Thrombin-mediated Focal Adhesion Plaque Reorganization in Endothelium: Role of Protein Phosphorylation

Kane L. Schaphorst, Fredrick Pavalko, Carolyn E. Patterson, Joe G N Garcia

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.

Original languageEnglish (US)
Pages (from-to)443-455
Number of pages13
JournalAmerican Journal of Respiratory Cell and Molecular Biology
Volume17
Issue number4
StatePublished - 1997
Externally publishedYes

Fingerprint

Phosphorylation
Focal Adhesions
Endothelial cells
Thrombin
Endothelium
Focal Adhesion Protein-Tyrosine Kinases
Adhesion
Endothelial Cells
Vinculin
Talin
Phosphotyrosine
Proteins
Myosin-Light-Chain Kinase
Ionomycin
thrombin receptor peptide SFLLRNP
Cell adhesion
Myosins
Immunoblotting
Cell Adhesion
Tyrosine

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Pulmonary and Respiratory Medicine

Cite this

Thrombin-mediated Focal Adhesion Plaque Reorganization in Endothelium : Role of Protein Phosphorylation. / Schaphorst, Kane L.; Pavalko, Fredrick; Patterson, Carolyn E.; Garcia, Joe G N.

In: American Journal of Respiratory Cell and Molecular Biology, Vol. 17, No. 4, 1997, p. 443-455.

Research output: Contribution to journalArticle

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