Difmup

Plasmids for expression of glutathione S-transferase (GST)-PTP fusion proteins of human Shp2 (residues 205–597) were constructed in pGEX-4T1 by PCR subcloning techniques. All constructs were verified by DNA sequencing. GST- Shp2 fusion proteins were expressed in Escherichia coli BL21 and affinity purified with glutathione Sepharose. After elution from glutathione affinity columns, GST-fusion proteins were dialyzed with dialysis buffer (12.5 mM Tris-Cl, pH 7.5, 25 mM NaCl, 1 mM dithiothreitol, and 0.1% β-mercaptoethanol) at 4 °C over night and then stored in dialysis buffer plus 20% glycerol at −80 °C.
Shp2 activity was measured as described previously6 (link). Using the 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP; Invitrogen, Carlsbad, CA) as the substrate, reaction buffer contained 50 mM Hepes (pH 7.0), 150 mM NaCl, 0.05% Tween 20, 2 mM dithiothreitol, 1 mM EDTA, 20 μM DiFMUP, 0.1 μM GST-Shp2, and different concentrations of test compound or dimethyl sulfoxide (solvent) in a total reaction volume of 100 μL in black 96-well plates. Reaction was initiated by addition of DiFMUP, and the incubation time was 30 min at 37 °C. DiFMUP fluorescence signal was measured at an excitation of 355 nm and an emission of 455 nm with a plate reader (Thermo Scientific Verioskan Flash).

The coding sequence of human PPP1CA was expressed as a maltose binding protein fusion in a BL-21 strain of E. coli and purified as previously described (55 (link)). Phosphohistone phosphatase assays were performed as previously described (55 (link), 56 (link)). Briefly, LB-100, at the indicated concentrations, or vehicle control (H₂O) was added to enzyme/buffer aliquots about 10 minutes prior to starting assays by the addition of [³²P]-phosphohistone substrate (to a final assay concentration of 300 nM incorporated phosphate). [³²P]-phosphohistone was prepared by the phosphorylation of bovine brain histone (MilliporeSigma, type-2AS) with cAMP-dependent protein kinase (PKA) in the presence of cAMP and [³²P]-ATP using established methods (56 (link), 57 (link)). Phosphatase activity was measured by the quantitation of [³²P]-labeled orthophosphate liberated from the substrate using established protocols (57 (link)). 6,8-Difluoro-4-methylumbelliferyl phosphate–based (DiFMUP-based) inhibition assays were conducted as described (56 (link), 57 (link)), in a 96-well format using DiFMUP (Invitrogen) (100 μM final assay concentration). IC₅₀ values were calculated from a 10-point concentration/dose response curve by a 4-parameter logistic fit of the data, using 3–8 replicates per concentration.