Proline-rich Tyrosine Kinase 2 (Pyk2) Mediates Vascular Endothelial-Cadherin-based Cell-Cell Adhesion by Regulating β-Catenin Tyrosine Phosphorylation

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.

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A Pyk2–Vav1 complex is recruited to β3-adhesion sites to initiate Rho activation

Biochemical Journal ◽

10.1042/bj20090037 ◽

2009 ◽

Vol 420 (1) ◽

pp. 49-56 ◽

Cited By ~ 13

Author(s):

Chunlei Gao ◽

Scott D. Blystone

Keyword(s):

Cell Adhesion ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Fluorescent Protein ◽

Rho Gtpase ◽

Adaptor Protein ◽

Direct Interaction ◽

Nucleotide Exchange ◽

Tyrosine Kinase 2

Integrin αvβ3-mediated adhesion of haemopoietic cells to vitronectin results in β3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with αvβ3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with αvβ3. The association of Pyk2 and Vav1 with β3 relies on the presence of Tyr747 in β3, the primary site of β3 phosphorylation. However, association of Pyk2 with Vav1 is independent of β3 tyrosine phosphorylation. Formation of a Pyk2–Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)–Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and β3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of β3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.

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Vascular Endothelial Cadherin (VE-Cadherin): Cloning and Role in Endothelial Cell-Cell Adhesion

Microcirculation ◽

10.3109/10739689709146790 ◽

1997 ◽

Vol 4 (2) ◽

pp. 267-277 ◽

Cited By ~ 46

Author(s):

Jahanara Ali ◽

Fang Liao ◽

Eric Martens ◽

William A. Muller

Keyword(s):

Cell Adhesion ◽

Endothelial Cell ◽

Vascular Endothelial ◽

Vascular Endothelial Cadherin ◽

Cell Cell

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Vascular Endothelial Cadherin-mediated Cell-cell Adhesion Regulated by a Small GTPase, Rap1

BMB Reports ◽

10.5483/bmbrep.2006.39.2.132 ◽

2006 ◽

Vol 39 (2) ◽

pp. 132-139 ◽

Cited By ~ 21

Author(s):

Shigetomo Fukuhra ◽

Atsuko Sakurai ◽

Akiko Yamagishi ◽

Keisuke Sako ◽

Naoki Mochizuki

Keyword(s):

Cell Adhesion ◽

Small Gtpase ◽

Vascular Endothelial ◽

Vascular Endothelial Cadherin ◽

Cell Cell

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MAGI-1 Is Required for Rap1 Activation upon Cell-Cell Contact and for Enhancement of Vascular Endothelial Cadherin-mediated Cell Adhesion

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0647 ◽

2006 ◽

Vol 17 (2) ◽

pp. 966-976 ◽

Cited By ~ 108

Author(s):

Atsuko Sakurai ◽

Shigetomo Fukuhara ◽

Akiko Yamagishi ◽

Keisuke Sako ◽

Yuji Kamioka ◽

...

Keyword(s):

Cell Adhesion ◽

Adherens Junction ◽

Small Gtpase ◽

Cell Contact ◽

Vascular Endothelial ◽

Nucleotide Exchange ◽

Cell Contacts ◽

Vascular Endothelial Cadherin ◽

Rap1 Activation ◽

Cell Cell

Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherin-dependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with β-catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca2+ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca2+ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion.

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Fyn tyrosine kinase is a downstream mediator of Rho/PRK2 function in keratinocyte cell–cell adhesion

The Journal of Cell Biology ◽

10.1083/jcb.200105140 ◽

2002 ◽

Vol 156 (1) ◽

pp. 137-148 ◽

Cited By ~ 118

Author(s):

Enzo Calautti ◽

Maddalena Grossi ◽

Cristina Mammucari ◽

Yumi Aoyama ◽

Maria Pirro ◽

...

Keyword(s):

Cell Adhesion ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Kinase Activation ◽

Positive Effects ◽

Fyn Kinase ◽

Keratinocyte Cell ◽

Downstream Mediator ◽

Cell Cell ◽

Fyn Tyrosine Kinase

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell–cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of β- and γ-catenin, which is associated with the establishment of keratinocyte cell–cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell–cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of β- and γ-catenin and p120ctn. The positive effects of activated Rho on cell–cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell–cell adhesion and induces tyrosine phosphorylation of β- and γ-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell–cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.

