A Role for P21-Activated Kinase in Endothelial Cell Migration

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and in-creased stress fibers and focal adhesions. The DN effect mapped to the most NH2-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged α-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.

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Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

10.1101/2021.04.01.438077 ◽

2021 ◽

Author(s):

Erik S Linklater ◽

Emily Duncan ◽

Ke Jun Han ◽

Algirdas Kaupinis ◽

Mindaugas Valius ◽

...

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Focal Adhesion ◽

Focal Adhesions ◽

Leading Edge ◽

Stress Fibers ◽

Actin Dynamics ◽

Migration And Invasion ◽

Matrix Remodeling ◽

Cell Migration And Invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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Hic-5 promotes endothelial cell migration to lysophosphatidic acid

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00728.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. H193-H203 ◽

Cited By ~ 23

Author(s):

C. Avraamides ◽

M. E. Bromberg ◽

J. P. Gaughan ◽

S. M. Thomas ◽

A. Y. Tsygankov ◽

...

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Focal Adhesion ◽

Lysophosphatidic Acid ◽

Fluorescent Protein ◽

Focal Adhesions ◽

Endothelial Cell Migration ◽

Erk Phosphorylation ◽

Green Fluorescent

Endothelial cell migration is critical for proper blood vessel development. Signals from growth factors and matrix proteins are integrated through focal adhesion proteins to alter cell migration. Hydrogen peroxide-inducible clone 5 (Hic-5), a paxillin family member, is enriched in the focal adhesions in bovine pulmonary artery endothelial (BPAE) cells, which migrate to lysophosphatidic acid (LPA) on denatured collagen. In this study, we investigate the role of Hic-5 in LPA-stimulated endothelial cell migration. LPA recruits Hic-5 to the focal adhesions and to the pseudopodia in BPAE cells plated on collagen, suggesting that recruitment of Hic-5 to focal adhesions is associated with endothelial cell migration. Knockdown of endogenous Hic-5 significantly decreases migration toward LPA, confirming involvement of Hic-5 in migration. To address the role of Hic-5 in endothelial cell migration, we exogenously expressed wild-type (WT) Hic-5 and green fluorescent protein Hic-5 C369A/C372A (LIM3 mutant) constructs in BPAE cells. WT Hic-5 expression increases chemotaxis of BPAE cells to LPA, whereas migration toward LPA of the green fluorescent protein Hic-5 C369A/C372A-expressing cells is similar to that shown in vector control cells. Additionally, ERK phosphorylation is enhanced in the presence of LPA in WT Hic-5 cells. A pharmacological inhibitor of MEK activity inhibits LPA-stimulated WT Hic-5 cell migration and ERK phosphorylation, suggesting Hic-5 enhances migration via MEK activation of ERK. Together, these studies indicate that Hic-5, a focal adhesion protein in endothelial cells, is recruited to the pseudopodia in the presence of LPA and enhances migration.

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Vascular Endothelial Growth Factor (VEGF)-Induced Endothelial Cell Migration Requires Urokinase Receptor (uPAR) for Integrin Redistribution.

Blood ◽

10.1182/blood.v104.11.846.846 ◽

2004 ◽

Vol 104 (11) ◽

pp. 846-846

Author(s):

