Phylogenetic and genetic linkage between novel atypical dual-specificity phosphatases from non-metazoan organisms

Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFA-DSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including parasite organisms responsible for infectious human 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.     

Visualizing active-site dynamics in single crystals of HePTP: opening of the WPD loop involves coordinated movement of the E loop

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loop in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an ‘atypically open’ conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.     

PTP inhibitor IV protects JNK kinase activity by inhibiting dual-specificity phosphatase 14 (DUSP14)

Protein phosphorylation plays critical roles in the regulation of protein activity and cell signaling. The level of protein phosphorylation is controlled by protein kinases and protein tyrosine phosphatases (PTPs). Disturbance of the equilibrium between protein kinase and PTP activities results in abnormal protein phosphorylation, which has been linked to the etiology of several diseases, including cancer. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by bis (4-trifluoromethyl-sulfonamidophenyl, TFMS)-1,4-diisopropylbenzene (PTP inhibitor IV). PTP inhibitor IV inhibited DUSP14 phosphatase activity. Kinetic studies with PTP inhibitor IV and DUSP14 revealed a competitive inhibition, suggesting that PTP inhibitor IV binds to the catalytic site of DUSP14. PTP inhibitor IV effectively and specifically inhibited DUSP14-mediated dephosphorylation of JNK, a member of the mitogen-activated protein kinase (MAPK) family.     

Crystal structure of human dual specificity phosphatase, JNK stimulatory phosphatase-1, at 1.5 A resolution

Human JNK stimulatory phosphatase-1 (JSP-1) is a novel member of dual specificity phosphatases. A C-terminus truncated JSP-1 was expressed in Escherichia coli and was crystallized using the sitting-drop vapor diffusion method. Thin-plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit-cell parameters a = 84.0 A, b = 49.3 A, c = 47.3 A, and beta = 119.5 degrees , and diffract up to 1.5 A resolution at 100 K. The structure of JSP-1 has a single compact (alpha/beta) domain, which consists of six alpha-helices and five beta-strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP-loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP-1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP-1 has no loop corresponding to the Lys120-loop of PTP1B, and tryptophan residue corresponding to the substrate-stacking in PTP1B is substituted by alanine residue in JSP-1.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase

Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity.     

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine.

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

Conformational basis for substrate recruitment in protein tyrosine phosphatase 10D

The coordinated activity of protein tyrosine phosphatases (PTPs) is crucial for the initiation, modulation, and termination of diverse cellular processes. The catalytic activity of this protein depends on a nucleophilic cysteine at the active site that mediates the hydrolysis of the incoming phosphotyrosine substrate. While the role of conserved residues in the catalytic mechanism of PTPs has been extensively examined, the diversity in the mechanisms of substrate recognition and modulation of catalytic activity suggests that other, less conserved sequence and structural features could contribute to this process. Here we describe the crystal structures of Drosophila melanogaster PTP10D in the apo form as well as in a complex with a substrate peptide and an inhibitor. These studies reveal the role of aromatic ring stacking interactions at the boundary of the active site of PTPs in mediating substrate recruitment. We note that phenylalanine 76, of the so-called KNRY loop, is crucial for orienting the phosphotyrosine residue toward the nucleophilic cysteine. Mutation of phenylalanine 76 to leucine results in a 60-fold decrease in the catalytic efficiency of the enzyme. Fluorescence measurements with a competitive inhibitor, p-nitrocatechol sulfate, suggest that Phe76 also influences the formation of the enzyme-substrate intermediate. The structural and biochemical data for PTP10D thus highlight the role of relatively less conserved residues in PTP domains in both substrate recruitment and modulation of reaction kinetics.     

Combinatorial control of the specificity of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs), the enzymes that dephosphorylate tyrosyl phosphoproteins, were initially believed to be few in number and serve a 'housekeeping' role in signal transduction. Recent work indicates that this is totally incorrect. Instead, PTPs comprise a large superfamily whose members play critical roles in a wide variety of cellular processes. Moreover, PTPs exhibit exquisite substrate specificity in vivo. Recent evidence has led us to propose that members of the PTP family achieve selectivity through different combinations of specific targeting strategies and intrinsic catalytic domain specificity.     

