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Binding of EGF to its receptor induces dimerization of the normally monomeric receptor. Activation of its intracellular tyrosine kinase then occurs through the formation of an asymmetric kinase dimer in which one subunit, termed the “receiver” kinase, is activated by interaction with the other subunit, termed the “activator” kinase [Zhang, et al. (2006) Cell 125: 1137–1149]. Although there is significant experimental support for this model, the relationship between ligand binding and the mechanics of kinase activation are not known. Here we use luciferase fragment complementation in EGF receptor (EGFR)/ErbB2 heterodimers to probe the mechanics of ErbB kinase activation. Our data support a model in which ligand binding causes the cis -kinase (the EGFR) to adopt the receiver position in the asymmetric dimer and to be activated first. If the EGF receptor is kinase active, this results in the phosphorylation of the trans -kinase (ErbB2). However, if the EGF receptor kinase is kinase dead, the ErbB2 kinase is never activated. Thus, activation of the kinases in the EGFR/ErbB2 asymmetric dimer occurs in a specific sequence and depends on the kinase activity of the EGF receptor.

A431 cell membranes phosphorylate a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gly) in which residues 2--12 correspond to the sequence of the reported site of tyrosine phosphorylation in pp60src. Epidermal growth factor stimulates the phosphorylation of this peptide 2-fold over basal levels in a dose-dependent fashion. Phosphorylation is linear for approximately 3 min at 30 degrees C and occurs on the tyrosine residue. Kinetic analysis of the phosphorylation reaction indicates that epidermal growth factor increases the average Vmax from 3.8 to 7.5 nmol/min per mg and slightly decreases the average Km from 0.53 mM to 0.28 mM. A number of other peptides analogous to this tridecapeptide are also phosphorylated by A431 membranes. The data suggest that peptides with sequences similar to the site of tyrosine phosphorylation in pp60src are preferred substrates for the kinase in these membranes. Thus, the epidermal growth factor-stimulated protein kinase has the potential to interact with and phosphorylate pp60src. However, the A431 membranes also phosphorylate a tyrosine-containing peptide of totally unrelated sequence, suggesting that the kinase possesses a broad specificity for peptide phosphorylation that may not reflect its specificity with protein substrates.