Tyrphostins disrupt stress fibers and cellular attachments in endothelial monolayers.
Endothelial permeability, which plays a critical role in many physiologic and pathologic processes, depends on the integrity of intercellular and cell-substrate attachments and the actin cytoskeleton. The proteins located at the cytoplasmic face of adherens and focal contact junctions are rich in sites of tyrosine phosphorylation. To better understand the role of tyrosine phosphorylation in regulating endothelial cell shape, actin stress fibers, and cell junctions, we treated confluent calf pulmonary artery endothelial cells with 14 different tyrphostins, a class of specific tyrosine kinase inhibitors. Using immunofluorescence microscopy to assess cell shape, phosphotyrosine levels, actin stress fibers, and focal contact and junctional proteins, we found that the effects of the tyrphostins could be grouped into three categories. Four tyrphostins had no discernible effect on stress fibers or cell attachments. Seven tyrphostins produced cell retraction with concomitant disruption of both stress fibers and cell-substrate attachments. One member of this group, tyrphostin 25, showed greater specificity for cell-cell junctions than the others, causing cell separation without significantly affecting actin stress fibers or focal contacts. The third group of tyrphostins had the opposite effect, completely disrupting stress fibers and focal contacts without causing cell separation. The ability of specific tyrphostins to disrupt cell-cell or cell-substrate attachments and/or actin stress fibers implies that a certain steady-state level of tyrosine phosphorylation is necessary to maintain these structures and that there may be independent tyrosine kinase signaling pathways controlling them. Comparison of the phosphotyrosinated proteins affected by each group of tyrphostins should provide a useful new approach toward understanding the regulation of endothelial cell-cell and cell-substrate junctions.
Duke University School of Medicine, Durham, North Carolina, 27708, USA.
A Z
AZ
Farooki
D L
DL
Epstein
E T
ET
O'Brien
D000199:Actins / Q000187:drug effects*; D000818:Animals; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D007365:Intercellular Junctions / Q000187:drug effects; D008856:Microscopy, Fluorescence; D009570:Nitriles / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D013997:Time Factors
D000199:Actins; D003598:Cytoskeletal Proteins; D009570:Nitriles; D011505:Protein-Tyrosine Kinases