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.     

Substrate-trapping techniques in the identification of cellular PTP targets

Tyrosine phosphorylation is negatively regulated by the protein-tyrosine phosphatases (PTPs). In order to find the physiological substrates of these enzymes, diverse PTP mutants that do not possess any catalytic activities but appear to bind tightly to their tyrosine phosphorylated substrates have been designed. Hence, they can be used as tools to pull out their respective substrates from heterogeneous extracts. Named PTP "substrate-trapping" mutants by the Tonks laboratory, they represent a diverse variety of defective PTPs that are epitomized by the Cys to Ser mutant (C/S) where the active cysteine residue of the signature motif is mutated to a serine residue. In addition, new mutants have been developed which are expected to help characterize novel and less abundant substrates. In this article, we review and describe all the different substrate-trapping mutants that have successfully been used or that hold interesting promises. We present their methodology to identify substrates in vivo (co-immunoprecipitation) and in vitro (GST pulldown), and provide a current list of substrates that have been identified using these technologies.     

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.     

A one-step method to identify MAP kinase residues involved in inactivation by tyrosine- and dual-specificity protein phosphatases

MAP kinases (MAPKs) are enzymes directly involved in the control of cellular homeostasis in response to external cues, from differentiation and developmental processes to cell transformation. The activation status of MAPKs, both in magnitude and in duration, reflects the balance of phosphorylation at their Thr and Tyr regulatory residues by specific MAPK kinases and their dephosphorylation by inactivating protein serine/threonine phosphatases (PPs) and protein tyrosine phosphatases (PTPs). The dephosphorylation of MAPKs by PTPs relies on molecular docking between the two enzymes at specific interaction sites. Here we outline a one-step method to identify ERK1/2 and p38α mutations that prevent binding and inactivation by PTPs (tyrosine- or dual-specificity phosphatases) based on the use of anti-pTyr antibodies and cell lysis buffers lacking or containing the broad PTP inhibitor sodium orthovanadate (Na₃VO₄).     

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.     

Structure of the hematopoietic tyrosine phosphatase (HePTP) catalytic domain: structure of a KIM phosphatase with phosphate bound at the active site

Hematopoietic tyrosine phosphatase (HePTP) is a 38kDa class I non-receptor protein tyrosine phosphatase (PTP) that is strongly expressed in T cells. It is composed of a C-terminal classical PTP domain (residues 44-339) and a short N-terminal extension (residues 1-43) that functions to direct HePTP to its physiological substrates. Moreover, HePTP is a member of a recently identified family of PTPs that has a major role in regulating the activity and translocation of the MAP kinases Erk and p38. HePTP binds Erk and p38 via a short, highly conserved motif in its N terminus, termed the kinase interaction motif (KIM). Association of HePTP with Erk via the KIM results in an unusual, reciprocal interaction between the two proteins. First, Erk phosphorylates HePTP at residues Thr45 and Ser72. Second, HePTP dephosphorylates Erk at PTyr185. In order to gain further insight into the interaction of HePTP with Erk, we determined the structure of the PTP catalytic domain of HePTP, residues 44-339. The HePTP catalytic phosphatase domain displays the classical PTP1B fold and superimposes well with PTP-SL, the first KIM-containing phosphatase solved to high resolution. In contrast to the PTP-SL structure, however, HePTP crystallized with a well-ordered phosphate ion bound at the active site. This resulted in the closure of the catalytically important WPD loop, and thus, HePTP represents the first KIM-containing phosphatase solved in the closed conformation. Finally, using this structure of the HePTP catalytic domain, we show that both the phosphorylation of HePTP at Thr45 and Ser72 by Erk2 and the dephosphorylation of Erk2 at Tyr185 by HePTP require significant conformational changes in both proteins.