scholarly journals EVL is a novel focal adhesion protein involved in the regulation of cytoskeletal dynamics and vascular permeability

2021 ◽  
Vol 11 (4) ◽  
pp. 204589402110490
Author(s):  
Joseph B. Mascarenhas ◽  
Amir A. Gaber ◽  
Tania M. Larrinaga ◽  
Rachel Mayfield ◽  
Stefanie Novak ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Actin Cytoskeleton ◽  
Focal Adhesion Kinase ◽  
Vascular Permeability ◽  
Focal Adhesion ◽  
Regulatory Protein ◽  
Focal Adhesions ◽  
Adhesion Protein ◽  
Cytoskeletal Responses ◽  
Focal Adhesion Protein

Increases in lung vascular permeability is a cardinal feature of inflammatory disease and represents an imbalance in vascular contractile forces and barrier-restorative forces, with both forces highly dependent upon the actin cytoskeleton. The current study investigates the role of Ena-VASP-like (EVL), a member of the Ena-VASP family known to regulate the actin cytoskeleton, in regulating vascular permeability responses and lung endothelial cell barrier integrity. Utilizing changes in transendothelial electricial resistance (TEER) to measure endothelial cell barrier responses, we demonstrate that EVL expression regulates endothelial cell responses to both sphingosine-1-phospate (S1P), a vascular barrier-enhancing agonist, and to thrombin, a barrier-disrupting stimulus. Total internal reflection fluorescence demonstrates that EVL is present in endothelial cell focal adhesions and impacts focal adhesion size, distribution, and the number of focal adhesions generated in response to S1P and thrombin challenge, with the focal adhesion kinase (FAK) a key contributor in S1P-stimulated EVL-transduced endothelial cell but a limited role in thrombin-induced focal adhesion rearrangements. In summary, these data indicate that EVL is a focal adhesion protein intimately involved in regulation of cytoskeletal responses to endothelial cell barrier-altering stimuli. Keywords: cytoskeleton, vascular barrier, sphingosine-1-phosphate, thrombin, focal adhesion kinase (FAK), Ena-VASP like protein (EVL), cytoskeletal regulatory protein

scholarly journals RANTES induces tyrosine kinase activity of stably complexed p125FAK and ZAP-70 in human T cells.

1996 ◽  
Vol 184 (3) ◽  
pp. 873-882 ◽  
Author(s):  
K B Bacon ◽  
M C Szabo ◽  
H Yssel ◽  
J B Bolen ◽  
T J Schall
Keyword(s):  
T Cells ◽  
Tyrosine Kinase ◽  
Focal Adhesion ◽  
Cell Activation ◽  
Focal Adhesions ◽  
Adhesion Protein ◽  
Human T Cells ◽  
Multiple Protein ◽  
Transduction Mechanisms ◽  
Focal Adhesion Protein

The chemokine RANTES is a chemoattractant and activating factor for T lymphocytes. Investigation of the signal transduction mechanisms induced by RANTES in T cells revealed tyrosine phosphorylation of multiple protein species with prominent bands at 70-85 and 120-130 kD. Immunoprecipitation and Western analyses revealed that a protein of 125 kD was identical to the focal adhesion kinase (FAK) pp125FAK. RANTES stimulated phosphorylation of FAK as early as 30 seconds and immunoblots using antiphosphotyrosine monoclonal antibodies revealed that there was consistent phosphorylation of a 68-70 kD species in the pp125FAK immunoprecipitates. Immunoblotting and kinase assays showed this to be two separate proteins, the tyrosine kinase zeta-associated protein (ZAP) 70, and the focal adhesion protein paxillin. These results indicate a potentially important role for RANTES in the generation of T cell focal adhesions and subsequent cell activation via a molecular complex containing FAK, ZAP-70, and paxillin.

scholarly journals Paxillin-dependent Paxillin Kinase Linker and p21-Activated Kinase Localization to Focal Adhesions Involves a Multistep Activation Pathway

2002 ◽  
Vol 13 (5) ◽  
pp. 1550-1565 ◽  
Author(s):  
Michael C. Brown ◽  
Kip A. West ◽  
Christopher E. Turner
Keyword(s):  
Focal Adhesion ◽  
Focal Adhesions ◽  
Identical Result ◽  
Cytoskeletal Reorganization ◽  
Serine Threonine Kinase ◽  
Adhesion Protein ◽  
Spatial Regulation ◽  
P21 Activated Kinase ◽  
Focal Adhesion Protein ◽  
Activation Cascade

