Interaction of epidermal growth factor-dependent protein kinase with endogenous membrane proteins and soluble peptide substrate

D. Cassel, L. J. Pike, G. A. Grant, E. G. Krebs, L. Glaser

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Abstract

In A431 membranes, epidermal growth factor (EGF) stimulates the phosphorylation of its membrane receptor as well as a synthetic peptide with a sequence related to that surrounding the phosphorylated tyrosine of pp60(src), the transforming protein encoded by Rous sarcoma virus. We have studied the relationship between these EGF-stimulated activities using both [γ- 32P]ATP and adenosine 5'-O-(3-[ 35S]thiotriphosphate) ([ 35S]ATPγS) as phosphate and thiophosphate donors, respectively. The phosphorylation and thiophosphorylation of the EGF receptor show a similar time course and maximal extent. In addition, the labeled receptor yields similar phosphorylated/thiophosphorylated tryptic peptides. By contrast, EGF-dependent phosphorylation of the synthetic peptide with [γ- 32P]ATP is 50-200 times faster than the thiophosphorylation with [ 35S]ATPγS. ATPγS acts as a competitive inhibitor of 32P incorporation from [γ- 32P]ATP into both the peptide and membrane proteins with similar concentration dependence. This suggests that while ATPγS can bind to the ATP site the interaction between ATPγS and the kinase is relatively nonproductive for the peptide thiophosphorylation reaction. The EGF-dependent phosphorylation of the peptide was not due to transfer of phosphate from the phosphorylated EGF receptor. Phosphorylation of the receptor seems unnecessary or insufficient for activation of the phosphorylation of the peptide since preincubation of membranes with ATPγS + EGF followed by removal of EGF does not increase peptide phosphorylation. The peptide inhibited the EGF-dependent phosphorylation of the EGF receptor. EGF-dependent phosphorylation of both endogenous membrane proteins and the peptide showed similar sensitivity to treatment with N-ethylmaleimide under conditions which preserve EGF binding to the receptor. These results suggest that the protein kinase functions of the EGF receptor represented by the phosphorylation of endogenous and exogenous substrates are closely related, and are likely to be mediated by the same receptor-associated kinase activity.

Original languageEnglish
Pages (from-to)2945-2950
Number of pages6
JournalJournal of Biological Chemistry
Volume258
Issue number5
StatePublished - 1983

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