Design of RapR-Shp2 and in vitro analysis of phosphatase activity. (A) Representation of RapR-Shp2. Insertion of iFKBP (blue) allosterically disrupts the phosphatase domain (peach). Activity is restored following rapamycin (orange) and FRB (purple) binding. Created using biorender.com. (B) Phosphatase domain of Shp2 (PDB accession no. 4DGP, beige) with indicated potential insertion sites (blue), WPD loop (magenta), and substrate binding loop containing KRNY sequence (orange). (C and D) Cell-free in vitro assay evaluating phosphatase activity through dephosphorylation of phospho-paxillin substrate. Activity analysis of immunoprecipitated constitutively active Shp2 (CA-Shp2), dominant negative Shp2 (DN-Shp2), and RapR-Shp2 constructs (iFKBP insertion sites Thr²⁸⁸, Lys³¹⁷, Val⁴⁰⁶) with and without rapamycin. Representative blots of N = 4 independent experiments shown with quantification of Paxillin (pY³¹) blot intensity (D). Error bars represent 90% confidence interval (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 insertion site with and without rapamycin. *P < 0.05, **P < 0.01.