The solution structure of Escherichia coli Wzb reveals a novel substrate recognition mechanism of prokaryotic low molecular weight protein-tyrosine phosphatases

Low molecular weight protein-tyrosine phosphatases (LMW-PTPs) are small enzymes that ubiquitously exist in various organisms and play important roles in many biological processes. In Escherichia coli, the LMW-PTP Wzb dephosphorylates the autokinase Wzc, and the Wzc/Wzb pair regulates colanic acid production. However, the substrate recognition mechanism of Wzb is still poorly understood thus far. To elucidate the molecular basis of the catalytic mechanism, we have determined the solution structure of Wzb at high resolution by NMR spectroscopy. The Wzb structure highly resembles that of the typical LMW-PTP fold, suggesting that Wzb may adopt a similar catalytic mechanism with other LMW-PTPs. Nevertheless, in comparison with eukaryotic LMW-PTPs, the absence of an aromatic amino acid at the bottom of the active site significantly alters the molecular surface and implicates Wzb may adopt a novel substrate recognition mechanism. Furthermore, a structure-based multiple sequence alignment suggests that a class of the prokaryotic LMW-PTPs may share a similar substrate recognition mechanism with Wzb. The current studies provide the structural basis for rational drug design against the pathogenic bacteria.     

Low molecular weight protein tyrosine phosphatase and caveolin-1: interaction and isoenzyme-dependent regulation

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are small enzymes that are ubiquitous in many organisms. They are important in biological processes such as cell proliferation, adhesion, migration, and invasiveness. LMW-PTP is expressed in mammalian cells as two isoforms (IF1 and IF2) originating through alternative splicing. We have previously shown that IF2 targets lipid rafts called caveolae and interacts with caveolin-1, their major structural protein. Caveolae are cholesterol- and sphingolipid-rich membrane microdomains that have been implicated in a variety of cellular functions, including signal transduction events. Caveolin-1 contains a scaffolding region that contributes to the binding of the protein to the plasma membrane and mediates protein omo- and etero-oligomerization. Interaction of many signaling molecules with the scaffolding domain sequesters them into caveolae and inhibits or suppresses their activities. Caveolin-interacting proteins usually have a typical sequence motif, also present in all the LMW-PTPs, which is characterized by aromatic or large hydrophobic residues in specific positions. We have examined here the interaction of the LMW-PTP isoforms with caveolin-1 and its molecular mechanism, together with the consequences for their tyrosine phosphatase activities. We found that IF1 and IF2 are both capable of interacting with defined regions of caveolin-1 and that their putative caveolin binding sequence motif is not responsible for the association. The formation of LMW-PTP/caveolin-1 complexes is accompanied by modulation of the enzyme activities, and the inhibitory effect elicited against IF1 is stronger than that against IF2. The caveolin scaffolding domain is directly involved in the observed phenomena.     

The Apo-structure of the Low Molecular Weight Protein-tyrosine Phosphatase A (MptpA) from Mycobacterium tuberculosis Allows for Better Target-specific Drug Development

Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX₅R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ∼10 Å, from an “open” to a “closed” conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.     

Synthesis,activity and molecular modeling of a new series of chromones as low molecular weight protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTP) are crucial elements in eukaryotic signal transduction. Several reports suggested that the LMW-PTP family has oncogenic relevance. Moreover, LMW-PTP has been recognized as a negative regulator of insulin-mediated mitotic and metabolic signaling. Thus, inhibition of the LMW-PTP can be considered an attractive approach for the design of new therapeutic agents for the treatment of type II diabetes and for new antitumoral drugs. To date very few (and weak) inhibitors of LMW-PTP have been identified. On the basis of the reported weak activity of some flavonoids on phosphatases, we discovered a lead that originated a new class of highly active LMW-PTP inhibitors; these compounds inhibit also PTP-1B and are active in cellular assays. Docking experiments and SAR highlighted the possible binding mode of these compounds to the enzyme, putting the background for the future optimization of their inhibitory activity and selectivity towards the closely related enzyme PTP-1B.     

