin Cytoskeleton ◽  
Critical Role ◽  
Large Diameter ◽  
Zebrafish Embryos ◽  
Downstream Target ◽  
Cytoskeleton Remodeling ◽  
Transgenic Embryos ◽  
Cytoskeleton Dynamics

Recent studies have focused on capillary pruning in various organs and species. However, the way in which large-diameter vessels are pruned remains unclear. Here we show that pruning of the zebrafish caudal vein (CV) from ventral capillaries of the CV plexus in different transgenic embryos is driven by endothelial cell (EC) rearrangement, which involves EC nucleus migration, junction remodeling, and actin cytoskeleton remodeling. Further observation reveals a growing difference in blood flow velocity between the two vessels in CV pruning in zebrafish embryos. With this model, we identify the critical role of Kruppel-like factor 6a (klf6a) in CV pruning. Disruption of klf6a functioning impairs CV pruning in zebrafish. klf6a is required for EC nucleus migration, junction remodeling, and actin cytoskeleton dynamics in zebrafish embryos. Moreover, actin-related protein transgelin 2 (tagln2) is a direct downstream target of klf6a in CV pruning in zebrafish embryos. Together these results demonstrate that the klf6a-tagln2 axis regulates CV pruning by promoting EC rearrangement.

scholarly journals Exploring the Possible Role of Lysine Acetylation onEntamoeba histolyticaVirulence: A Focus on the Dynamics of the Actin Cytoskeleton

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
L. López-Contreras ◽  
V. I. Hernández-Ramírez ◽  
A. E. Lagunes-Guillén ◽  
Sarita Montaño ◽  
B. Chávez-Munguía ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Effective Means ◽  
Cell Movement ◽  
Lysine Acetylation ◽  
Formation Processes ◽  
Severe Impairment ◽  
Cytoskeleton Remodeling ◽  
Cytoskeleton Dynamics

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins ofEntamoeba histolyticahas been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics andin vivovirulence ofE. histolytica. Using western blot, immunoprecipitation, microscopy assays, andin silicoanalysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

scholarly journals Phospholipase D Activity Is Required for Actin Stress Fiber Formation in Fibroblasts

2001 ◽  
Vol 21 (12) ◽  
pp. 4055-4066 ◽  
Author(s):  
Yoonseok Kam ◽  
John H. Exton
Keyword(s):  
Actin Cytoskeleton ◽  
Phospholipase D ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Dominant Negative Mutant ◽  
Actin Stress Fiber ◽  
Wild Type ◽  
Stress Fiber Formation ◽  
Significant Difference

ABSTRACT Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ∼50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of α-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by α-actinin.

scholarly journals In Vivo Rho GTPase-Activating Protein Activity of Pseudomonas aeruginosa Cytotoxin ExoS

2002 ◽  
Vol 70 (1) ◽  
pp. 360-367 ◽  
Author(s):  
Rebecca Krall ◽  
Jianjun Sun ◽  
Kristin J. Pederson ◽  
Joseph T. Barbieri
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Actin Cytoskeleton ◽  
Cho Cells ◽  
Rho Gtpases ◽  
Fluorescent Protein ◽  
Rho Gtpase ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Gtpase Activating Protein

ABSTRACT ExoS is a bifunctional type III cytotoxin secreted by Pseudomonas aeruginosa, which comprises a C-terminal ADP ribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain. In vitro, ExoS is a Rho GAP for Rho, Rac, and Cdc42; however, the in vivo modulation of Rho GTPases has not been addressed. Using a transient transfection system and delivery by P. aeruginosa, interactions were examined between the Rho GAP domain of ExoS and Rho GTPases in CHO cells. Rho GTPases were expressed as green fluorescent protein (GFP) fusion proteins to facilitate quantitation. GFP fusions of wild-type and dominant active Rho, Rac, and Cdc42 localized to discrete regions of CHO cells and appeared functional based upon their modulation of the actin cytoskeleton. Coexpression of the Rho GAP domain of ExoS changed the intracellular distribution of GFP-Rac and GFP-Cdc42 from a predominately membrane location to a cytosolic location. Coexpression of the Rho GAP domain of ExoS did not change the distribution of GFP-Rho, which was primarily in the cytosol. Coexpression of dominant active Rac (DARac) and DACdc42 inhibited actin reorganization by the Rho GAP domain but did not maintain the formation of actin stress fibers, which indicated that Rho had been inactivated. Similar results were observed when ExoS was delivered into CHO cells by P. aeruginosa. These data indicate that in vivo the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibition of Rho.

Sign in / Sign up

Export Citation Format

Share Document