Gerald W. Prager ◽

Johannes M. Breuss4 ◽

Patrick Brunner4 ◽

Bernd R. Binder4

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Focal Adhesions ◽

Specific Inhibitor ◽

Leading Edge ◽

Urokinase Receptor ◽

Endothelial Cell Migration ◽

Spatial Proximity

Abstract VEGF activates endothelial cells to migrate and invade surrounding tissues, an initial event in the angiogenic process. For invasion, the coordinated localized formation of a proteolytic repertoir is necessary. Focusing the urokinase receptor towards the leading edge of migrating cells provides such armor and inhibition of uPA binding to its receptor inhibits invasion of endothelial cells. In addition integrins continuously have to form focal contacts at the leading edge. Thus the spatial proximity between the localized proteases and the matrix seems to be essential for matrix degradation. In order to allow cell locomotion integrins have to release their ligands when they reach the trailing end and are subsequently endocytosed and redistributed to newly formed focal adhesions in a repetitive process. We here describe a new role of uPAR in regulating integrin redistribution. We have previously reported that stimulation of human endothelial cells by VEGF (50ng/ml) via its receptor flk-1 induces pro-uPA activation, when bound to uPAR. Subsequently a uPA/PAI-1/uPAR-complex is formed, which thereafter is endocytosed via a LDL-R family member. We now show that by this process beta-1 integrins are co-internalized in clathrin coated vesicles via a uPAR dependent mechanism. Subsequently, endocytosed uPAR recycles to focal adhesions where it co-localizes with integrin alpha-v/beta-3. Disrupting this chain of events, either by (1) RAP - a specific inhibitor of the LDL-R family - or by (2) uPAR depletion (using uPAR−/− cells or cleaving the GPI-anchor of uPAR by PI-PLC), beta-1 integrins are no longer internalized after VEGF stimulation. Under the same circumstances the migratory response of endothelial cells toward VEGF is impaired in vitro as shown by video-based migration assays and in vivo as demonstrated by matrigel angiogenesis assays. Next, we generated synthetic peptides interfering with uPAR/integrin interaction, which inhibit not only VEGF-induced integrin redistribution, but also diminish VEGF-induced endothelial cell migration, significantly. These data suggest that in VEGF-induced cell migration uPAR plays a central role not only in focusing proteolytic activity, but also in initial integrin redistribution. Interference with this process could be a therapeutic target for diseases depending on VEGF-induced angiogenesis.

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Rab40–Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration

The Journal of Cell Biology ◽

10.1083/jcb.202008060 ◽

2021 ◽

Vol 220 (7) ◽

Author(s):

Erik S. Linklater ◽

Emily D. Duncan ◽

Ke-Jun Han ◽

Algirdas Kaupinis ◽

Mindaugas Valius ◽

...

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Focal Adhesion ◽

Focal Adhesions ◽

Leading Edge ◽

Stress Fibers ◽

Actin Dynamics ◽

Migration And Invasion ◽

Matrix Remodeling ◽

Cell Migration And Invasion

Rab40b is a SOCS box–containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b–Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b–Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b–Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation

Molecular Biology of the Cell ◽

10.1091/mbc.e09-12-1061 ◽

2011 ◽

Vol 22 (15) ◽

pp. 2766-2776 ◽

Cited By ~ 111

Author(s):

Birger Herzog ◽

Caroline Pellet-Many ◽

Gary Britton ◽

Basil Hartzoulakis ◽

Ian C. Zachary

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Complex Formation ◽

Rational Design ◽

Cell Model ◽

Umbilical Vein ◽

Focal Adhesions ◽

Dominant Negative ◽

Endothelial Cell Migration

In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1.

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FAK Potentiates Rac1 Activation and Localization to Matrix Adhesion Sites: A Role for βPIX

Molecular Biology of the Cell ◽

10.1091/mbc.e06-03-0207 ◽

2007 ◽

Vol 18 (1) ◽

pp. 253-264 ◽

Cited By ~ 86

Author(s):

Fumin Chang ◽

Christopher A. Lemmon ◽

Dongeun Park ◽

Lewis H. Romer

Keyword(s):

Focal Adhesion ◽

Protein Tyrosine Kinase ◽

Cell Attachment ◽

Focal Adhesions ◽

Leading Edge ◽

Cell Spreading ◽

Subcellular Location ◽

Dominant Negative ◽

Membrane Ruffling ◽

Adhesion Sites

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosine-phosphorylated βPIX and thereby increased its binding to Rac1. In addition, βPIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of βPIX.

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ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The Journal of Cell Biology ◽

10.1083/jcb.200112107 ◽

2002 ◽

Vol 157 (5) ◽

pp. 819-830 ◽

Cited By ~ 146

Author(s):

Takahiro Tsuji ◽

Toshimasa Ishizaki ◽

Muneo Okamoto ◽

Chiharu Higashida ◽

Kazuhiro Kimura ◽

...