The Drosophila primo locus encodes two low-molecular-weight tyrosine phosphatases

The fine modulation of tyrosine phosphorylation by protein tyrosine phosphatases and protein tyrosine kinases is a key regulatory mechanism for many cell signaling pathways active during development. In a screen for genes with interesting expression patterns in the developing Drosophila pupal retina, we identified a novel pair of protein tyrosine phosphatases that exhibit an expression pattern suggesting a role in multiple steps of Drosophila neurogenesis. Together, these phosphatases define the primo locus. Their sequence is approx. 50% identical to each other and to low-molecular-weight protein tyrosine phosphatases (LMW-PTPs) identified in other species. Little is understood of the biological role of LMW-PTPs, and the powerful tools available in Drosophila should provide important insight into their role in signaling and development.     

Insight into the role of low molecular weight phosphotyrosine phosphatase (LMW-PTP) on platelet-derived growth factor receptor (PDGF-r) signaling. LMW-PTP controls PDGF-r kinase activity through TYR-857 dephosphorylation

Low molecular weight phosphotyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor-induced mitogenesis and cytoskeleton rearrangement. Our previous results demonstrated that LMW-PTP is able to bind and dephosphorylate activated platelet-derived growth factor receptor (PDGF-r), thus inhibiting cell proliferation. Here we revisit the role of LMW-PTP on activated PDGF-r dephosphorylation. We demonstrate that LMW-PTP preferentially acts on cell surface PDGF-r, excluding the internalized activated receptor pool. Many phosphotyrosine phosphatases act by site-selective dephosphorylation on several sites of PDGF-r, but until now, there has been no evidence of a direct involvement of a specific phosphotyrosine phosphatase in the dephosphorylation of the 857 kinase domain activation tyrosine. Here we report that LMW-PTP affects the kinase activity of the receptor through the binding and dephosphorylation of Tyr-857 and influences many of the signal outputs from the receptor. In particular, we demonstrate a down-regulation of phosphatidylinositol 3-kinase, Src homology phosphatase-2, and phospholipase C-gamma1 binding but not of MAPK activation. In addition, we report a slight action of LMW-PTP on Tyr-716, which directs MAPK activation through Grb2 binding. On the basis of these results, we propose a key role for LMW-PTP in PDGF-r down-regulation through the dephosphorylation of the activation loop Tyr-857, thus determining a general negative regulation of all downstream signals, with the exception of those elicited by internalized receptors.     

Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation

Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2.     

Tyrosine phosphorylation regulates alpha II spectrin cleavage by calpain

Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of alpha II-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The alpha II-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the alpha I-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in alpha II-spectrin. Western blotting showed that alpha II-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of alpha II-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.     

Many fructosamine 3-kinase homologues in bacteria are ribulosamine/erythrulosamine 3-kinases potentially involved in protein deglycation

The purpose of this work was to identify the function of bacterial homologues of fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate.     

Inhibitors of the Yersinia protein tyrosine phosphatase through high throughput and virtual screening approaches

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design.     

Up-regulated expression of low molecular weight protein tyrosine phosphatases in different human cancers

Protein tyrosine phosphorylation, mediated by the balanced action of tyrosine kinases and phosphatases, contributes to the regulation of the growth, migration, and invasion of normal and malignant cells. Among tyrosine phosphatases, low molecular weight protein tyrosine phosphatases (LMW-PTP) have been recognized as a possible "positive factor" in tumour onset and progression. The aim of this work was to assess whether LMW-PTP are differentially expressed in normal and malignant tissues. Using real-time PCR analysis we evaluated the expression levels of total LMW-PTP mRNA in surgical samples of breast, colon and lung cancers (63, 60, and 58, respectively), and in their paired adjacent not affected tissues. Moreover, the same analysis was carried out on a group of neuroblastomas (25 cases). Significant correlations between LMW-PTP overexpression and the most common clinical-pathological features of cancers exist. In colon cancer and neuroblastoma increased total LMW-PTP mRNA expression correlates with unfavourable outcome. While LMW-PTP mRNA expression increases in tumour samples, the relative contribution of the different isoforms does not change. Our findings indicate that LMW-PTP can be considered an oncogene as it is overexpressed in different tumour types and suggests that LMW-PTP enhanced expression is generally prognostic for a more aggressive cancer.     

