S-Nitrosylation of the epidermal growth factor receptor: a regulatory mechanism of receptor tyrosine kinase activity.
Nitric oxide (NO) donors inhibit the epidermal growth factor (EGF)-dependent auto(trans)phosphorylation of the EGF receptor (EGFR) in several cell types in which NO exerts antiproliferative effects. We demonstrate in this report that NO inhibits, whereas NO synthase inhibition potentiates, the EGFR tyrosine kinase activity in NO-producing cells, indicating that physiological concentrations of NO were able to regulate the receptor activity. Depletion of intracellular glutathione enhanced the inhibitory effect of the NO donor 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) on EGFR tyrosine kinase activity, supporting the notion that such inhibition was a consequence of an S-nitrosylation reaction. Addition of DEA/NO to cell lysates resulted in the S-nitrosylation of a large number of proteins including the EGFR, as confirmed by the chemical detection of nitrosothiol groups in the immunoprecipitated receptor. We prepared a set of seven EGFR(C --> S) substitution mutants and demonstrated in transfected cells that the tyrosine kinase activity of the EGFR(C166S) mutant was completely resistant to NO, whereas the EGFR(C305S) mutant was partially resistant. In the presence of EGF, DEA/NO significantly inhibited Akt phosphorylation in cells transfected with wild-type EGFR, but not in those transfected with C166S or C305S mutants. We conclude that the EGFR can be posttranslationally regulated by reversible S-nitrosylation of C166 and C305 in living cells.
Murillo-Carretero
Maribel
M
Area de Fisiología, Facultad de Medicina, Universidad de Cádiz, Cádiz, Spain.
Torroglosa
Ana
A
Castro
Carmen
C
Villalobo
Antonio
A
Estrada
Carmen
C
D020134:Catalytic Domain / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025461:Feedback, Physiological; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009447:Neuroblastoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D019076:Transgenes
D000644:Quaternary Ammonium Compounds; C406802:diethylammonium-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; D009569:Nitric Oxide; D004815:Epidermal Growth Factor; D019001:Nitric Oxide Synthase; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D003545:Cysteine