Activation of the purified protein tyrosine kinase domain of the epidermal growth factor receptor.
Biological responses to epidermal growth factor (EGF) depend on the ligand-stimulated protein tyrosine kinase activity of its receptor. To further characterize the enzymatic activity of the EGF receptor, the baculovirus expression system was used to express the cytoplasmic protein tyrosine kinase domain of the EGF receptor. Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus correctly expressed an active tyrosine kinase domain of the EGF receptor as demonstrated by 35S metabolic labeling, immunoblotting with anti-EGF receptor and anti-phosphotyrosine antibodies, and autophosphorylation analysis. The kinase domain (Mr 66,000) was purified to near homogeneity using a monoclonal anti-phosphotyrosine antibody column, providing 0.5 mg of kinase domain/liter of Sf9 cells (23% yield). The purified kinase domain exhibited a strong preference for Mn2+ compared to Mg2+. The specific activity of the kinase domain was low compared to purified, EGF-activated EGF receptor. However, the addition of sphingosine or ammonium sulfate greatly increased the activity of the kinase domain to equal or exceed the activity of ligand-activated holo EGF receptor. These results indicate that the addition of sphingosine or ammonium sulfate to the purified kinase domain can mimic the effect of EGF to induce a conformation of the holo EGF receptor which is optimal for tyrosine kinase activity. Deletion of the ligand binding domain, analogous to that which occurs in erb B, is not sufficient to fully activate the kinase, implying that EGF causes conformational changes additional to removal of an inhibitory constraint.
Wedegaertner
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PB
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Gill
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GN
D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007313:Insecta / Q000235:genetics; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D011487:Protein Conformation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology; D014162:Transfection; D014780:Viruses / Q000235:genetics
D002413:Cations, Divalent; D008345:Manganese; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D013110:Sphingosine; D000645:Ammonium Sulfate