Fig. 1. High-affinity bindings sites for the N-terminal SH2 domain of SHP-1 in the C-terminal region of Ros receptor tyrosine kinase. (A) Synthetic phosphopeptides representing sequences around Ros pY2267 and pY2327, and Epo pY429 were analyzed for binding of the N-terminal SH2 domain of SHP-1 by surface plasmon resonance. Representative experiments are shown (left panel). The data were fitted to determine the kinetic constants k_ass and k_diss depicted in Table 1. Signals at equilibrium were also determined and plotted against concentrations (right panel). Dissociation constants determined by this procedure are also given in Table 1. (B) The same set of peptides was used for activation experiments. Recombinant SHP-1 was incubated with different concentrations of the phosphopeptides and activity was then determined with pNPP as a substrate. (C) Wild-type EGFR and an EGFR with the sequence around Ros pY2267 (LNY₂₂₆₇MVL) inserted in place of the EGFR pY1173 (AEY₁₁₇₃LR) were overexpressed in HEK293 cells and cell lysates were subjected to pulldown with a GST-fusion protein of the SHP-1 N-terminal SH2 domain. Affinity-precipitated receptor is visualized by immunoblotting with anti-EGFR antibodies. (D) Phosphopeptides representing the sequence around pY2267 or activation loop phosphotyrosines pY2103, pY2107 and pY2108 were subjected to a phosphatase reaction with the catalytic domain of SHP-1. Phosphate release was determined with Malachit Green. Note that the more efficient dephosphorylation of the activation loop phosphopeptide is also obvious at matched concentrations of phosphotyrosine (100 µM activation loop phosphopeptide versus 300 µM pY2267 peptide).