SHP1 has a higher phosphatase activity than SHP2. (A) LUV FRET assays showing PTPase activity of SHP1^PTPase or SHP2^PTPase. Left: Cartoon depicting a FRET assay for measuring the recruitment of SC505*ZAP70^tSH2 to LUV-attached Lck-phosphorylated CD3ζ^INT in the presence of LUV-attached SHP1^PTPase or SHP2^PTPase (see Materials and methods). Right: Time courses of ATP-triggered quenching of SC505*ZAP70^tSH2 at increasing levels of SHP1^PTPase or SHP2^PTPase. (B) Relative CD3ζ phosphorylation, calculated by normalizing the extent of SC505*ZAP70^tSH2 quenching at 20 min in A to 0 nM SHP1^PTPase or SHP2^PTPase conditions, were plotted against [SHP1^PTPase] or [SHP2^PTPase]. Data were fit with “dose response—inhibition” to yield IC₅₀ values of SHP1^PTPase and SHP2^PTPase. (C and D) Same as A and B, except replacing CD3ζ^INT and SC505*ZAP70^tSH2 with equal concentrations of CD28^INT and SC505*P50. (E) Left: Cartoon depicting a FRET assay for probing the activities of SHP1^PTPase or SHP2^PTPase (50 nM) toward CD3ζ^INT. Experiments were set up as in A, except with an apyrase addition step. Right: Representative time courses of SC505 fluorescence intensity (FI) before and after addition of 1 mM ATP and 0.5 U apyrase. (F) FRET assays showing how membrane-attached BTLA^INT stimulated the ability of SHP1/2 to inhibit CD3ζ^INT phosphorylation. Cartoon on the left depicts the assay setup: Lck, CD3ζ^INT, and increasing BTLA^INT were attached to LUV while SC505*ZAP70^tSH2 and SHP1 or SHP2 were presented in the extravesicular solution. ATP-triggered phosphorylation of CD3ζ^INT and BTLA^INT. The prior phosphorylation caused recruitment of SC505*ZAP70^tSH2 and its quenching due to FRET; the latter phosphorylation led to recruitment of SHP1 or SHP2 that would potentially dephosphorylate CD3ζ^INT, measured by SC505*ZAP70^tSH2 dequenching. (G) Same as E, except replacing BTLA^INT with equal concentrations of PD-1^INT. Orange pentagons denote His₁₀ tags; green stars denote SC505; red stars indicate Rhod-PE; P in white circles indicate phosphotyrosine.