Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF

作者：Janice E. Buss, Jeffrey E. Kudlow, Cheri S. Lazar, Gordon N. Gill · 发表于：Proceedings of the National Academy of Sciences · 年份：1982 · DOI：10.1073/pnas.79.8.2574 · 被引用次数：95 · 研究领域：HER2/EGFR in Cancer Research、Monoclonal and Polyclonal Antibodies Research、Click Chemistry and Applications

To determine the role of epidermal growth factor (EGF)-stimulated protein kinase in the biological effects caused by EGF, tyrosine-specific kinase activity has been quantitated in A431 human epidermoid carcinoma cells and six variant cell lines. Because EGF inhibited proliferation of A431 cells, variants resistant to this inhibition were selected by treatment with mutagen and maintenance for 1 month in 0.1 muM EGF. After cloning and growth for 6-20 generations without EGF, the resistance of the variants to the growth-inhibitory effect of EGF was confirmed. Whereas EGF increased cellular phosphotyrosine content approximately 10-fold in parental A431 cells, EGF caused smaller or undetectable increases in the six variant cell lines. Solubilized membranes from the six variants displayed diminished EGF-stimulated phosphorylation of the EGF receptor and of antibodies to p60(src) (the product of the Rous sarcoma virus transforming gene), which act as an exogenous substrate. The decrease in EGF-stimulated tyrosine-specific protein kinase activity varied from approximately 40% (clone 16) to approximately 8% (clone 18) of parental A431 activity. Phosphorylated EGF receptors from parental and variant cells migrated identically on sodium dodecyl sulfate/polyacrylamide gels. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity, so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in the ...