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

• Autorzy: Al-Nedawi K. N. I. , Pawlowska Z. , Cierniewski C.S.
• Języki publikacji: EN

Abstrakty

EN

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [γ-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently 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 kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-β-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor α (TNFα) and interferon g (IFNγ) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFNγ bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFNγ it was associated with the appearance of a strongly poshosphorylated band of 17 kDa corresponding to IFNγ itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR - a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.

Słowa kluczowe

EN

plasma membrane; tumour necrosis factor-alpha; endothelial cell; 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole; interferon gamma; extracellular phosphorylation; protein kinase; membrane protein

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 1999
• Tom: 46
• Numer: 3
• Opis fizyczny: p.693-702,fig.

Twórcy

autor

Al-Nedawi K. N. I.

• Medical University, W.Lindleya 3, 90-131 Lodz, Poland

autor

Pawlowska Z.

autor

Cierniewski C.S.

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