Global analysis of protein tyrosine phosphatase activity with ultra-sensitive fluorescent probes

Protein tyrosine phosphatases (PTPs) consist of a large family of enzymes known to play important roles in controlling virtually all aspects of cellular processes. However, assigning functional significance of PTPs in normal physiology and in diseases remains a major challenge in cell signaling. Since the function of a PTP is directly associated with its intrinsic activity, which is subject to post-translational regulation, new tools are needed to monitor the dynamic activities of PTPs, rather than mere abundance, on a global scale within the physiologically relevant environment of cells. To meet this objective, we report the synthesis and characterization of two rhodamine-conjugated probes that covalently label the active site of the PTPs in an activity-dependent manner, thus providing a direct readout of PTP activity and superior sensitivity, robustness, and quantifiability to previously reported biotinylated probes. We present evidence that the fluorescent probes can be used to identify new PTP markers and targets for potential diagnosis and treatment of human diseases. We also show that the fluorescent probes are capable of monitoring H(2)O(2)-mediated PTP inactivation, which should facilitate the study of regulated H(2)O(2) production as a new tier of control over tyrosine phosphorylation-dependent signal transduction. 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 PTPs and contribute to the discovery of PTPs as novel therapeutic targets.     

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.     

Protein tyrosine phosphatases: structure-function relationships

Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.     

Protein Tyrosine Phosphatases: Emerging Regulators of Apoptosis

Apoptosis is a precisely controlled physiological mechanism that is required for the elimination of cells during embryonic development, in response to stress and infection as well as in the maintenance of homeostasis. Since the outcome of several of these biological processes is regulated by signaling events involving tyrosine phosphorylation, members of the protein tyrosine phosphatase (PTP) gene family are expected to be of primary importance. Here, we summarize the current literature linking the activities of classical PTPs with the regulation of apoptosis. The recent discovery of caspase-cleavage mediated modulation of a member of this family, PTP-PEST, indicates that other PTPs could be modulated in a similar manner. In light of this, we present an analysis of all murine and human PTPs gene for the presence of putative caspase cleavage motifs. Additional studies linking the activity of PTPs to their own regulation during programmed cell death initiation should provide important insight into the understanding of this fundamental physiological phenomenon.     

Protein tyrosine phosphatases as novel targets in breast cancer therapy

Breast cancer is linked to hyperactivation of protein tyrosine kinases (PTKs), and recent studies have unveiled that selective tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) of specific substrates, including PTKs, may activate or inactivate oncogenic pathways in human breast cancer cell growth-related processes. Here, we review the current knowledge on the involvement of PTPs in breast cancer, as major regulators of breast cancer therapy-targeted PTKs, such as HER1/EGFR, HER2/Neu, and Src. The functional interplay between PTKs and PTK-activating or -inactivating PTPs, and its implications in novel breast cancer therapies based on targeting of specific PTPs, are discussed.     

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.     

A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production from 3-nitrophosphotyrosine containing peptidic substrates, which are accepted by many PTPs. Compared to conventional chromogenic phosphate derivatives, the more realistic peptidic substrates allow evaluating substrate specificity. The assay’s applicability is demonstrated by determining kinetic parameters for several PTP-substrate combinations and inhibitor evaluation, as well as detection of PTP activity in lysates. The convenient new assay may assist further adoption of PTPs in drug development.     

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.     

Probing the molecular basis for potent and selective protein-tyrosine phosphatase 1B inhibition

Protein-tyrosine phosphatases (PTPs) are important for the control of proper cellular tyrosine phosphorylation. Despite the large number of PTPs encoded in the human genome and the emerging roles played by PTPs in human diseases, a detailed understanding of the role played by PTPs in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific inhibitors. Such inhibitors could serve as useful tools for determining the physiological functions of PTPs and may constitute valuable therapeutics in the treatment of several human diseases. However, because of the highly conserved nature of the active site, it has been difficult to develop selective PTP inhibitors. By taking an approach to tether together two small ligands that can interact simultaneously with the active site and a unique proximal noncatalytic site, we have recently acquired Compound 2 (see Fig. 1), the most potent and selective PTP1B inhibitor identified to date, which exhibits several orders of magnitude selectivity in favor of PTP1B against a panel of PTPs. We describe an evaluation of the interaction between 2 and its analogs with PTP1B and its site-directed mutants selected based on hydrogen/deuterium exchange of PTP1B backbone amides in the presence and absence of 2. We have established the binding mode of Compound 2 and identified 12 PTP1B residues that are important for the potency and selectivity of Compound 2. Although many of the residues important for Compound 2 binding are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which suggest that the binding surface defined by these residues in individual PTPs determines inhibitor selectivity. Our results provide structural information toward understanding of the molecular basis for potent and selective PTP1B inhibition and further establish the feasibility of acquiring potent, yet highly selective, PTP inhibitory agents.     

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.     