5-Arylidene-2,4-thiazolidinediones as inhibitors of protein tyrosine phosphatases

4-(5-Arylidene-2,4-dioxothiazolidin-3-yl)methylbenzoic acids (2) were synthesized and evaluated in vitro as inhibitors of PTP1B and LMW-PTP, two protein tyrosine phosphatases (PTPs) which act as negative regulators of the metabolic and mitotic signalling of insulin. The synthesis of compounds 2 represents an example of utilizing phosphotyrosine-mimetics to identify effective low molecular weight nonphosphorus inhibitors of PTPs. Several thiazolidinediones 2 exhibited PTP1B inhibitory activity in the low micromolar range with moderate selectivity for human PTP1B and IF1 isoform of human LMW-PTP compared with other related PTPs.     

Identification of a novel phosphatase sequence motif.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structure-function analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.     

Solution structure of the MAPK phosphatase PAC-1 catalytic domain. Insights into substrate-induced enzymatic activation of MKP

Inactivation of mitogen-activated protein kinases (MAPKs) by MAPK phosphatases (MKPs) is accomplished via substrate-induced activation of the latter enzymes; however, the structural basis for the underlying mechanism remains elusive. Here, we report the three-dimensional solution structure of the C-terminal phosphatase domain of the prototypical MKP PAC-1, determined when bound to phosphate. Structural and biochemical analyses reveal unique active site geometry of the enzyme important for binding to phosphorylated threonine and tyrosine of MAPK ERK2. Our study further demonstrates that the dynamic interaction between the N-terminal kinase binding domain and the C-terminal phosphatase domain of an MKP is directly coupled to MAPK-induced conformational change of the phosphatase active site, which is essential for eliciting its full enzymatic activity.     

Analyzing protein tyrosine phosphatases by phosphotyrosine analog integration

Reversible protein phosphorylation plays a central role in cellular signal transduction and is a focus of biomedical studies. However, it is a challenging task to study the effects of protein phosphorylation in the presence of protein phosphatase activities, especially for protein tyrosine phosphatases SHP1, SHP2 and LMW-PTP, which are themselves regulated by protein tyrosine phosphorylation. Expressed protein ligation, by combining chemical peptide synthesis with recombinant protein expression, allows for site-specific unnatural modifications of semisynthetic proteins. In this review, we describe how semisynthetic proteins were prepared to incorporate nonhydrolyzable phosphotyrosine analogs, and utilized in combination with site-directed mutagenesis and other means to elucidate regulatory mechanisms of protein tyrosine phosphatases.     

Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes

Listeria monocytogenes is a pathogen that causes listeriosis, a severe food-borne infection. This bacterium, in order to survive and grow in the multiple conditions encountered in the host and the environment, has evolved a large number of regulatory elements, in particular many signal transduction systems based on reversible phosphorylation. The genome sequence has revealed genes for 16 putative two-component systems, four putative tyrosine phosphatases, three putative serine-threonine kinases and two putative serine-threonine phosphatases. We found that one of the latter genes, stp, encodes a functional Mn(2+)-dependent serine-threonine phosphatase similar to PPM eukaryotic phosphatases (Mg(2+)-or Mn(2+)-dependent protein phosphatase) and is required for growth of L. monocytogenes in a murine model of infection. We identified as the first target for Stp, the elongation factor EF-Tu. Post-translational phosphorylation of EF-Tu had been shown to prevent its binding to amino-acylated transfer RNA as well as to kirromycin, an antibiotic known to inhibit EF-Tu function. Accordingly, an stp deletion mutant is less sensitive to kirromycin. These results suggest an important role for Stp in regulating EF-Tu and controlling bacterial survival in the infected host.     

