Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endotheli al cells. Extracellular phosphorylation was detected by incubation of prima ry endothelial cells (HUVEC's) and endothelial cell Line EA.hy 926 with [g- P-32]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five protein s with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were promin ently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinas e C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic p rotein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) cas ein kinase II inhibitor 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole (DRB ). Stimulation of endothelial cells with tumor necrosis factor a (TNFa) and interferon g (IFNg) is associated with 20-80% reduction of extracellular p hosphorylation of all membrane proteins. IFNg bound to membrane receptors b ecomes rapidly phosphorylated. Only in the case of IFNg it was associated w ith the appearance of a strongly phosphorylated band of 17 kDa correspondin g to IFNg, itself. Phosphorylation of this 17 kDa exogenous substrate was p revented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphop rotein phosphatase activity in endothelial cells was evidenced by testing t he effect of microcystin LR - a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylat ion of 19 kDa and 110 kDa phosphoproteins significantly increased in the pr esence of microcystin. Our results suggest the presence of at least two ect o-kinase activities on endothelial cells that may play a significant role(s ) in the regulation of cytokines function.