Polar tube proteins of microsporidia of the family encephalitozoonidae

Encephalitozoonidae are microsporidia associated with human infections including hepatitis, encephalitis, conjunctivitis, and disseminated disease. Microsporidia produce a small resistant spore containing a polar tube which serves as a unique vehicle of infection. Polar tube proteins (PTPs) from Encephalitozoon hellem. Encephalitozoon (Septata) intestinalis, and Encephalitozoon cuniculi were purified to homogeneity by HPLC. By SDS-PAGE, the Mr of E. hellem PTP was 55 kDa, while the Mr of E. intestinalis and E. cuniculi PTP was 45 kDa. Polyclonal rabbit antiserum to these purified PTPs localized to polar filaments by immunogold electron microscopy and immunofluorescence, and demonstrated cross-reactivity by both immunoblotting and immunogold electron microscopy. These PTPs have similar solubility properties, hydrophobicity, and proline content to a 43-kDa PTP we have previously purified from Glugea americanus, a fish microsporidium. As the polar tube is critical in the transmission of this organism, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.     

Redox regulation of PTEN and protein tyrosine phosphatases in H(2)O(2) mediated cell signaling

Protein tyrosine phosphatase (PTP) is a family of enzymes important for regulating cellular phosphorylation state. The oxidation and consequent inactivation of several PTPs by H₂O₂ are well demonstrated. It is also shown that recovery of enzymatic activity depends on the availability of cellular reductants. Among these redox-regulated PTPs, PTEN, Cdc25 and low molecular weight PTP are known to form a disulfide bond between two cysteines, one in the active site and the other nearby, during oxidation by H₂O₂. The disulfide bond likely confers efficiency in the redox regulation of the PTPs and protects cysteine-sulfenic acid of PTPs from further oxidation. In this review, through a comparative analysis of the oxidation process of Yap1 and PTPs, we propose the mechanism of disulfide bond formation in the PTPs.     

Generation of inhibitor-sensitive protein tyrosine phosphatases via active-site mutations

Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of phosphotyrosine, a central control element in mammalian signal transduction. Small-molecule inhibitors that are specific for each cellular PTP would be valuable tools in dissecting phosphorylation networks and for validating PTPs as therapeutic targets. However, the common architecture of PTP active sites impedes the discovery of selective PTP inhibitors. Our laboratory has recently used enzyme/inhibitor-interface engineering to generate selective PTP inhibitors. The crux of the strategy resides in the design of "inhibitor-sensitized" PTPs through protein engineering of a novel binding pocket in the target PTP. "Allele-specific" inhibitors that selectively target the sensitized PTP can be synthesized by modifying broad-specificity inhibitors with bulky chemical groups that are incompatible with wild-type PTP active sites; alternatively, specific inhibitors that serendipitously recognize the sensitized PTP's non-natural pocket may be discovered from panels of "non-rationally" designed compounds. In this review, we describe the current state of the PTP-sensitization strategy, with emphases on the methodology of identifying PTP-sensitizing mutations and synthesizing the compounds that have been found to target PTPs in an allele-specific manner. Moreover, we discuss the scope of PTP sensitization in regard to the potential application of the approach across the family of classical PTPs.     

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.     

Selective regulation of hydrogen peroxide signaling by receptor tyrosine phosphatase-alpha

Reactive oxygen species (ROS) are constantly produced in the human body and are involved in the pathogenesis of aging, cardiovascular diseases, and cancer. Emerging evidence indicates that oxidation and inhibition of protein tyrosine phosphatases (PTPs) are critical for ROS signal transduction. However, the role of individual PTPs in ROS signaling remains unclear. Here, we demonstrated that the receptor-like PTP alpha (RPTP alpha) was an effector of H2O2, the most stable form of ROS. H2O2 at nontoxic concentration rapidly induced the association of RPTP alpha with Src family kinases, platelet-derived growth factor receptor-beta, and protein kinase D in various cultured cells, although it markedly suppressed RPTP alpha phosphorylation on Tyr-789. We further identified that RPTP alpha selectively regulated the signal transduction pathways induced by H2O2. Particularly, RPTP alpha was required for the activation of protein kinase D and for the modulation of p130Cas tyrosine phosphorylation in response to H2O2. In contrast, the H2O2-induced inactivation of Src family kinases and suppression of paxillin phosphorylation on Tyr-118 were both largely independent of RPTP alpha. Our findings indicate that H2O2 signaling pathways are selectively regulated by RPTP alpha in cells, which may provide new insights into the functional regulation of ROS signal transduction by PTPs.     

Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied to the members of large protein families. Here we report that protein tyrosine phosphatases can be systematically 'sensitized' to target-specific inhibition by a cell-permeable small molecule, Fluorescein Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain's WPD loop confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all cases, FlAsH-mediated PTP inhibition was target specific and potent, with inhibition constants for the seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or model-organism-based signaling studies.     

Structure and catalytic mechanism of human protein tyrosine phosphatome

Together with protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs) serve as hallmarks in cellular signal transduction by controlling the reversible phosphorylation of their substrates. The human genome is estimated to encode more than 100 PTPs, which can be divided into eleven sub-groups according to their structural and functional characteristics. All the crystal structures of catalytic domains of sub-groups have been elucidated, enabling us to understand their precise catalytic mechanism and to compare their structures across all sub-groups. In this review, I describe the structure and mechanism of catalytic domains of PTPs in the structural context. [BMB Reports 2012; 45(12): 693-699]     

Protein tyrosine phosphatases: from genes, to function, to disease

The protein tyrosine phosphatase (PTP) superfamily of enzymes functions in a coordinated manner with protein tyrosine kinases to control signalling pathways that underlie a broad spectrum of fundamental physiological processes. In this review, I describe recent breakthroughs in our understanding of the role of the PTPs in the regulation of signal transduction and the aetiology of human disease.     

Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs.     

Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts

The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR.