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Growth of a selected variant of A431 cells (clone 29) is stimulated by epidermal growth factor (EGF) in contrast to the growth inhibition caused by EGF in an unselected clone, A431(8). Twelve phosphoproteins from each clone were compared to determine whether unique EGF-dependent substrate phosphorylations might explain the cells' differing growth responses to EGF. Treatment of both clone 29 and A431(8) cells with EGF increased phosphorylation of the EGF receptor/kinase and six cellular proteins identified on 2-dimensional polyacrylamide gels. Four of these proteins (the EGF receptor/kinase and proteins of 36, 70, and 81 kd) contained phosphotyrosine in both clone 29 and A431(8) cells, indicating that the same modification of several proteins occurred in cells which have totally different growth responses to EGF. Two proteins were identified whose phosphorylation was EGF dependent and which were unique to clone 29 cells; however, EGF increased phosphorylation of only serine residues in these proteins. This indicates that these proteins are not primary targets of the EGF-dependent tyrosine-specific protein kinase, but rather are substrates for serine-specific kinase(s) activated as a consequence of EGF:receptor interaction. cAMP, which inhibited growth of both clones, was utilized to compare the effects of EGF when the growth response of both cell lines was similar. In the presence of cAMP, EGF increased A431(8) cellular phosphotyrosine content and the phosphorylation of the same phosphotyrosine-containing proteins of both clone 29 and A431(8) cells. The in vivo activity of a second tyrosine-specific protein kinase, p60V -src in B77 Rous sarcoma virus (RSV)-transformed newborn rat kidney (NRK) cells, was also unaffected by cAMP. Thus cAMP did not block the in vivo activity of two tyrosine-specific kinases or the tyrosine phosphorylation of three specific protein substrates. A threshold model of tyrosine kinase activity is proposed as an alternative explanation for the differing growth responses to EGF.

Human epidermoid carcinoma A431 cell clones have been obtained whose growth is inhibited, stimulated, or unaffected by epidermal growth factor (EGF). In clones exhibiting each type of growth response, EGF induced similar morphologic changes consisting of aggregation of cells into dense clusters with baring of large areas of the culture dish. The similarity of the clones' morphologic responses, despite their differing growth responses, indicates that the effects of EGF on morphology are distinct from effects on growth. Cells whose growth was inhibited by EGF contained high numbers of EGF receptors, whereas the concentration of EGF receptors was reduced in cells whose growth was stimulated or unaffected by EGF. There were, however, no consistent differences in EGF receptor concentrations between stimulated or null clones. Cells that exhibited each type of growth response displayed similar rates of EGF binding to receptors, rates of internalization of EGF, and rates and extent of EGF-induced receptor down-regulation. Changes in EGF-stimulated tyrosine-specific protein kinase activity paralleled changes in EGF receptors, both between clones and upon down-regulation. These studies indicate that a reduction in the concentration of EGF receptors in A431 cells allows escape from the growth inhibitory effects of EGF, but suggest that the pattern of growth response depends on biochemical events subsequent to EGF-receptor metabolism and activation of tyrosine-specific protein kinase.