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首页> 外文期刊>Biochemistry >Effects of sulfhydryl modification reagents on the kinase activity of the epidermal growth factor receptor
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Effects of sulfhydryl modification reagents on the kinase activity of the epidermal growth factor receptor

机译：巯基修饰剂对表皮生长因子受体激酶活性的影响

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摘要

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.
机译：较早的报道表明，表皮生长因子（EGF）受体自磷酸化被认为是受体跨膜信号传导的关键步骤，可以用相对巯基特异性试剂N-乙基马来酰亚胺（NEM）灭活。但是，没有Cys残基参与该激酶的催化机制。为了解决NEM抑制的机制，我们研究了几种巯基修饰剂对EGF受体自身磷酸化和受体对外源肽底物的激酶活性的影响。激酶活性对碘乙酸（IAAcid）和碘乙酰胺（IAAmide）相对不敏感，尽管相对于未经处理的受体，IAAmide处理的受体相对于ATP显示出更高的Km（app）。相反，即使是低浓度的非常特异的巯基试剂对氯mercuribenzoic酸（PCMB）也会使受体激酶失活。用IAAmide（而不是IAAcid）对受体进行预处理，可为激酶提供保护，使其免受NEM的后续处理，并提供一定程度的保护，使其免受PCMB的后续处理。此外，通过将受体与抗水解的ATP类似物AMP-PNP共同温育，可以抑制NEM的失活，以及程度较小的PCMB。当IAAmide治疗期间存在AMP-PNP时，NEA灭活对IAAmide的保护作用也会消失。用IAAcid预处理受体对随后的IAAmide修饰没有影响。这些结果加在一起表明，NEM，PCMB和IAAmide（而不是IAAcid）修饰了EGF受体激酶的Cys残基，该残基在结合核苷酸时不可访问。用大体积试剂（NEM，PCMB）修饰此残基会通过空间机制使激酶失活，而使用较小试剂（IAAmide）修饰会导致活性酶对ATP的亲和力发生变化。此外，PCMB似乎与另外的Cys残基（或多个残基）反应，也导致空间失活。

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