EGFR kinase possesses a broad specificity for ErbB phosphorylation sites, and ligand increases catalytic-centre activity without affecting substrate binding affinity

We previously found that EGF (epidermal growth factor) increases the EGFR (EGF receptor) kinase-binding affinity towards the major tyrosine phosphorylation sites in downstream adaptor proteins such as Gab1 (Grb2-associated binding protein 1) and Shc [Src homology 2 (SH2) domain and collagen containing protein], but not that towards EGFR autophosphorylation sites [Fan, Wong, Deb and Johnson (2004) J. Biol. Chem. 279, 38143–38150]. EGFR activation can also result in transphosphorylation of tyrosine resides in the C-terminal region of the related receptors ErbB2, ErbB3 and ErbB4 in heterodimers which are formed upon ligand stimulation. In the present study, we investigated the specificity of EGFR kinase by comparing the steady state kinetic parameters for peptides derived from all four ErbBs in the absence or presence of EGF. Our results demonstrated that (i) EGFR kinase can efficiently phosphorylate a broad range of diverse peptide sequences representing ErbB sites; (ii) certain ErbB2, ErbB3 and ErbB4 sites had higher specificity constants than any EGFR sequence and (iii) EGF stimulation consistently increases the k_cat approx. 5-fold, but does not significantly alter the K_m for any ErbB peptides. Furthermore, peptides containing lysine at position −2 or −3 N-terminal to the target tyrosine were found to be poor EGFR kinase substrates, and substitution of these lysines with glutamine decreased the K_m and increased the k_cat for these substrates. We conclude that EGFR kinase-mediated ErbB transphosphorylations are mostly controlled at the level of oligomerization, and not by a preference of the EGFR kinase for phosphorylation sites in any particular ErbB. The results also demonstrated that, unlike phosphorylation sites in select downstream targets, EGF does not regulate the recognition of phosphorylation sites in the C-terminal region of any of the ErbBs.