epidermal-growth-factor and 5-iodoacetamidofluorescein

The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. These observations identify the carboxyl-terminal selenocysteine of TR1 as a cellular redox sensor and support an essential role for mammalian TR isozymes in redox-regulated cell signaling.

Topics: Amino Acid Sequence; Animals; Dinitrochlorobenzene; Epidermal Growth Factor; Fluoresceins; Gene Expression Regulation, Enzymologic; Humans; Hydrogen Peroxide; Isoenzymes; Liver; Male; Mass Spectrometry; Mice; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments; Peroxidases; Reactive Oxygen Species; Recombinant Proteins; RNA, Messenger; Selenocysteine; Signal Transduction; Testis; Thioredoxin-Disulfide Reductase; Tumor Cells, Cultured

Recent studies suggest that H2O2, at subtoxic concentrations generated in response to the activation of a variety of cell surface receptors, functions as an intracellular messenger. However, the intracellular targets of H2O2 action have not been identified. A procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H2O2 is now described. This approach is based on the labeling of proteinaceous cysteine with 5-iodoacetamidofluorescein at pH 5.5 and immunoblot analysis of the labeled proteins with antibodies specific to fluorescein. With this procedure, many proteins in human A431 cells were shown to contain reactive cysteines and to be readily oxidized by H2O2 generated in response to cellular stimulation with epidermal growth factor. One of these H2O2-sensitive proteins was identified as protein tyrosine phosphatase 1B.

Topics: Antibodies; Blotting, Western; Catecholamines; Cysteine; Epidermal Growth Factor; Fluoresceins; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Imidazolines; Molecular Weight; Oxidants; Oxidation-Reduction; Papain; Phosphorylation; Phosphotyrosine; Precipitin Tests; Protein Tyrosine Phosphatases; Proteins; Substrate Specificity; Tumor Cells, Cultured

Epidermal growth factor (EGF) stimulates cellular mitogenesis by binding to and activating its membrane-associated receptor. An important component of signal transduction by the activated receptor is the stimulation of an intrinsic tyrosyl residue-specific protein kinase, which selectively phosphorylates tyrosyl residues in the cytoplasmic tail of the receptor and in other cytoplasmic substrates. A recent study utilizing tyrsub, a new high affinity synthetic peptide substrate for the EGF receptor kinase, provided evidence that in peptide substrate binding, the tyrosyl residue plays the central role in recognition, with residues surrounding the tyrosyl residue contributing to stabilization of docking [Guyer et al. (1994) Arch. Biochem. Biophys. 312, 573-578]. A large body of previous work had identified acidic residues near the site of phosphorylation as most important for binding; therefore, other residues in tyrsub appeared to be promising sites for locating spectroscopic reporter groups. Since tyrsub has neutral residues -4 and +4 residues from the site of phosphorylation, we prepared two analogs of tyrsub, in each of which one of those residues was substituted with Cys. These cystyrsubs were found to be effectively phosphorylated by EGF receptor prepared from A431 cells, on stimulation with EGF, with high affinities [Km(app) = 40-50 microM.] Modification of the cystyrsubs with iodoacetamide had no deleterious effect on the ability of the peptide to be phosphorylated by the EGF receptor kinase, while the labeling by 5-iodoacetimidofluorescein completely abolished the productive interaction between the peptide and the EGF receptor. This unexpected failure of the fluorescently labeled peptides to be phosphorylated does, however, provide information on steric limitations to recognition of substrates by the EGF receptor kinase.

Topics: Binding Sites; Cysteine; Epidermal Growth Factor; ErbB Receptors; Fluoresceins; Fluorescent Dyes; Iodoacetamide; Kinetics; Mass Spectrometry; Molecular Weight; Oligopeptides; Phosphorylation; Protein Conformation; Signal Transduction; Substrate Specificity; Tyrosine