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Stealthy Nanoparticles Protecting Endothelium Barrier from Leakiness by Resisting the Absorption of VE-cadherin

Nanoscale ◽

10.1039/d1nr03155d ◽

2021 ◽

Author(s):

Yuan Huang ◽

Suxiao Wang ◽

Jin-Zhi Zhang ◽

Hang-Xing Wang ◽

Qichao Zou ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Interstitial Space ◽

Cell Interactions ◽

Vascular Endothelial ◽

Vascular Endothelial Cadherin ◽

Cell Cell

Nanomaterial induced endothelial cells leakiness (NanoEL) is caused because nanomaterials enter the interstitial space of endothelial cells and disrupt the endothelial cell-cell interactions by interacting with vascular endothelial cadherin (VE-cad)....

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sFLT-1 inhibits proliferation, migration, and invasion of colorectal cancer SW480 cells through vascular mimicry formation suppression

Tumor Biology ◽

10.1177/1010428317698339 ◽

2017 ◽

Vol 39 (5) ◽

pp. 101042831769833 ◽

Cited By ~ 3

Author(s):

Wang Jinjun ◽

Wang Zhaowei ◽

Li Qiang ◽

Xue Zhijun ◽

Zhang Juanzi ◽

...

Keyword(s):

Colorectal Cancer ◽

Tyrosine Kinase ◽

Three Dimensional ◽

New Drugs ◽

Migration And Invasion ◽

Vascular Endothelial ◽

Three Dimensional Model ◽

Vascular Endothelial Cadherin ◽

Sw480 Cells ◽

Vascular Mimicry

To investigate the effects of soluble fms-like tyrosine kinase-1 on the vascular mimicry formation, proliferation, migration, and invasion of colorectal cancer SW480 cells. The recombinant plasmid pBLAST49-sFLT-1 or pBLAST49 control plasmid was transfected into SW480 cells to obtain hsFLT-1-SW480 or Ctrl-SW480 cells. The three-dimensional model culture, sulforhodamine B assay, scratch assay, and Transwell assay were performed to detect the vascular mimicry formation, proliferation, migration, and invasion of colorectal cancer SW480 cells, respectively. Western blotting was used to detect the expression of vascular endothelial–cadherin protein. Compared with Ctrl-SW480 cells, vascular mimicry formation ((0.85 ± 0.04) vs (7.40 ± 0.69), p < 0.05) and vascular endothelial–cadherin expression ((1.25 ± 0.08) vs (1.89 ± 0.03), p < 0.05) were significantly decreased, and the growth rate was also significantly decreased in hsFLT-1-SW480 cells ((32.54 ± 5.12) vs (88.13 ± 11.52), p < 0.05). Moreover, the migration ((0.46 ± 0.08) vs (0.94 ± 0.03), p < 0.05) and invasion capacity ((59.14 ± 3.64) vs (134.85 ± 10.16), p < 0.05) of SW480 cells were significantly inhibited upon soluble fms-like tyrosine kinase-1 transfection. soluble fms-like tyrosine kinase-1 inhibits cell proliferation, migration, and invasion of colorectal cancer SW480 cells through suppression of vascular mimicry formation, which provides a good basis for the development of new drugs for the treatment of colorectal cancer by targeting both angiogenesis and vascular mimicry formation.

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Vascular-endothelial-cadherin modulates endothelial monolayer permeability

Journal of Cell Science ◽

10.1242/jcs.112.12.1915 ◽

1999 ◽

Vol 112 (12) ◽

pp. 1915-1923 ◽

Cited By ~ 3

Author(s):

P.L. Hordijk ◽

E. Anthony ◽

F.P. Mul ◽

R. Rientsma ◽

L.C. Oomen ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Transendothelial Migration ◽

Stress Fibers ◽

Vascular Endothelial ◽

Monolayer Permeability ◽

Actin Stress Fibers ◽

Cell Cell

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than ‘jagged’, VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.

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