The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein paxillin kinase linker (PKL) and PIX/Cool suggested a mechanism. In this report, we demonstrate an essential role for a paxillin–PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulated the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1–329) or the autoinhibitory domain (AID 83–149) induced PKL, PIX, and PAK localization to focal adhesions, indicating a role for PAK scaffold activation. We show PIX, but not NCK, binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL, consistent with a PAK–PIX–PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino terminus, containing the PIX-binding site, but lacking paxillin-binding subdomain 2 (PBS2), was unable to localize to focal adhesions and also abrogated PAK localization. An identical result was obtained after PKLΔPBS2 expression. Finally, neither PAK nor PKL was capable of localizing to focal adhesions in cells overexpressing paxillinΔLD4, confirming a requirement for this motif in recruitment of the PAK–PIX–PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK, which through interaction with PIX provokes a functional PKL PBS2–paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is probably critical for the correct subcellular positioning of PAK, thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility.

scholarly journals The Focal Adhesion Analysis Server: a web tool for analyzing focal adhesion dynamics

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 68 ◽  
Author(s):  
Matthew E Berginski ◽  
Shawn M Gomez
Keyword(s):  
Computer Vision ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Dynamic Measurements ◽  
Web Tool ◽  
Web Based ◽  
Adhesion Protein ◽  
Adhesion Behavior ◽  
Focal Adhesion Protein ◽  
Over Time

The Focal Adhesion Analysis Server (FAAS) is a web-based implementation of a set of computer vision algorithms designed to quantify the behavior of focal adhesions in cells imaged in 2D cultures. The input consists of one or more images of a labeled focal adhesion protein. The outputs of the system include a range of static and dynamic measurements for the adhesions present in each image as well as how these properties change over time. The user is able to adjust several parameters important for proper focal adhesion identification. This system provides a straightforward tool for the global, unbiased assessment of focal adhesion behavior common in optical microscopy studies. The webserver is available at: http://faas.bme.unc.edu/.

scholarly journals Actopaxin, a New Focal Adhesion Protein That Binds Paxillin Ld Motifs and Actin and Regulates Cell Adhesion

2000 ◽  
Vol 151 (7) ◽  
pp. 1435-1448 ◽  
Author(s):  
Sotiris N. Nikolopoulos ◽  
Christopher E. Turner
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Substantial Reduction ◽  
Ectopic Expression ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Types ◽  
Focal Adhesion Protein

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor– and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M.C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851–863). In this study, we report the cloning and initial characterization of a new paxillin LD motif–binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell–cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.

β1 and β3 integrins disassemble from basal focal adhesions and β3 integrin is later localised to the apical plasma membrane of rat uterine luminal epithelial cells at the time of implantation

2011 ◽  
Vol 23 (3) ◽  
pp. 481 ◽  
Author(s):  
Yui Kaneko ◽  
Laura Lecce ◽  
Margot L. Day ◽  
Christopher R. Murphy
Keyword(s):  
Epithelial Cells ◽  
Focal Adhesion ◽  
Apical Membrane ◽  
Focal Adhesions ◽  
Endometrial Receptivity ◽  
Apical Plasma Membrane ◽  
Adhesion Protein ◽  
Β3 Integrin ◽  
Focal Adhesion Protein ◽  
Luminal Epithelial Cells

The present study investigated the expression of integrin subunits that are known to be associated with focal adhesions, namely β1 and β3 integrins in rat uterine luminal epithelial cells during early pregnancy. The β1 and β3 integrins were concentrated along the basal cell surface and were colocalised and structurally interacted with talin, a principal focal adhesion protein, on Day 1 of pregnancy. At the time of implantation, β1 and β3 integrins disassembled from the site of focal adhesions, facilitating the removal of uterine luminal epithelial cells for embryo invasion. Also at this time, β3 integrin markedly increased along the apical membrane, suggesting a role in embryo attachment. This distributional change in β1 and β3 integrins seen at the time of implantation was predominantly under the influence of progesterone. Taken together, β1 and β3 integrin disassembly from focal adhesions and the increase in β3 integrin apically are key components of hormonally regulated endometrial receptivity.

scholarly journals Paxillin: a new vinculin-binding protein present in focal adhesions.

