Hubler L, Kher U, Bertics P J
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Biochim Biophys Acta. 1992 Feb 3;1133(3):307-15. doi: 10.1016/0167-4889(92)90052-d.
The protein-tyrosine kinase activity of the epidermal growth factor (EGF) receptor is critical for EGF-stimulated cell growth, although little is known about the molecular details of its enzymatic activity. Previous studies have found that EGF receptor kinase activity can be stimulated by factors such as ammonium sulfate ((NH4)2SO4), but the manner in which (NH4)2SO4 induces this effect is unclear. Therefore, we have explored the processes by which (NH4)2SO4 potentiated tyrosine kinase activity to better understand not only the molecular events involved in (NH4)2SO4 activation, but also the kinetic properties and mechanism of the EGF receptor. In this study, the addition of an optimum concentration of (NH4)2SO4 (250 mM) resulted in a 5-fold stimulation of kinase activity toward the peptide substrate, angiotensin II. The sulfate group is primarily involved in this action, since other salts containing SO4(2-) increased kinase activity similarly, whereas salts containing Cl- and F- had less of an effect, and divalent salts such as HPO4(2-) and NaVO4(2-) were inhibitory at doses of 1 mM or more. In addition, EGF receptor kinase activation by (NH4)2SO4 did not strictly correlate with changes in the ionic strength or conductivity of the solution. However, several lines of evidence suggest that SO4(2-) directly alters the kinetic properties of the EGF receptor kinase: (1) the maximum velocity (Vmax) and Km (ATP) for EGF receptor phosphorylation of angiotensin II were substantially higher in the presence of (NH4)2SO4. (2) EGF receptor kinase activity in the absence of (NH4)2SO4 required either Mn2+ or Mg2+, yet in the presence of (NH4)2SO4, only Mn2+ supported the increase in kinase activity. (3) Ammonium sulfate addition altered the product inhibition pattern of ADP versus angiotensin II, suggesting that an enzyme-angiotensin II-ADP complex can form in the presence of (NH4)2SO4 but not in its absence. (4) The near-maximal rate of self-phosphorylation was not affected by (NH4)2SO4 but the apparent Km (ATP) was greatly increased. From these results, we propose a model for (NH4)2SO4 stimulation of EGF receptor kinase activity in which SO4(2-) interacts directly with the receptor or receptor-Mn(2+)-ATP complex and alters reactant binding and the catalytic efficiency of the tyrosine kinase.
表皮生长因子（EGF）受体的蛋白酪氨酸激酶活性对于EGF刺激的细胞生长至关重要，尽管其酶活性的分子细节仍知之甚少。先前的研究发现，EGF受体激酶活性可被硫酸铵（(NH4)2SO4）等因子刺激，但(NH4)2SO4诱导这种效应的方式尚不清楚。因此，我们探究了(NH4)2SO4增强酪氨酸激酶活性的过程，以便不仅更好地理解(NH4)2SO4激活所涉及的分子事件，还能了解EGF受体的动力学特性和机制。在本研究中，添加最佳浓度的(NH4)2SO4（250 mM）导致对肽底物血管紧张素II的激酶活性增强了5倍。硫酸根主要参与此作用，因为其他含SO4(2-)的盐同样增加了激酶活性，而含Cl-和F-的盐影响较小，并且HPO4(2-)和NaVO4(2-)等二价盐在1 mM或更高剂量时具有抑制作用。此外，(NH4)2SO4对EGF受体激酶的激活与溶液的离子强度或电导率变化并不严格相关。然而，几条证据表明SO4(2-)直接改变了EGF受体激酶的动力学特性：（1）在存在(NH4)2SO4的情况下，血管紧张素II的EGF受体磷酸化的最大速度（Vmax）和Km（ATP）显著更高。（2）在不存在(NH4)2SO4的情况下，EGF受体激酶活性需要Mn2+或Mg2+，但在存在(NH4)2SO4的情况下，只有Mn2+能支持激酶活性的增加。（3）添加硫酸铵改变了ADP对血管紧张素II的产物抑制模式，表明在存在(NH4)2SO4的情况下可形成酶 - 血管紧张素II - ADP复合物，而在不存在(NH4)2SO4的情况下则不能。（4）自磷酸化的近最大速率不受(NH4)2SO4影响，但表观Km（ATP）大大增加。根据这些结果，我们提出了一个(NH4)2SO4刺激EGF受体激酶活性的模型，其中SO4(2-)直接与受体或受体 - Mn(2+) - ATP复合物相互作用，并改变反应物结合和酪氨酸激酶的催化效率。
