Potentiation of epidermal growth factor receptor protein-tyrosine kinase activity by sulfate.
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.
Hubler
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L
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Kher
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Bertics
P J
PJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000838:Anions / Q000494:pharmacology*; D002712:Chlorides / Q000494:pharmacology; D066246:ErbB Receptors / Q000187:drug effects* / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D008954:Models, Biological; D010710:Phosphates / Q000494:pharmacology; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000187:drug effects*; D013379:Substrate Specificity; D013431:Sulfates / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D010710:Phosphates; D013431:Sulfates; D000804:Angiotensin II; D014638:Vanadates; D011092:Polyethylene Glycols; D008345:Manganese; D000244:Adenosine Diphosphate; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D005459:Fluorides