Development of a robust scintillation proximity assay for protein tyrosinephosphatase 1B using the catalytically inactive (C215S) mutant

Protein tyrosine phosphatases are a class of enzymes that function to modul ate tyrosine phosphorylation of cellular proteins and play an essential rol e in regulating cell function. PTP1B has been implicated in the negative re gulation of the insulin signaling pathway by dephosphorylating the activate d insulin receptor. Inhibiting this phosphatase and preventing the insulin- receptor downregulation has been suggested as a target for the treatment of Type II diabetes. A high-throughput screen for inhibitors of PTP1B was dev eloped using a scintillation proximity assay (SPA) with GST- or FLAG-PTP1B( (1-320)) and a potent [H-3]-tripeptide inhibitor, The problem of interferen ce from extraneous oxidizing and alkylating agents which react with the cys teine active-site nucleophile was overcome by the use of the catalytically inactive C215S form of the native enzyme (GST-PTP1B(C215S)). The GST-PTP1B was linked to the protein A scintillation bead via GST antibody. The radiol abeled inhibitor when bound to the enzyme gave a radioactive signal that wa s competed away by the unknown competitive compounds. Further utility of PT P1B(C215S) was demonstrated by mixing in the same well both the catalytical ly inactive GST-PTP1B(C215S) and the catalytically active FLAG-CD45 with an inhibitor. Both a binding and kinetic assay was then performed in the same 96-well plate with the inhibition results determined for the PTP1B(C215S) (binding assay) and CD45 (activity assay). In this way inhibitors could be differentiated between the two phosphatases under identical assay condition s in one 96-well assay plate. The use of a mutant to reduce interference in a binding assay and compare with activity assays is also amenable for most cysteine active-site proteases, (C) 2001 Academic Press.