Design and Characterization of an Improved Protein Tyrosine Phosphatase Substrate-Trapping Mutant

Abstract

Although members of the protein tyrosine phosphatase (PTPase) family share a common mechanism of action (hydrolysis of phosphotyrosine), the cellular processes in which they are involved can be both highly specialized and fundamentally important. Identification of cellular PTPase substrates will help elucidate the biological functions of individual PTPases. Two types of substrate-trapping mutants are being used to isolate PTPase substrates. In the first, the active site Cys residue is replaced by a Ser (e.g., PTP1B/C215S) while in the second, the general acid Asp residue is substituted by an Ala (e.g., PTP1B/D181A). Unfortunately, only a limited number of PTPase substrates have been identified with these two mutants, which are usually relatively abundant cellular proteins. Based on mechanistic considerations, we seek to create novel PTPase mutants with improved substrate-trapping properties. Kinetic and thermodynamic characterization of the newly designed PTP1B mutants indicates that PTP1B/D181A/Q262A displays lower catalytic activity than that of D181A. In addition, D181A/Q262A also possesses 6- and 28-fold higher substrate-binding affinity than those of D181A and C215S, respectively. In vivo substrate-trapping experiments indicate that D181A/Q262A exhibits much higher affinity than both D181A and C215S for a bona fide PTP1B substrate, the epidermal growth factor receptor. Moreover, D181A/Q262A can also identify novel, less abundant substrates, that are missed by D181A. Thus, this newly developed and improved substrate-trapping mutant can serve as a powerful affinity reagent to isolate and purify both high- and low-abundant protein substrates. Given that both Asp181 and Gln262 are invariant among the PTPase family, it is predicted that this improved substrate-trapping mutant would be applicable to all members of PTPases for substrate identification.

The Role of Protein-tyrosine Phosphatase 1B in Integrin Signaling

“…To provide further evidence that Src is a substrate for PTP1B, we performed substrate-trapping experiments using PTP1B mutants that are capable of substrate binding but unable to carry out the dephosphorylation reaction (8,49). PTP1B substrates identified using this approach include insulin receptor, (50), epidermal growth factor receptor (8, 49), and JAK2 (51).…”

“…The D181A mutant has been used to trap the epidermal growth factor receptor as a PTP1B substrate (8). The D181A/Q262A mutant exhibits an affinity for substrates that is severalfold higher than that of D181A and is thus a more superior PTP1B substrate-trapping mutant (49). After transfection, cells were serum-starved and applied to fibronectincoated dishes.…”

A Novel Substrate of Receptor Tyrosine Phosphatase PTPRO Is Required for Nerve Growth Factor-Induced Process Outgrowth

“…A catalytically inactive mutant of PTPRO was created by site-directed mutagenesis of two invariant residues (Xie et al, 2002) of the wild-type intracellular domain of PTPRO (GeneEditor; Promega, Madison, WI). cDNAs encoding the wild-type and the mutant PTPRO were inserted in frame into pGEX6P vector (Amersham Biosciences, Arlington Heights, IL) to make glutathione S-transferase (GST)-PTPRO fusion proteins.…”

“…This suggests that NPCD can be dephosphorylated by PTPRO. To test this idea, we performed GST pulldowns with a catalytically inactive form of GST-PTPRO (Flint et al, 1997;Xie et al, 2002). A quantitatively similar amount of the 41 kDa NPCD protein associated with the mutant PTPRO as with wild-type PTPRO, as assessed by anti-Ptx Western blotting.…”

An affinity-based fluorescence polarization assay for protein tyrosine phosphatases

“…To eliminate false positives due to covalent modifications of the active site Cys, we have developed a competitive, homogeneous, high-throughput fluorescence polarization assay to identify high affinity active site binders to the PTPs. This assay is based on the observations that the catalytically inactive Cys to Ser mutant PTP (e.g., PTP1B/C215S) retains the ability to bind substrates [10,11], and with the same affinity as the wild-type enzyme [12][13][14]. Compounds that are capable of displacing a fluorescently labeled ligand from the active site of the Cys to Ser mutant PTP are likely competitive PTP inhibitors.…”