Tong K, Guyer C A, Staros J V
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee, USA.
Int J Pept Protein Res. 1996 Mar;47(3):219-26. doi: 10.1111/j.1399-3011.1996.tb01348.x.
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.
表皮生长因子（EGF）通过与膜相关受体结合并激活该受体来刺激细胞有丝分裂。活化受体信号转导的一个重要组成部分是对内在的酪氨酸残基特异性蛋白激酶的刺激，该激酶选择性地使受体细胞质尾部和其他细胞质底物中的酪氨酸残基磷酸化。最近一项利用tyrsub（一种用于EGF受体激酶的新型高亲和力合成肽底物）的研究表明，在肽底物结合中，酪氨酸残基在识别中起核心作用，酪氨酸残基周围的残基有助于对接的稳定[Guyer等人（1994年）《生物化学与生物物理学报》312卷，573 - 578页]。此前大量的工作已确定磷酸化位点附近的酸性残基对结合最为重要；因此，tyrsub中的其他残基似乎是定位光谱报告基团的有前景的位点。由于tyrsub在磷酸化位点的 - 4和 + 4位有中性残基，我们制备了两种tyrsub类似物，每种类似物中这些残基之一被半胱氨酸取代。发现这些半胱氨酸tyrsub在受到EGF刺激时，能被从A431细胞制备的EGF受体高效磷酸化，亲和力高[表观米氏常数（Km(app)）= 40 - 50微摩尔]。用碘乙酰胺修饰半胱氨酸tyrsub对肽被EGF受体激酶磷酸化的能力没有有害影响，而用5 - 碘乙酰胺基荧光素标记则完全消除了肽与EGF受体之间的有效相互作用。然而，这种荧光标记肽不能被磷酸化的意外结果确实提供了关于EGF受体激酶识别底物的空间限制的信息。
