Implementation of Engineered Allosteric regulation in PTP1B and PTP-PEST. (A) Depiction of Shp2 (purple), PTP1B (green), and PTP-PEST (magenta) with the homologous insertion sites to Val⁴⁰⁶ in Shp2 highlighted in yellow (arrows). (right) Amino acid sequences of the regions adjacent to the insertion site in Shp2, PTP1B, and PTP-PEST (catalytically important WPD loop is bolded). (B–E) Cell-free in vitro assay evaluating phosphatase activity. Indicated PTP1B-flag and PTP-PEST-GFP constructs were coexpressed with mVenus-ipep-FRB (B and C) or mCherry-ipep-FRB (D and E) in HEK293 cells, activated with rapamycin, immunoprecipitated and incubated with phosphorylated paxillin for 30 min. (B and C) Analysis of activity for wild-type PTP1B-flag (WT PTP1B), dominant negative PTP1B (PTP1B[C215S]), and three linker variations (G, GPG, or GPGGSG) of RapR-PTP1B with iFKBP inserted at Thr¹⁶⁴. (C) Analysis of activity for wild-type PTP-PEST (PTP-PEST WT), three linker variations (G, GPG, or GPGGSG) of RapR-PTP-PEST-GFP with iFKBP inserted at Phe¹⁸², and nontransfected control immunoprecipitated using anti-GFP antibody. Representative blots of N = 3 independent experiments shown with quantification (C and E). Error bars show 90% CI. Analysis of statistical significance at the 0.05 level was determined by two-tailed Student’s t test for independent pairwise comparisons of each linker with and without rapamycin. *P < 0.05.