Evolution of the multifunctional protein tyrosine phosphatase family

The protein tyrosine phosphatase (PTP) family plays a central role in signal transduction pathways by controlling the phosphorylation state of serine, threonine, and tyrosine residues. PTPs can be divided into dual specificity phosphatases and the classical PTPs, which can comprise of one or two phosphatase domains. We studied amino acid substitutions at functional sites in the phosphatase domain and identified putative noncatalytic phosphatase domains in all subclasses of the PTP family. The presence of inactive phosphatase domains in all subclasses indicates that they were invented multiple times in evolution. Depending on the domain composition, loss of catalytic activity can result in different consequences for the function of the protein. Inactive single-domain phosphatases can still specifically bind substrate and protect it from dephosphorylation by other phosphatases. The inactive domains of tandem phosphatases can be further subdivided. The first class is more conserved, still able to bind phosphorylated tyrosine residues and might recruit multiphosphorylated substrates for the adjacent active domain. The second has accumulated several variable amino acid substitutions in the catalytic center, indicating a complete loss of tyrosine-binding capabilities. To study the impact of substitutions in the catalytic center to the evolution of the whole domain, we examined the evolutionary rates for each individual site and compared them between the classes. This analysis revealed a release of evolutionary constraint for multiple sites surrounding the catalytic center only in the second class, emphasizing its difference in function compared with the first class. Furthermore, we found a region of higher conservation common to both domain classes, suggesting a new regulatory center. We discuss the influence of evolutionary forces on the development of the phosphatase domain, which has led to additional functions, such as the specific protection of phosphorylated tyrosine residues, substrate recruitment, and regulation of the catalytic activity of adjacent domains.     

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.     

Computational analysis of protein tyrosine phosphatases: practical guide to bioinformatics and data resources

The exponential growth of sequence data has become a challenge to database curators and end-users alike and biologists seeking to utilize the data effectively are faced with numerous analysis methods. Here, with practical examples from our bioinformatics analysis of the protein tyrosine phosphatases (PTPs), we show how computational analysis can be exploited to fuel hypothesis-driven experimental research through the exploration of online databases. We cover the following elements: (i) similarity searches and strategies to collect a non-redundant database of tyrosine-specific PTP domains; (ii) utilization of this database to classify human, fly, and worm PTPs (based on alignments and phylogenetic analysis); (iii) three-dimensional structural analysis to identify conserved regions (structure-function) and non-conserved selectivity-determining regions (substrate specificity); and (iv) genomic analysis, including mapping of exon structure, identification of pseudogenes, and exploration of disease databases. We discuss the importance of manual curation, illustrating examples in which pseudogenes give rise to predicted proteins in GenBank and note that domain servers, such as PFAM and SMART, erroneously include dual-specificity and lipid phosphatases in their collection of tyrosine-specific PTPs. To capitalize on our annotated set of 402 PTP domains (from 47 species and five phyla), we identify sequence conservation across taxonomic categories and explore structure-function relationships among tandem domain receptor-like PTPs. We define three Src homology 2 domain-containing PTP genes in stingray, zebrafish, and fugu and speculate on their evolutionary relationship with human pseudogenes. Our annotated sequences, along with a web service for phylogenetic classification of PTP domains, are available online (http://ptp.cshl.edu and http://science.novonordisk.com/ptp).     

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.     

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA

Myosin VIIs provide motor function for a wide range of eukaryotic processes. We demonstrate that mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. The ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult "escapers" that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. We generate a homology model for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, we establish that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the approximately 100 and approximately 75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, our data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability.     

