NITRIC-OXIDE REVERSIBLY INHIBITS THE EPIDERMAL GROWTH-FACTOR RECEPTORTYROSINE KINASE

Although it has been demonstrated that NO inhibits the proliferation o f different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interac tion of NO with the epidermal growth factor receptor (EGFR), using tra nsfected fibroblasts which overexpress the human EGFR. The NO donors S -nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitroso hydrazine (DEA-NO) and droxy-2-nitrosohydrazino]butyl}propane-1,3-diam ine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the pr esence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-H-3]thymidine incorporation. Neither 8 -bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicke d this effect, suggesting that NO is unlikely to inhibit cell prolifer ation via a cCMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhi bited the transphosphorylation of the EGFR and its tyrosine kinase act ivity toward the exogenous substrate poly-L-(Glu-Tyr), as measured in permeabilized cells using [gamma-P-32]ATP as phosphate donor. In contr ast, 3-[morpholinosydnonimine hydrochloride] (SIN-I), a peroxynitrite- forming compound, did not significantly inhibit either DNA synthesis o r the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO o n the EGFR tyrosine kinase was prevented by haemoglobin, an NO scaveng er, but not by superoxide dismutase, and was reversed by dithiothreito l. The binding of EGF to its receptor was unaffected by DEA-NO. The in hibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot anal ysis using an anti-phosphotyrosine antibody. Taken together, these res ults suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the rec eptor.