Epidermal growth factor-receptor-protein kinase interactions.
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-32]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating specific endogenous membrane proteins, as well as exogenously added histone. The binding of EGF to membranes in vitro resulted in a several-fold stimulation of the phosphorylation reaction. The phosphorylation reaction was not dependent on cyclic AMP or cyclic GMP. 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. The activation of the membrane-associated protein kinase by EGF appears to be a reversible phenomenon. The membrane preparation could be solubilized by a number of non-ionic detergents with the retention of both 125I-labeled EGF binding activity and EGF-enhanced phosphorylation of specific membrane proteins. The solubilized membrane preparation was purified by affinity chromatography. The purified preparation retained both EGF-binding activity and EGF-enhanced phosphorylation activity. Analysis of the affinity-purified preparation by SDS gel electrophoresis indicated 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. The EGF-stimulated phosphorylation reaction appears to be specific for tyrosine residues and in this regard resembles the src protein kinase.
D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*
D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D066246:ErbB Receptors