Figure 6. T42A-dependent changes in the activation of full-length SHP2.

(A) SHP2 activation is measured by incubation with phosphopeptide ligands, followed by monitoring dephosphorylation of the small-molecule substrate DiFMUP to generate fluorescent DiFMU. (B) Representative activation curves for SHP2^WT. N = 3–17 independent titrations of protein, peptide, and DiFMUP. (C) Correlation between the EC₅₀ of SHP2^WT activation by phosphopeptides and the K_D of those phosphopeptides for the N-SH2^WT domain. For EC₅₀ values in (B)-(C), N = 3 independent titrations of protein, peptide, and DiFMUP.(D) Correlation between activation EC₅₀ values for SHP2^WT and SHP2^R138Q. For SHP2^R138Q EC₅₀ values, N = 3–5 independent titrations of protein, peptide, and DiFMUP. (E) Comparison of SHP2^WT and SHP2^T42A activation curves for the PD-1 pTyr248 peptide. N = 3–4 independent titrations of protein, peptide, and DiFMUP. (F) Comparison of SHP2^WT and SHP2^T42A activation curves for the Imhof-9 peptide. N = 6–17 independent titrations of protein, peptide, and DiFMUP. (G) Bubble plot juxtaposing the EC₅₀ values for activation of SHP2^WT and SHP2^T42A by nine peptides, alongside the fold-change in K_D for binding of those peptides to N-SH2^WT vs N-SH2^T42A. The dotted line indicates where EC₅₀ for SHP2^WT equals EC₅₀ for SHP2^T42A. Peptides with a large fold-change in binding affinity (larger bubble) have a large fold-change in EC₅₀ values for SHP2^T42A over SHP2^WT (distance from dotted line). All EC₅₀ values can be found in Table S5.