The molecular details of WPD-loop movement differ in the protein-tyrosine phosphatases YopH and PTP1B

The movement of a conserved protein loop (the WPD-loop) is important in catalysis by protein tyrosine phosphatases (PTPs). Using kinetics, isotope effects, and X-ray crystallography, the different effects arising from mutation of the conserved tryptophan in the WPD-loop were compared in two PTPs, the human PTP1B, and the bacterial YopH from Yersinia. Mutation of the conserved tryptophan in the WPD-loop to phenylalanine has a negligible effect on k(cat) in PTP1B and full loop movement is maintained. In contrast, the corresponding mutation in YopH reduces k(cat) by two orders of magnitude and the WPD loop locks in an intermediate position, disabling general acid catalysis. During loop movement the indole moiety of the WPD-loop tryptophan moves in opposite directions in the two enzymes. Comparisons of mammalian and bacterial PTPs reveal differences in the residues forming the hydrophobic pocket surrounding the conserved tryptophan. Thus, although WPD-loop movement is a conserved feature in PTPs, differences exist in the molecular details, and in the tolerance to mutation, in PTP1B compared to YopH. Despite high structural similarity of the active sites in both WPD-loop open and closed conformations, differences are identified in the molecular details associated with loop movement in PTPs from different organisms.     

Reversible oxidation of the membrane distal domain of receptor PTPalpha is mediated by a cyclic sulfenamide

Protein tyrosine phosphatases (PTPs) are fundamental to the regulation of cellular signalling cascades triggered by protein tyrosine kinases. Most receptor-like PTPs (RPTPs) comprise two tandem PTP domains, with only the membrane proximal domains (D1) having significant phosphatase activity; the membrane distal domains (D2) display little to no catalytic activity. Intriguingly, however, many RPTP D2s share the catalytically essential Cys and Arg residues of D1s. D2 of RPTPalpha may function as a redox sensor that mediates regulation of D1 via reactive oxygen species. Oxidation of Cys723 of RPTPalpha D2 (equivalent to PTP catalytic Cys residues) stabilizes RPTPalpha dimers, induces rotational coupling, and is required for inactivation of D1 phosphatase activity. Here, we investigated the structural consequences of RPTPalpha D2 oxidation. Exposure of RPTPalpha D2 to oxidants promotes formation of a cyclic sulfenamide species, a reversibly oxidized state of Cys723, accompanied by conformational changes of the D2 catalytic site. The cyclic sulfenamide is highly resistant to terminal oxidation to sulfinic and sulfonic acids. Conformational changes associated with RPTPalpha D2 oxidation have implications for RPTPalpha quaternary structure and allosteric regulation of D1 phosphatase activity.     

Inter-domain interactions influence the stability and catalytic activity of the bi-domain protein tyrosine phosphatase PTP99A

The two protein tyrosine phosphatase (PTP) domains in bi-domain PTPs share high sequence and structural similarity. However, only one of the two PTP domains is catalytically active. Here we describe biochemical studies on the two tandem PTP domains of the bi-domain PTP, PTP99A. Phosphatase activity, monitored using small molecule as well as peptide substrates, revealed that the inactive (D2) domain activates the catalytic (D1) domain. Thermodynamic measurements suggest that the inactive D2 domain stabilizes the bi-domain (D1-D2) protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by few interactions at the inter-domain interface. In particular, mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent location to the so-called allosteric site of the canonical single domain PTP, PTP1B. These observations suggest functional optimization in bi-domain PTPs whereby the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme.     

Dock/Nck facilitates PTP61F/PTP1B regulation of insulin signalling

PTP1B (protein tyrosine phosphatase 1B) is a negative regulator of IR (insulin receptor) activation and glucose homoeostasis, but the precise molecular mechanisms governing PTP1B substrate selectivity and the regulation of insulin signalling remain unclear. In the present study we have taken advantage of Drosophila as a model organism to establish the role of the SH3 (Src homology 3)/SH2 adaptor protein Dock (Dreadlocks) and its mammalian counterpart Nck in IR regulation by PTPs. We demonstrate that the PTP1B orthologue PTP61F dephosphorylates the Drosophila IR in S2 cells in vitro and attenuates IR-induced eye overgrowth in vivo. Our studies indicate that Dock forms a stable complex with PTP61F and that Dock/PTP61F associate with the IR in response to insulin. We report that Dock is required for effective IR dephosphorylation and inactivation by PTP61F in vitro and in vivo. Furthermore, we demonstrate that Nck interacts with PTP1B and that the Nck/PTP1B complex inducibly associates with the IR for the attenuation of IR activation in mammalian cells. Our studies reveal for the first time that the adaptor protein Dock/Nck attenuates insulin signalling by recruiting PTP61F/PTP1B to its substrate, the IR.     

