scholarly journals Identification of Fer Tyrosine Kinase Localized on Microtubules as a Platelet Endothelial Cell Adhesion Molecule-1 Phosphorylating Kinase in Vascular Endothelial Cells

2003 ◽  
Vol 14 (9) ◽  
pp. 3553-3564 ◽  
Author(s):  
Naoko Kogata ◽  
Michitaka Masuda ◽  
Yuji Kamioka ◽  
Akiko Yamagishi ◽  
Akira Endo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Adhesion Molecule ◽  
Intracellular Signaling ◽  
Vascular Endothelial Cells ◽  
Expression Cloning ◽  
Vascular Endothelial ◽  
Cell Contacts ◽  
Cell Cell

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.

Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine

2001 ◽  
Vol 281 (1) ◽  
pp. H266-H274 ◽  
Author(s):  
Yoshiyuki Rikitake ◽  
Seinosuke Kawashima ◽  
Tomosaburo Takahashi ◽  
Tomomi Ueyama ◽  
Satoshi Ishido ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Phorbol Esters ◽  
Vascular Endothelial Cells ◽  
Biological Effects ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Vascular Endothelial ◽  
Acetoxymethyl Ester

Lysophosphatidylcholine (LPC), a component of oxidized low-density lipoprotein, exerts various biological effects on vascular endothelial cells. However, the intracellular signaling of LPC is poorly understood. In this study, we investigated the involvement of proline-rich tyrosine kinase (PYK2) in LPC signaling in cultured bovine aortic endothelial cells by immunoprecipitation and Western blotting assays. Treatment of cells with LPC promoted a rapid increase in tyrosine phosphorylation of PYK2. LPC-stimulated PYK2 phosphorylation was inhibited by calcium chelators, 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-acetoxymethyl ester, EGTA, protein kinase C (PKC) inhibitor, GF-109203X, or PKC depletion by phorbol esters. PYK2 phosphorylation was inhibited by treatment with cytochalasin D but with neither botulinum C3 transferase nor overexpression of a dominant negative mutant of Rho A. LPC stimulated the association of Shc with PYK2, Shc tyrosine phosphorylation, and Grb2 binding to Shc and induced Ras activation. These results provide evidence that 1) LPC tyrosine phosphorylates PYK2 by calcium- and PKC-dependent mechanisms, 2) the intact cytoskeleton is required for LPC-stimulated PYK2 phosphorylation, and 3) LPC-activated Ras via the PYK2/Shc/Grb2 signaling.

scholarly journals Junctional adhesion molecule-C regulates vascular endothelial permeability by modulating VE-cadherin–mediated cell–cell contacts

2006 ◽  
Vol 203 (12) ◽  
pp. 2703-2714 ◽  
Author(s):  
Valeria V. Orlova ◽  
Matina Economopoulou ◽  
Florea Lupu ◽  
Sentot Santoso ◽  
Triantafyllos Chavakis
Keyword(s):  
Endothelial Cells ◽  
Vascular Permeability ◽  
Adhesion Molecule ◽  
Endothelial Permeability ◽  
Fiber Formation ◽  
Vascular Endothelial ◽  
Cell Contacts ◽  
Microvascular Endothelial ◽  
Cell Cell

We recently reported that junctional adhesion molecule (JAM)-C plays a role in leukocyte transendothelial migration. Here, the role of JAM-C in vascular permeability was investigated in vitro and in vivo. As opposed to macrovascular endothelial cells that constitutively expressed JAM-C in cell–cell contacts, in quiescent microvascular endothelial cells, JAM-C localized mainly intracellularly, and was recruited to junctions upon short-term stimulation with vascular endothelial growth factor (VEGF) or histamine. Strikingly, disruption of JAM-C function decreased basal permeability and prevented the VEGF- and histamine-induced increases in human dermal microvascular endothelial cell permeability in vitro and skin permeability in mice. Permeability increases are essential in angiogenesis, and JAM-C blockade reduced hyperpermeability and neovascularization in hypoxia-induced retinal angiogenesis in mice. The underlying mechanisms of the JAM-C–mediated increase in endothelial permeability were studied. JAM-C was essential for the regulation of endothelial actomyosin, as revealed by decreased F-actin, reduced myosin light chain phosphorylation, and actin stress fiber formation due to JAM-C knockdown. Moreover, the loss of JAM-C expression resulted in stabilization of VE-cadherin–mediated interendothelial adhesion in a manner dependent on the small GTPase Rap1. Together, through modulation of endothelial contractility and VE-cadherin–mediated adhesion, JAM-C helps to regulate vascular permeability and pathologic angiogenesis.

