Potentiation of epidermal growth factor receptor protein-tyrosine kinase activity by sulfate

Abstract

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 ((NH 4) 2SO 4), but the manner in which (NH 4) 2SO 4 induces this effect is unclear. Therefore, we have explored the processes by which (NH 4) 2SO 4 potentiated tyrosine kinase activity to better understand not only the molecular events involved in (NH 4) 2SO 4 activation, but also the kinetic properties and mechanism of the EGF receptor. In this study, the addition of an optimum concentration of (NH 4) 2SO 4 (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 SO 4 2− increased kinase activity similarly, whereas salts containing Cl − and F − had less of an effect, and divalent salts such as HPO 4 2− were inhibitory at doses of 1 mM or more. In addition, EGF receptor kinase activation by (NH 4) 2SO 4 did not strictly correlate with changes in the ionic strength or conductivity of the solution. However, several lines of evidence suggest that SO 4 2− directly alters the kinetic properties of the EGF receptor kinase: (1) the maximum velocity ( V max) and K m (ATP) for EGF receptor phosphorylation of angiotensin II were substantially higher in the presence of (NH 4) 2SO 4. (2) EGF receptor kinase activity in the absence of (NH 4) 2SO 4 required either Mn 2+ or Mg 2+, yet in the presence of (NH 4) 2SO 4, only Mn 2+ 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 (NH 4) 2SO 4 but not in its absence. (4) The near-maximal rate of self-phosphorylation was not affected by (NH 4) 2SO 4, but the apparent K m(ATP) was greatly increased. From these results, we propose a model for (NH 4) 2SO 4 stimulation of EGF receptor kinase activity in which SO 4 2− interacts directly with the receptor or receptor-Mn 2+-ATP complex and alters reactant binding and the catalytic efficiency of the tyrosine kinase.