Identification of tyrosine phosphatases that dephosphorylate the insulin receptor. A brute force approach based on "substrate-trapping" mutants

Many pharmacologically important receptors, including all cytokine receptors, signal via tyrosine (auto)phosphorylation, followed by resetting to their original state through the action of protein tyrosine phosphatases (PTPs). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics. We have set up a systematic approach for finding PTPs that are specific for a receptor and have validated this approach with the insulin receptor kinase. We have tested nearly all known human PTPs (45) in a membrane binding assay, using "substrate-trapping" PTP mutants. These results, combined with secondary dephosphorylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstrate that this approach can rapidly reduce the number of PTPs that have a particular receptor or other phosphoprotein as their substrate.     

Design and characterization of an improved protein tyrosine phosphatase substrate-trapping mutant

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.     

Regulation of insulin signaling through reversible oxidation of the protein-tyrosine phosphatases TC45 and PTP1B

Many studies have illustrated that the production of reactive oxygen species (ROS) is important for optimal tyrosine phosphorylation and signaling in response to diverse stimuli. Protein-tyrosine phosphatases (PTPs), which are important regulators of signal transduction, are exquisitely sensitive to inhibition after generation of ROS, and reversible oxidation is becoming recognized as a general physiological mechanism for regulation of PTP function. Thus, production of ROS facilitates a tyrosine phosphorylation-dependent cellular signaling response by transiently inactivating those PTPs that normally suppress the signal. In this study, we have explored the importance of reversible PTP oxidation in the signaling response to insulin. Using a modified ingel PTP assay, we show that stimulation of cells with insulin resulted in the rapid and transient oxidation and inhibition of two distinct PTPs, which we have identified as PTP1B and TC45, the 45-kDa spliced variant of the T cell protein-tyrosine phosphatase. We investigated further the role of TC45 as a regulator of insulin signaling by combining RNA interference and the use of substrate-trapping mutants. We have shown that TC45 is an inhibitor of insulin signaling, recognizing the beta-subunit of the insulin receptor as a substrate. The data also suggest that this strategy, using ligand-induced oxidation to tag specific PTPs and using interference RNA and substrate-trapping mutants to illustrate their role as regulators of particular signal transduction pathways, may be applied broadly across the PTP family to explore function.     

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.     

Identification of protein tyrosine phosphatases with specificity for the ligand-activated growth hormone receptor

Protein tyrosine phosphatases (PTPs) play key roles in switching off tyrosine phosphorylation cascades, such as initiated by cytokine receptors. We have used substrate-trapping mutants of a large set of PTPs to identify members of the PTP family that have substrate specificity for the phosphorylated human GH receptor (GHR) intracellular domain. Among 31 PTPs tested, T cell (TC)-PTP, PTP-beta, PTP1B, stomach cancer-associated PTP 1 (SAP-1), Pyst-2, Meg-2, and PTP-H1 showed specificity for phosphorylated GHR that had been produced by coexpression with a kinase in bacteria. We then used GH-induced, phosphorylated GH receptor, purified from overexpressing mammalian cells, in a Far Western-based approach to test whether these seven PTPs were also capable of recognizing ligand-induced, physiologically phosphorylated GHR. In this assay, only TC-PTP, PTP1B, PTP-H1, and SAP-1 interacted with the mature form of the phosphorylated GHR. In parallel, we show that these PTPs recognize very different subsets of the seven GHR tyrosines that are potentially phosphorylated. Finally, mRNA tissue distribution of these PTPs by RT-PCR analysis and coexpression of the wild-type PTPs to test their ability to dephosphorylate ligand-activated GHR suggest PTP-H1 and PTP1B as potential candidates involved in GHR signaling.     

The SPOT technique as a tool for studying protein tyrosine phosphatase substrate specificities