Opsin of Calliphora peripheral photoreceptors R1-6. Homology with Drosophila Rh1 and posttranslational processing.

The rhodopsin and metarhodopsin states of two very distantly related fly species (Calliphora and Drosophila) are found to exhibit no species-specific differences in their absorbance spectra. Isolation and characterization of cDNAs encoding the major opsin of Calliphora reveal a high (86%) degree of amino acid identity with the corresponding Drosophila visual pigment. Completely conserved is the third cytoplasmic loop which displays the major structural differences with the vertebrate photopigments. Other conserved motifs are six potential phosphorylation sites in the C-terminal region of the molecule and two potential glycosylation sites in the extracellular domains at positions Asn18 and Asn194, respectively. Interestingly, unlike vertebrate visual pigments, only newly synthesized fly opsin is N-glycosylated, while the mature protein is not. The conserved structure of the cytoplasmic loops suggests that the molecular mechanism for the activation of the transduction cascade is precisely the same in Drosophila and in Calliphora. Thus, data obtained by investigating the biochemistry of rhodopsin-related processes in larger flies may be integrated with the results of genetic experiments in Drosophila into a common model of invertebrate phototransduction.     

Differential oxidation of protein-tyrosine phosphatases

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPalpha-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPalpha-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPalpha-D1 and RPTPalpha-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPmu-D1, with a similar conformation as RPTPalpha-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H(2)O(2) concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.     

Steric hindrance as a basis for structure-based design of selective inhibitors of protein-tyrosine phosphatases.

Utilizing structure-based design, we have previously demonstrated that it is possible to obtain selective inhibitors of protein-tyrosine phosphatase 1B (PTP1B). A basic nitrogen was introduced into a general PTP inhibitor to form a salt bridge to Asp48 in PTP1B and simultaneously cause repulsion in PTPs containing an asparagine in the equivalent position [Iversen, L. F., et al. (2000) J. Biol. Chem. 275, 10300-10307]. Further, we have recently demonstrated that Gly259 in PTP1B forms the bottom of a gateway that allows easy access to the active site for a broad range of substrates, while bulky residues in the same position in other PTPs cause steric hindrance and reduced substrate recognition capacity [Peters, G. H., et al. (2000) J. Biol. Chem. 275, 18201-18209]. The current study was undertaken to investigate the feasibility of structure-based design, utilizing these differences in accessibility to the active site among various PTPs. We show that a general, low-molecular weight PTP inhibitor can be developed into a highly selective inhibitor for PTP1B and TC-PTP by introducing a substituent, which is designed to address the region around residues 258 and 259. Detailed enzyme kinetic analysis with a set of wild-type and mutant PTPs, X-ray protein crystallography, and molecular modeling studies confirmed that selectivity for PTP1B and TC-PTP was achieved due to steric hindrance imposed by bulky position 259 residues in other PTPs.     

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.     

A gatekeeper residue for inhibitor sensitization of protein tyrosine phosphatases

Allele-specific enzyme inhibitors are powerful tools in chemical biology. However, few general approaches for the discovery of such inhibitors have been described. Herein is reported a method for the sensitization of protein tyrosine phosphatases (PTPs) to small-molecule inhibition. It is shown that mutation of an active-site isoleucine to alanine (I219A) sensitizes PTP1B to inhibition by a class of thiophene-based inhibitors. This sensitization strategy succeeds for both 'orthogonal' inhibitors, designed to be incompatible with wild-type PTP active sites, and previously optimized wild-type PTP inhibitors. The finding that the I219A mutation sensitizes phosphatase domains to a variety of compounds suggests that isoleucine 219 may act as a 'gatekeeper' residue that can be widely exploited for the chemical-genetic analysis of PTP function.     

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.     

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.