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Fig. 6
Binding of the CF-OP peptide to the whole SHP2 protein (wild type and pathogenic mutants) and activation of the phosphatase activity. (A) Relative catalytic activity of the wild-type protein and selected pathogenic mutants, under basal conditions (filled bars) and after stimulation with 10 μM BTAM peptide (empty bars) or 10 μM OP (dashed bars). All values were normalized to the basal activity of the wild-type protein. Each experiment was performed in triplicate. Binding to phosphorylated sequences induces activation. All mutations cause an increase in basal activity by destabilizing the autoinhibited conformation, and a concomitant enhanced responsiveness to activating phosphopeptides. (B) Curves for binding of the CF-OP peptide to the wild-type protein and selected mutants, obtained from fluorescence anisotropy experiments (1.0 nM CF-OP). Independent replicate experiments (n = 4 for E76K, n = 3 for E76V, F71L, D61G, and wild-type N-SH2, and n = 2 for the wild type, A72S, D61H) were fitted collectively. (C) Correlation between the relative basal activity of the various mutants (as reported in panel A) and their binding affinity (association constant, i.e., 1/Kd) for CF-OP. An enhanced basal activation of the protein, caused by destabilization of the autoinhibited state, is accompanied by an increased affinity for the CF-OP peptide. Therefore, CF-OP binds more tightly to the most activating mutants, which also cause the strongest pathogenic effects. Error bars represent standard deviations.