Guyer C A, Woltjer R L, Coker K J, Staros J V
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.
Arch Biochem Biophys. 1994 Aug 1;312(2):573-8. doi: 10.1006/abbi.1994.1347.
The epidermal growth factor (EGF) receptor, like other protein tyrosine kinases, shows a preference for substrates having acidic residues in the vicinity of the tyrosyl residue that undergoes phosphorylation. We have developed a peptide substrate for the EGF receptor, termed tyrsub, which is based upon the highly acidic amino terminal sequence of human erythrocyte Band 3. Tyrsub possesses the lowest apparent Km(Km(app) = 32 microM) for phosphorylation by the EGF receptor of any peptide substrate reported to date. Using tyrsub, as well as analogs containing either Ser (sersub) or Phe (phesub) in place of Tyr, we investigated the relative importance of characteristics of the hydroxyaminoacyl residue in substrate recognition. Sersub was unable either to act as a substrate or serve as an effective inhibitor of tyrsub phosphorylation by the EGF receptor. Phesub was also unable to inhibit EGF-stimulable tyrsub phosphorylation, suggesting that the phenolic hydroxyl of the tyrosyl residue, rather than the aromatic ring, predominates in substrate recognition. These results indicate that for peptide substrates, at least, binding consists of two steps, recognition, in which the tyrosyl side chain plays the central role, and docking, in which residues surrounding the tyrosyl residue contribute to stabilizing binding interactions.
表皮生长因子（EGF）受体与其他蛋白酪氨酸激酶一样，对在发生磷酸化的酪氨酰残基附近含有酸性残基的底物表现出偏好。我们开发了一种用于EGF受体的肽底物，称为tyrsub，它基于人红细胞带3的高度酸性氨基末端序列。tyrsub是迄今为止报道的任何肽底物中，对EGF受体磷酸化具有最低表观Km（Km(app)=32 microM）的底物。使用tyrsub以及含有Ser（sersub）或Phe（phesub）取代Tyr的类似物，我们研究了羟基氨基酰基残基的特性在底物识别中的相对重要性。Sersub既不能作为底物，也不能作为EGF受体对tyrsub磷酸化的有效抑制剂。Phesub也不能抑制EGF刺激的tyrsub磷酸化，这表明酪氨酰残基的酚羟基而非芳香环在底物识别中起主要作用。这些结果表明，至少对于肽底物而言，结合包括两个步骤，即识别（其中酪氨酰侧链起核心作用）和对接（其中酪氨酰残基周围的残基有助于稳定结合相互作用）。