The activity of protein tyrosine phosphatases (PTPs) is restricted by their substrate specificities. The analysis of PTP specificity was greatly helped by the discovery that "substrate-trapping" PTP mutants, such as PTP-1B D181A, stably and specifically bind their substrates. We have set up a PTP substrate specificity assay based on the SPOT technique, which involves the microsynthesis of (phospho)peptides on membranes. To validate this approach, substrate trapping PTP-1B was tested on its cognate ligand, the autophosphorylated insulin receptor (IR). On SPOT membranes, IR peptides with phosphotyrosine 1163 were efficiently bound by PTP1B D181A, and dephosphorylated by PTP-1B. Phosphotyrosine 1163 was preferred over the neighboring 1158 and 1162 phosphotyrosines. PTP-1B also recognized IR-like motifs in Trk autophosphorylation domains, and STAT 5 phosphopeptides. Using a gridded 20-by-20 SPOT library, we show that peptides with the YZM motif (Z: phosphotyrosine) are the strongest ligands for PTP-1B D181A, but not the optimal substrates for dephosphorylation by wild-type PTP1B. In addition we show that PTP-1B and PTP-beta dephosphorylation efficiency is strongly modulated by the introduction of phospho-serine or phospho-threonine in their cognate phospho-tyrosine substrates. Altogether our data illustrate that the SPOT technique is a highly efficient tool for the study of PTP substrate specificity.     

Proteomic approaches to studying protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) constitute a large family of enzymes that play key roles in cell signaling. Deregulation of PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. Since phosphate removal by the PTPs can both enhance and antagonize cellular signaling, it is essential to elucidate the physiological context in which PTPs operate. Two powerful proteomic approaches have been developed to rapidly establish the exact functional roles for every PTP, both in normal cellular physiology and in pathogenic conditions. In the first, an affinity-based substrate-trapping approach has been employed for PTP substrate identification. Identification and characterization of specific PTP-substrate interactions will associate functions with PTP as well as implicate PTP to specific signaling pathways. In the second, a number of activity-based PTP probes have been developed that can provide a direct readout of the functional state of the PTPs in complex proteomes. The ability to profile the entire PTP family on the basis of changes in their activity is expected to yield new functional insights into pathways regulated by the PTPs and contribute to the discovery of PTPs as novel therapeutic targets. Effective application of these proteomic techniques will accelerate the functional characterization of PTPs, thereby facilitating our understanding of PTPs in cell signaling and in diseases.     

Probing protein-tyrosine phosphatase substrate specificity using a phosphotyrosine-containing phage library

Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and Km determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-beta, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.     

Investigation of protein-tyrosine phosphatase 1B function by quantitative proteomics

Because of their antagonistic catalytic functions, protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases act together to control phosphotyrosine-mediated signaling processes in mammalian cells. However, unlike for protein-tyrosine kinases, little is known about the cellular substrate specificity of many PTPs because of the lack of appropriate methods for the systematic and detailed analysis of cellular PTP function. Even for the most intensely studied, prototypic family member PTP1B many of its physiological functions cannot be explained by its known substrates. To gain better insights into cellular PTP1B function, we used quantitative MS to monitor alterations in the global tyrosine phosphorylation of PTP1B-deficient mouse embryonic fibroblasts in comparison with their wild-type counterparts. In total, we quantified 124 proteins containing 301 phosphotyrosine sites under basal, epidermal growth factor-, or platelet-derived growth factor-stimulated conditions. A subset of 18 proteins was found to harbor hyperphosphorylated phosphotyrosine sites in knock-out cells and was functionally linked to PTP1B. Among these proteins, regulators of cell motility and adhesion are overrepresented, such as cortactin, lipoma-preferred partner, ZO-1, or p120ctn. In addition, regulators of proliferation like p62DOK or p120RasGAP also showed increased cellular tyrosine phosphorylation. Physical interactions of these proteins with PTP1B were further demonstrated by using phosphatase-inactive substrate-trapping mutants in a parallel MS-based analysis. Our results correlate well with the described phenotype of PTP1B-deficient fibroblasts that is characterized by an increase in motility and reduced cell proliferation. The presented study provides a broad overview about phosphotyrosine signaling processes in mouse fibroblasts and, supported by the identification of various new potential substrate proteins, indicates a central role of PTP1B within cellular signaling networks. Importantly the MS-based strategies described here are entirely generic and can be used to address the poorly understood aspects of cellular PTP function.     

Protein tyrosine phosphatase function: the substrate perspective

It is now well established that the members of the PTP (protein tyrosine phosphatase) superfamily play critical roles in fundamental biological processes. Although there has been much progress in defining the function of PTPs, the task of identifying substrates for these enzymes still presents a challenge. Many PTPs have yet to have their physiological substrates identified. The focus of this review will be on the current state of knowledge of PTP substrates and the approaches used to identify them. We propose experimental criteria that should be satisfied in order to rigorously assign PTP substrates as bona fide. Finally, the progress that has been made in defining the biological roles of PTPs through the identification of their substrates will be discussed.     

