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

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Abstract
To determine the role of epidermal growth factor (EGF)-stimulated protein kinase in the biological effects caused by EGF, tyrosine-specific kinase activity was quantitated in A431 human epidermoid carcinoma cells and 6 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 mo. in 0.1 .mu.M 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 .apprx. 10-fold in parental A431 cells, EGF caused smaller or undetectable increases in the 6 variant cell lines. Solubilized membranes from the 6 variants displayed diminished EGF-stimulated phosphorylation of the EGF receptor and of antibodies to p60src (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 .apprx. 40% (clone 16) to .apprx. 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 6 variant cell lines with reductions in both activities to as low as 10%. This tyrosine-specific protein kinase activity mediates the growth-inhibitory effect of EGF on A431 cells and both EGF binding and kinase activities reside in the same or tightly associated molecules.