Cyclic changes in the organization of cell adhesions and the associated cytoskeleton, induced by stimulation of tyrosine phosphorylation in bovine aortic endothelial cells

Abstract

In this study we have investigated the relationships between the stimulation of tyrosine-specific protein phosphorylation and the state of assembly of cell-cell and cell-matrix adherens-type junctions. Bovine aortic endothelial (BAE) cells were treated with either the phosphotyrosine phosphatase inhibitor pervanadate or with epidermal growth factor (EGF), and the effect of the treatment on the organization of cell contacts and the actin cytoskeleton was evaluated by digital immunomicroscopy. We show here that pervanadate induced a dramatic (about 40-fold) increase in the level of phosphotyrosine labeling of cell-cell junctions, which reached maximal values following 20 minutes of incubation. Concomitantly, the junctional levels of vinculin, actin and plakoglobin increased, followed by a slower recruitment of cadherins to these sites. Upon longer incubation cell-cell junctions deteriorated and stress fibers and focal adhesions were formed. EGF stimulation of serum-starved BAE cells induced a rapid ‘wave’ of junctional tyrosine phosphorylation, followed by cyclic changes in the local levels of phosphotyrosine labeling. Periodic changes were also found in the intensity of labeling of junctional actin, vinculin and cadherins. These results suggest that tyrosine phosphorylation and the assembly of cell-cell adherens junctions are interdependent processes, and raise the possibility that the cross-talk between the two is responsible both for the regulation of junction formation and for adhesion-mediated signaling.