Keyword(s):

Focal Adhesions ◽

Small Gtpase ◽

Dominant Negative ◽

Stress Fibers ◽

Induced Membrane ◽

3T3 Fibroblasts ◽

C3 Exoenzyme ◽

Swiss 3T3 ◽

Swiss 3T3 Fibroblasts ◽

Membrane Ruffle

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632–induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632–induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.

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R-Ras Controls Membrane Protrusion and Cell Migration through the Spatial Regulation of Rac and Rho

Molecular Biology of the Cell ◽

10.1091/mbc.e04-04-0277 ◽

2005 ◽

Vol 16 (1) ◽

pp. 84-96 ◽

Cited By ~ 63

Author(s):

Michele A. Wozniak ◽

Lina Kwong ◽

David Chodniewicz ◽

Richard L. Klemke ◽

Patricia J. Keely

Keyword(s):

Cell Migration ◽

Molecular Mechanisms ◽

Leading Edge ◽

Pi3 Kinase ◽

Dominant Negative ◽

Cell Periphery ◽

Membrane Protrusion ◽

Spatial Regulation ◽

Entire Cell ◽

Migratory Cells

Although it is known that the spatial coordination of Rac and Rho activity is essential for cell migration, the molecular mechanisms regulating these GTPases during migration are unknown. We found that the expression of constitutively activated R-Ras (38V) blocked membrane protrusion and random migration. In contrast, expression of dominant negative R-Ras (41A) enhanced migrational persistence and membrane protrusion. Endogenous R-Ras is necessary for cell migration, as cells that were transfected with siRNA for R-Ras did not migrate. Expression of R-Ras (38V) decreased Rac activity and increased Rho activity around the entire cell periphery, whereas expression of dominant negative R-Ras (41A) showed the converse, suggesting that R-Ras can spatially activate Rho and inactivate Rac. Consistent with this role, endogenous R-Ras localized and was preferentially activated at the leading edge of migratory cells in response to adhesion. The effects of R-Ras on cell migration are mediated by PI3-Kinase, as an effector mutant that uncouples PI3-Kinase binding from R-Ras (38V) rescued migration. From these data, we hypothesize that R-Ras plays a key role in cell migration by locally regulating the switch from Rac to Rho activity after membrane protrusion and adhesion.

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Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

Experimental Biology and Medicine ◽

10.1177/153537020222700607 ◽

2002 ◽

Vol 227 (6) ◽

pp. 412-424 ◽

Cited By ~ 20

Author(s):

Imre L. Szabó ◽

Rama Pai ◽

Michael K. Jones ◽

George R. Ehring ◽

Hirofumi Kawanaka ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

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Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.6.c1669 ◽

2001 ◽

Vol 280 (6) ◽

pp. C1669-C1679 ◽

Cited By ~ 163

Author(s):

Kazuo Katoh ◽

Yumiko Kano ◽

Mutsuki Amano ◽

Kozo Kaibuchi ◽

Keigi Fujiwara

Keyword(s):

Kinase Inhibitors ◽

Fluorescent Protein ◽

Human Fibroblasts ◽

Rho Kinase ◽

Focal Adhesions ◽

Stress Fiber ◽

Stress Fibers ◽

Fiber System ◽

Rho Kinase Inhibitors ◽

Calmodulin Inhibitors

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal adhesions in the central portion of the cell within 1 h. However, stress fibers located in the periphery of the cell were not severely affected by the Rho-kinase inhibitors. When these cells were washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused disruption and/or shortening of peripheral stress fibers, leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indicating that the peripheral fibers play a major role in keeping the flattened state of the cell. When stress fiber models were reactivated, the peripheral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The central stress fiber system is dependent more on the activity of Rho-kinase than on that of MLCK, while the peripheral stress fiber system depends on MLCK.

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