Engineered inhibitor sensitivity in the WPD loop of a protein tyrosine phosphatase

Small molecules that can be used to turn off the activities of specific cellular proteins are essential tools for chemical biology. Few such compounds are known, however, and they are particularly difficult to identify for members of large protein families. Here, we present a method for insertion of a chemical "off switch" into a catalytically essential loop region (the "WPD loop") of a protein tyrosine phosphatase (PTP). Using a combination of point mutations and amino acid insertions, we have engineered variants of T-cell PTP (TCPTP) that possess cysteine-rich WPD loops. The engineered WPD loops, which contain sequences that appear in no wild-type PTP, confer upon TCPTP the ability to bind a cell-permeable small molecule (the biarsenical fluorescein derivative, FlAsH) that is not an inhibitor of wild-type PTPs. We have identified sites in TCPTP's WPD loop that can be modified to display FlAsH-binding cysteine residues without disrupting TCPTP's inherent PTP activity, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. Upon addition of the FlAsH ligand, however, the activities of the mutants drop dramatically. Inhibition of the FlAsH-sensitized TCPTP mutants is rapid and specific; and strong FlAsH sensitivity was observed in mutants that contain as few as two cysteine point mutations in their engineered WPD loops. Our results show that relatively conservative substitutions can be used to engineer precise small-molecule control of PTP activity. Moreover, since all known classical PTPs utilize the WPD-loop mechanism targeted in this study, it is likely that a substantial fraction of the PTP superfamily can be sensitized through an analogous approach.     

Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.     

Free-energy profiles for catalysis by dual-specificity phosphatases

PTPs (protein tyrosine phosphatases) are fundamental enzymes for cell signalling and have been linked to the pathogenesis of several diseases, including cancer. Hence, PTPs are potential drug targets and inhibitors have been designed as possible therapeutic agents for Type II diabetes and obesity. However, a complete understanding of the detailed catalytic mechanism in PTPs is still lacking. Free-energy profiles, obtained by computer simulations of catalysis by a dual-specificity PTP, are shown in the present study and are used to shed light on the catalytic mechanism. A highly accurate hybrid potential of quantum mechanics/molecular mechanics calibrated specifically for PTP reactions was used. Reactions of alkyl and aryl substrates, with different protonation states and PTP active-site mutations, were simulated. Calculated reaction barriers agree well with experimental rate measurements. Results show the PTP substrate reacts as a bi-anion, with an ionized nucleophile. This protonation state has been a matter of debate in the literature. The inactivity of Cys-->Ser active-site mutants is also not fully understood. It is shown that mutants are inactive because the serine nucleophile is protonated. Results also clarify the interpretation of experimental data, particularly kinetic isotope effects. The simulated mechanisms presented here are better examples of the catalysis carried out by PTPs.     

Application of hydrogen/deuterium exchange mass spectrometry to study protein tyrosine phosphatase dynamics, ligand binding, and substrate specificity

Protein tyrosine phosphatases (PTPs) are signaling enzymes that control a diverse array of cellular processes. Further insight into the specific functional roles of PTPs in cellular signaling requires detailed understanding of the molecular basis for substrate recognition by the PTPs. A central question is how PTPs discriminate between multiple structurally diverse substrates that they encounter in the cell. Although X-ray crystallography is capable of revealing the intimate structural details for molecular interaction, structures of higher order PTP.substrate complexes are often difficult to obtain. Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) is a powerful tool for mapping protein-protein interfaces, as well as identifying conformational and dynamic perturbations in proteins. In addition, H/DX-MS enables analysis of large protein complexes at physiological concentrations and provides insight into the solution behavior of these complexes that can not be gleaned from crystal structures. We have utilized H/DX-MS to probe PTP dynamics, ligand binding, and the structural basis of substrate recognition. In this article, we review general principles of H/DX-MS technology as applied to study protein-protein interactions and dynamics. We also provide protocols for H/DX-MS successfully used in our laboratory to define the molecular basis of ERK2 substrate recognition by MKP3. Many of the aspects that we cover in detail should be applicable to the study of other PTPs with their specific targets.     

A new highly efficient substrate-trapping mutant of protein tyrosine phosphatase 1B (PTP1B) reveals full autoactivation of the insulin receptor precursor

PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I(125) insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates.     