1990 ◽  
Vol 111 (3) ◽  
pp. 1059-1068 ◽  
Author(s):  
C E Turner ◽  
J R Glenney ◽  
K Burridge
Keyword(s):  
Focal Adhesion ◽  
Specific Interaction ◽  
Focal Adhesions ◽  
Adhesion Protein ◽  
Rous Sarcoma ◽  
Sds Page ◽  
Cytoskeletal Component ◽  
Sarcoma Virus ◽  
Focal Adhesion Protein

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.

scholarly journals Hic-5 Communicates between Focal Adhesions and the Nucleus through Oxidant-Sensitive Nuclear Export Signal

2003 ◽  
Vol 14 (3) ◽  
pp. 1158-1171 ◽  
Author(s):  
Motoko Shibanuma ◽  
Joo-ri Kim-Kaneyama ◽  
Keiko Ishino ◽  
Nobuko Sakamoto ◽  
Tomoko Hishiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Focal Adhesion ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Adhesion Protein ◽  
Focal Adhesion Protein ◽  
Export Signal

hic-5 was originally isolated as an H2O2-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H2O2. Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H2O2 treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.

scholarly journals Staurosporine induces endothelial cell apoptosis via focal adhesion kinase dephosphorylation and focal adhesion disassembly independent of focal adhesion kinase proteolysis

2002 ◽  
Vol 367 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Jahangir KABIR ◽  
Melvin LOBO ◽  
Ian ZACHARY
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cell Apoptosis ◽  
Focal Adhesions ◽  
Cell Types ◽  
Protein Synthesis Inhibitor ◽  
Endothelial Cell Apoptosis ◽  
Synthesis Inhibitor

The survival of endothelial cells is dependent on interactions between the matrix and integrins mediated through focal adhesions. Focal adhesion kinase (FAK) is thought to play a key role in maintaining focal adhesion function and cell survival, whereas caspase-mediated FAK proteolysis is implicated in focal adhesion disassembly during apoptosis. We examined the relationship between changes in FAK phosphorylation and proteolysis during apoptosis of primary porcine aortic endothelial cells (PAEC) induced by staurosporine, a widely used apoptogenic agent in diverse cell types. Staurosporine-induced PAEC apoptosis was detected after 1h and was preceded by disruption and loss of FAK localization to focal adhesions within a few minutes, whereas staurosporine-induced cleavage of FAK occurred only after 8—24h. Staurosporine induced a very rapid dephosphorylation of FAK at Tyr861 and Tyr397 and caused dissociation of phosphorylated FAK from focal adhesions as early as 30s. The effect of staurosporine was very potent with striking inhibition of Tyr861 and Tyr397 phosphorylation and focal adhesion disruption occurring in the range 10—100nM. Selective inhibition of a known target of staurosporine, protein kinase C, using GF109203X, and of phosphoinositide 3′-kinase using wortmannin, did not reduce FAK tyrosine phosphorylation at Tyr861 and Tyr397, or cause disruption of focal adhesions. Cycloheximide, the protein synthesis inhibitor, induced PAEC apoptosis more slowly than staurosporine, but did not induce FAK dephosphorylation or rapid focal adhesion disruption, and instead caused a slower loss of focal adhesions and a marked increase in FAK proteolysis. These studies show that FAK dephosphorylation and focal adhesion disassembly are very early events mediating the onset of staurosporine-induced endothelial cell apoptosis and are dissociated from FAK proteolysis. Cycloheximide induces apoptosis through a pathway involving FAK proteolysis without dephosphorylation.

scholarly journals The focal adhesion protein Testin modulates KCNE2 potassium channel β subunit activity

Channels ◽  
2021 ◽  
Vol 15 (1) ◽  
pp. 229-238
Author(s):  
Maria Papanikolaou ◽  
Shawn M. Crump ◽  
Geoffrey W. Abbott
Keyword(s):  
Potassium Channel ◽  
Focal Adhesion ◽  
Β Subunit ◽  
Adhesion Protein ◽  
Focal Adhesion Protein
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