Lloyd E. King

Membranes may be prepared from A-431 human epidermoid carcinoma cells which have the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner and which, in the presence of EGF, catalyze the phosphorylation of a number of endogenous membrane proteins. The activation of the membrane associated protein kinase by EGF appears to be a reversible phenomenon. The membrane preparation may be solubilized by a number of nonionic detergents with the retention of both 125I-labeled EGF-binding activity and EGF-enhanced phosphorylation of specific membrane proteins. The solubilized membrane preparation may be purified by affinity chromatography using EGF covalently linked to Affi-Gel. The purified preparation retains both EGF-binding activity and EGF-enhanced phosphorylation activity. Analysis of the affinity-purified preparation by sodium dodecyl sulfate-gel electrophoresis indicates the presence of one major protein band of molecular weight 150,000 and several trace bands. The evidence suggests that the major 150,000 protein band is the receptor for EGF and is a substrate of the phosphorylation reaction. The co-purification of EGF-binding activity and EGF-stimulated phosphorylation activity suggests an inherent close relationship.

Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32P]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating endogenous membrane components, including membrane-associated proteins; the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. The binding of EGF to these membranes in vitro resulted in a severalfold stimulation of the phosphorylation reaction; again, the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. Membrane-associated dephosphorylation reactions did not appear to be affected by EGF. The phosphorylation reaction was not stimulated by cyclic AMP or cyclic GMP in the absence or presence of EGF. The phosphorylation system of the membrane was able to utilize [gamma-32P]GTP in both the basal and EGF-stimulated reactions. The enhanced membrane phosphorylation was specific for EGF and its derivatives; a wide variety of other peptide hormones were ineffective. The A-431 membrane preparation also was capable of phosphorylating exogenous proteins, such as histone, phosvitin, and ribonuclease, by a process which was stimulated by EGF. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system.