Nitric oxide stimulates tyrosine phosphorylation in murine fibroblasts in the absence and presence of epidermal growth factor.
In the present study, utilizing anti-phosphotyrosine monoclonal antibodies, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) as sources of NO and murine fibroblasts expressing the human epidermal growth factor (EGF) receptor (HER14 cells), we showed that tyrosine phosphorylation of a set of proteins (126, 56 and 43 kDa) was stimulated when cells were incubated with either SNP or SNAP and abolished by Methylene Blue and oxyhaemoglobin. Inhibition by Methylene Blue suggested an involvement of cyclic GMP in the process, which was evidenced by the effects of 8-bromo cyclic GMP. This analogue of cyclic GMP stimulated tyrosine phosphorylation of the same set of proteins phosphorylated after incubation with the NO source. Tyrosine phosphorylation of the same set of proteins was stimulated when cells were incubated simultaneously with SNP and EGF, showing that NO also potentiates EGF-evoked tyrosine kinase activity in HER14 cells. However, stimulation of the autophosphorylation of the EGF receptor, above the levels obtained for EGF alone, was not observed under those conditions. Additionally, we investigated the effects of NO on EGF-receptor tyrosine phosphatase activities in HER14 cells. Increasing concentrations of NO correlate with a gradual inhibition of these activities in HER14 cells, either in intact cells or in cell lysates. Taken together, these observations suggest that NO modulates tyrosine phosphorylation in HER14 cells.
Peranovich
T M
TM
Fundação Hemocentro São Paulo, Brazil.
da Silva
A M
AM
Fries
D M
DM
Stern
A
A
Monteiro
H P
HP
D000818:Animals; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008751:Methylene Blue / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000494:pharmacology*; D009599:Nitroprusside; D010084:Oxidation-Reduction; D010108:Oxyhemoglobins / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives; D010750:Phosphoproteins / Q000096:biosynthesis*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis
D010108:Oxyhemoglobins; D010750:Phosphoproteins; D009599:Nitroprusside; D019000:Phosphotyrosine; D009569:Nitric Oxide; D014443:Tyrosine; D004815:Epidermal Growth Factor; D026423:S-Nitroso-N-Acetylpenicillamine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D010396:Penicillamine; D008751:Methylene Blue