Effects of sulfhydryl modification reagents on the kinase activity of the epidermal growth factor receptor.

ABSTRACT

Earlier reports have indicated that epidermal growth factor (EGF) receptor autophosphorylation, thought to be a key step in receptor transmembrane signaling, can be inactivated with the relatively sulfhydryl-specific reagent N-ethylmaleimide (NEM); however, no Cys residue has been implicated in the catalytic mechanism of the kinase. In an effort to address the mechanism of inhibition by NEM, we have investigated effects of several sulfhydryl-modifying reagents on EGF receptor autophosphorylation and on the kinase activity of the receptor toward an exogenous peptide substrate. Kinase activity is relatively insensitive to iodoacetic acid (IAAcid) and iodoacetamide (IAAmide), though IAAmide-treated receptor displays a higher Km(app) with respect to ATP, relative to untreated receptor. In contrast, even low concentrations of the very specific sulfhydryl reagent p-chloromercuribenzoic acid (PCMB) inactivate the receptor kinase. Pretreatment of the receptor with IAAmide, but not IAAcid, provides substantial protection of the kinase from subsequent treatment with NEM and a degree of protection from subsequent treatment with PCMB. Further, inactivation by NEM, and to a lesser extent PCMB, is inhibited by coincubation of the receptor with the hydrolysis-resistant ATP analog AMP-PNP. The protective effect of IAAmide from inactivation by NEM is also lost when AMP-PNP is present during the IAAmide treatment. Pretreatment of receptor with IAAcid has no effect on subsequent modification by IAAmide. These results, taken together, suggest that NEM, PCMB, and IAAmide, but not IAAcid, modify a Cys residue of the EGF receptor kinase that is inaccessible when nucleotide is bound. Modification of this residue by a bulky reagent (NEM, PCMB) inactivates the kinase by a steric mechanism, while modification with the smaller reagent (IAAmide) results in an active enzyme with altered affinity for ATP. Further, PCMB appears to react with an additional Cys residue (or residues), also resulting in steric inactivation.