Hubler L, Leventhal P S, Bertics P J
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Biochem J. 1992 Jan 1;281 ( Pt 1)(Pt 1):107-14. doi: 10.1042/bj2810107.
Previous studies have shown that lysine- and arginine-rich proteins can enhance the activity of tyrosine and serine/threonine protein kinases. However, the kinetics and mechanism of this activation are not fully understood. Therefore we investigated the ability of poly(amino acids) and the arginine-rich protein, protamine, to alter the kinetic properties of epidermal growth factor (EGF) receptor protein-tyrosine kinase activity using immunoaffinity-purified receptor isolated from human epidermoid carcinoma (A431) cells. Poly(L-lysine), poly(L-arginine) and protamine stimulated EGF receptor kinase activity by 3-5-fold at non-saturating doses of ATP and peptide substrate, while poly(L-glutamate) had no effect. Initial kinetic studies demonstrated an increase in the maximum velocity and a decrease in the apparent Km for the peptide substrate angiotensin II in the presence of the basic effectors. Further analysis of the kinetic mechanism by product inhibition revealed that protamine altered the pattern of ADP inhibition towards the peptide substrate but not towards ATP. The change was indicative of the receptor's ability to form an enzyme-angiotensin II-ADP ternary complex in the presence of protamine but not in its absence. In addition, the basic effectors had a substantially decreased influence on the kinase activity of a C-terminally truncated form of the EGF receptor. Thus the changes in kinase activity may be partially mediated by the C-terminal region of the receptor, which contains the sites of receptor self-phosphorylation. These results suggest that the basic domains of proteins can interact with the EGF receptor to induce changes in its kinetic properties, especially with regard to reactant recognition and binding.
先前的研究表明，富含赖氨酸和精氨酸的蛋白质可以增强酪氨酸和丝氨酸/苏氨酸蛋白激酶的活性。然而，这种激活的动力学和机制尚未完全了解。因此，我们使用从人表皮样癌（A431）细胞中分离的免疫亲和纯化受体，研究了聚氨基酸和富含精氨酸的蛋白质鱼精蛋白改变表皮生长因子（EGF）受体蛋白酪氨酸激酶活性动力学特性的能力。在ATP和肽底物非饱和剂量下，聚-L-赖氨酸、聚-L-精氨酸和鱼精蛋白可使EGF受体激酶活性提高3至5倍，而聚-L-谷氨酸则无作用。初始动力学研究表明，在存在碱性效应物的情况下，肽底物血管紧张素II的最大速度增加，表观Km降低。通过产物抑制对动力学机制的进一步分析表明，鱼精蛋白改变了ADP对肽底物的抑制模式，但对ATP没有影响。这种变化表明受体在存在鱼精蛋白时能够形成酶-血管紧张素II-ADP三元复合物，而在不存在鱼精蛋白时则不能。此外，碱性效应物对EGF受体C末端截短形式的激酶活性影响大幅降低。因此，激酶活性的变化可能部分由受体的C末端区域介导，该区域包含受体自身磷酸化的位点。这些结果表明，蛋白质的碱性结构域可以与EGF受体相互作用，诱导其动力学特性发生变化，特别是在反应物识别和结合方面。