Glycocalyx modulates the motility and proliferative response of vascular endothelium to fluid shear stress

2007 ◽  
Vol 293 (2) ◽  
pp. H1023-H1030 ◽  
Author(s):  
Yu Yao ◽  
Aleksandr Rabodzey ◽  
C. Forbes Dewey
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelium ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Surface Profile ◽  
Cell Junctions ◽  
Vascular Endothelial ◽  
Fluid Shear ◽  
New Perspective ◽  
Cell Cell

Flow-induced mechanotransduction in vascular endothelial cells has been studied over the years with a major focus on putative connections between disturbed flow and atherosclerosis. Recent studies have brought in a new perspective that the glycocalyx, a structure decorating the luminal surface of vascular endothelium, may play an important role in the mechanotransduction. This study reports that modifying the amount of the glycocalyx affects both short-term and long-term shear responses significantly. It is well established that after 24 h of laminar flow, endothelial cells align in the direction of flow and their proliferation is suppressed. We report here that by removing the glycocalyx by using the specific enzyme heparinase III, endothelial cells no longer align under flow after 24 h and they proliferate as if there were no flow present. In addition, confluent endothelial cells respond rapidly to flow by decreasing their migration speed by 40% and increasing the amount of vascular endothelial cadherin in the cell-cell junctions. These responses are not observed in the cells treated with heparinase III. Heparan sulfate proteoglycans (a major component of the glycocalyx) redistribute after 24 h of flow application from a uniform surface profile to a distinct peripheral pattern with most molecules detected above cell-cell junctions. We conclude that the presence of the glycocalyx is necessary for the endothelial cells to respond to fluid shear, and the glycocalyx itself is modulated by the flow. The redistribution of the glycocalyx also appears to serve as a cell-adaptive mechanism by reducing the shear gradients that the cell surface experiences.

The GPI-anchored adhesion molecule F3 induces tyrosine phosphorylation: involvement of the FNIII repeats

1996 ◽  
Vol 109 (3) ◽  
pp. 699-704 ◽  
Author(s):  
M. Cervello ◽  
V. Matranga ◽  
P. Durbec ◽  
G. Rougon ◽  
S. Gomez
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Intracellular Signaling ◽  
Protein Tyrosine Kinase ◽  
Cross Linking ◽  
Protein Tyrosine ◽  
Intracellular Signaling Pathway ◽  
Type Iii ◽  
Cell Cell

The glycosyl-phosphatidylinositol (GPI)-anchored F3 molecule, a member of the Ig superfamily made up of Ig and FNIII-like domains, is involved in cell-cell adhesion, neuronal pathfinding and fasciculation. Little is known about the mechanism(s) that governs the F3-mediated cell-cell recognition. In particular, it is not known whether F3 transduces signals across the membrane. Here we show that in F3-transfected CHO cells (1A cells) an increase in tyrosine phosphorylation occurs during F3-mediated aggregation. Moreover, under aggregation conditions F3 immunoprecipitated from 32P-metabolically labeled 1A cells associated with three major phosphorylated proteins. Interestingly, genistein inhibited the F3-mediated aggregation. Increased tyrosine phosphorylation was also observed using antibody-mediated F3-cross-linking. Furthermore, F3 expressed both in 1A cells and in post-natal mouse cerebellum forms non-covalent soluble complexes with protein tyrosine kinase(s). In cerebellum the F3-associated kinase was identified as fyn. By contrast, a truncated F3 protein, expressed in CHO cells, from which all the FN type III repeats have been deleted, does not associate with a kinase. Cross-linking of the F3-truncated form does not induce modulation of tyrosine phosphorylation. Taken together these data demonstrate that F3 is a molecule that transduces signals through both association with protein tyrosine kinase and modulation of protein tyrosine phosphorylation. The presence of FN type III domains is essential for the activation of the intracellular signaling pathway.

Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells

1997 ◽  
Vol 110 (17) ◽  
pp. 2065-2077 ◽  
Author(s):  
M.G. Lampugnani ◽  
M. Corada ◽  
P. Andriopoulou ◽  
S. Esser ◽  
W. Risau ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Adherens Junction ◽  
Cell Junctions ◽  
Molecular Organization ◽  
Beta Catenin ◽  
Cell Contacts ◽  
Cell Type Specific ◽  
Dynamic Structures ◽  
Cell Cell

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.

Sign in / Sign up

Export Citation Format

Share Document