Protein phosphorylation on tyrosine in bacteria

Protein phosphorylation on tyrosine has been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein-tyrosine kinases that exhibit structural and functional features similar, but not identical, to those of their eukaryotic counterparts. The reversibility of the reaction is effected by two main classes of protein-tyrosine phosphatases: one includes conventional eukaryotic-like phosphatases and dual-specific phosphatases, and the other comprises acidic phosphatases of low molecular weight. Less frequently, a third class concerns enzymes of the polymerase-histidinol phosphatase type. In terms of genomic organization, the genes encoding a protein-tyrosine phosphatase and a protein-tyrosine kinase in a bacterial species are most often located next to each other on the chromosome. In addition, these genes are generally part of large operons that direct the coordinate synthesis of proteins involved in the production or regulation of exopolysaccharides and capsular polysaccharides. Recent data provide evidence that there exists a direct relationship between the reversible phosphorylation of proteins on tyrosine and the production of these polysaccharidic polymers, which are also known to be important virulence factors. Therefore, a new concept has emerged suggesting the existence of a biological link between protein-tyrosine phosphorylation and bacterial pathogenicity.     

Solution structure of the low-molecular-weight protein tyrosine phosphatase from Tritrichomonas foetus reveals a flexible phosphate binding loop

Eukaryotic low-molecular-weight protein tyrosine phosphatases (LMW PTPs) contain a conserved serine, a histidine with an elevated pKa, and an active site asparagine that together form a highly conserved hydrogen bonding network. This network stabilizes the active site phosphate binding loop for optimal substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus (TPTP), both the conserved serine (S37) and asparagine (N14) are present, but the conserved histidine has been replaced by a glutamine residue (Q67). Site-directed mutagenesis, kinetic, and spectroscopic experiments suggest that Q67 is located near the active site and is important for optimal catalytic activity. Kinetic experiments also suggest that S37 participates in the active site/hydrogen bonding network. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structure of the TPTP enzyme and to further examine the roles of S37 and Q67. The backbone conformation of the TPTP phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases, with N14 existing in a strained, left-handed conformation that is a hallmark of the active site hydrogen bonding network in the LMW PTPs. As expected, both S37 and Q67 are located at the active site, but in the consensus structure they are not within hydrogen bonding distance of N14. The hydrogen bond interactions that are observed in X-ray structures of LMW PTPs may in fact be transient in solution. Protein dynamics within the active site hydrogen bonding network appear to be affected by the presence of substrate or bound inhibitors such as inorganic phosphate.     

3-D Model of the bee venom acid phosphatase: Insights into allergenicity

The acid phosphatase Api m 3 is the major allergen of the honeybee venom. Except for the amino acid sequence, no other structural information for the enzyme is available. We applied homology modeling to assign the three-dimensional structure of Api m 3. The structure of the homodimeric human prostatic acid phosphatase was used to model the Api m 3 tertiary structure. IgE epitopes and antigenic sites were predicted using programs based on the structure of known epitopes and analysis of the 3-D model. The model of Api m 3 revealed an active site similar to those of the histidine—type acid phosphatases with conservation of the catalytically important residues. The observed substitutions in the phosphate ion binding site suggest differences in the substrate specificity in comparison to other acid phosphatases. The analysis of the Api m 3 three-dimensional model revealed a very likely mechanism of enzyme action.     

The structural basis for the selectivity of benzotriazole inhibitors of PTP1B

Protein tyrosine phosphatase 1B (PTP1B) has been implicated in the regulation of the insulin signaling pathway and represents an attractive target for the design of inhibitors in the treatment of type 2 diabetes and obesity. Inspection of the structure of PTP1B indicates that potent PTP1B inhibitors may be obtained by targeting a secondary aryl phosphate-binding site as well as the catalytic site. We report here the crystal structures of PTP1B in complex with first and second generation aryldifluoromethyl-phosphonic acid inhibitors. While all compounds bind in a previously unexploited binding pocket near the primary binding site, the second generation compounds also reach into the secondary binding site, and exhibit moderate selectivity for PTP1B over the closely related T-cell phosphatase. The molecular basis for the selectivity has been confirmed by single point mutation at position 52, where the two phosphatases differ by a phenylalanine-to-tyrosine switch. These compounds present a novel platform for the development of potent and selective PTP1B inhibitors.