Development of a modified in-gel assay to identify protein tyrosine phosphatases that are oxidized and inactivated in vivo

Protein tyrosine phosphatases (PTPs) comprise a superfamily of enzymes that control a diverse array of signal transduction pathways. However, the function and regulation of many of these enzymes remain undefined. Previous studies have shown that the optimal tyrosine phosphorylation response to various exogenous stimuli requires the production of reactive oxygen species (ROS). It has been proposed that ROS might transiently inactivate inhibitory PTPs, thus facilitating tyrosine phosphorylation-dependent signaling. Interestingly, the unique chemistry of the invariant, active site Cys residue located in the signature motif renders it highly susceptible to oxidation, leading to the inactivation of PTPs. We have developed a novel strategy to identify those PTPs that are oxidized and therefore, inactivated in response to extracellular stimuli. Iodoacetic acid (IAA) was used to alkylate selectively the thiolate anion of the active site Cys in the reduced PTPs. In contrast, any PTPs in which the active site Cys had been oxidized in response to the stimulus were resistant to alkylation. Following this key step to differentiate between the two pools of PTPs, the oxidized phosphatases were reduced back to the active state during the process of a standard in-gel PTP activity assay. This novel technique revealed, for the first time, that multiple cellular PTPs were indeed oxidized and inactivated in response to exogenous hydrogen peroxide. We have used this technique extensively to show that the ligand-stimulated production of intracellular hydrogen peroxide reversibly regulates the activity of specific PTPs in vivo. By defining the precise PTP targets of intracellular oxidants, the mechanistic details of signal transduction can be delineated. Due to the potential use of this method in finding the molecular targets of intracellular oxidants in diverse signaling pathways, we describe here the theoretical background and the detailed protocols of the modified in-gel PTP assay.     

Protein tyrosine phosphatase PTP20 induces actin cytoskeleton reorganization by dephosphorylating p190 RhoGAP in rat ovarian granulosa cells stimulated with follicle-stimulating hormone

We identified 25 protein tyrosine phosphatases (PTPs) expressed in rat ovarian granulosa cells. Of these PTPs, the expression levels of at least PTP20, PTP-MEG1, PTPepsilonM, and PTPepsilonC significantly changed during the estrous cycle. We examined the cellular functions of PTP20 in granulosa cells by expressing the wild type, a catalytically inactive CS mutant in which Cys229 of PTP20 was changed to Ser, or a substrate-trapping DA mutant in which Asp197 was mutated to Ala, using an adenovirus vector. Overexpression of the wild type, but not of the CS mutant, induced retraction of the cell body with the extension of long, dendritic-like processes after stimulation with FSH, a critical factor for the survival and differentiation of these cells. In addition, cell adhesion to the substratum decreased in an FSH-dependent manner. Inhibiting Rho GTPase activity with C3 botulinum toxin caused similar morphological changes. The FSH-enhanced phosphotyrosine (p-Tyr) level of p190 RhoGAP was selectively reduced by the overexpressed wild type, but not by mutated PTP20. Although p190 RhoGAP is tyrosine phosphorylated by c-Src via the tyrosine kinase Pyk2, wild-type PTP20 had little effect on p-Tyr418 of c-Src and no effect on p-Tyr402 of Pyk2, which are required for full c-Src activity and for interacting between Pyk2 and c-Src, respectively. The CS and DA mutants as well as the wild type reduced the formation of p190 RhoGAP-p120 RasGAP complexes. Confocal microscopy analysis revealed that PTP20 intracellularly colocalizes with p190 RhoGAP. These results demonstrate that PTP20 regulates the functions of granulosa cells in an FSH-dependent manner by dephosphorylating p190 RhoGAP and subsequently inducing reorganization of the actin cytoskeleton. Moreover, our data suggest that PTPs play significant roles in controlling the dynamics of ovarian functions.     

Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases

A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of these substrates with PTPs were performed and Michaelis-Menten parameters were obtained. These designed substrates have Km 0.03-0. 35 mM, kcat/Km of 3-100 mM-1 s-1 with CD45 and PTP1B. The results showed that the substrates with negative charge groups on the fluorescein have higher affinities for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observed. Since FMSP showed large increases in both absorption and fluorescence upon dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, stable and high affinity substrates reported for PTPs.