The most significant trait of the protein tyrosine phosphatase (PTP) superfamily is conservation of the signature motif CX 5 R, which forms the phosphate-binding loop in the active site (known as the P-loop or PTPloop). Despite relatively large sequence variations in the X 5 segment, the conformation of the P-loop is strictly conserved and can be easily superimposed from different PTP structures, with minor deviations in the Ca tracing (< 1 Å ). This structurally conserved arrangement ensures that the catalytic Cys, the nucleophile in catalysis, and the Arg, involved in phosphate binding, remain in close proximity and form a cradle to hold the phosphate group of the substrate in place for nucleophilic attack. The cysteine Sc-atom is the nucleophile that attacks the substrate phosphorus atom leading to the cysteinyl-phosphate reaction intermediate. The arginine is involved both in substrate binding and in stabilization of the reaction intermediate [1]. Further to this, the amide groups in the P-loop point towards the interior of the cradle and form a network of hydrogen bonds to the phosphate oxygens (Fig. 1A). A conserved Ser ⁄ Thr residue in the P-loop has been proposed to play an important role in the stabilization of the thiolate group in the transition state facilitating the breakdown of the phosphoenzyme intermediate [2] (Scheme 1).The catalytic mechanism of PTP reaction requires the participation of a general acid and a general base. 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 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, 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.Abbreviations KIM, kinase interaction motif; LMW-PTP, low molecular weight protein tyrosine phosphatase; N-SH2, N-terminal SH2 domain; PTP, protein tyrosine phosphatase; RPTP, receptor protein tyrosine phosphatase;...

Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.

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The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTP belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTP. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTP ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTP ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTP at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.The phosphorylation state of numerous signaling proteins is controlled by opposing activities of protein-tyrosine kinases and protein-tyrosine phosphatases (PTP) 1 (1). The family of PTPs consists of soluble and receptor-like PTPs (RPTPs) (2). Whereas the intracellular region of RPTPs is relatively similar in all representatives containing either a single or two PTP domains, the extracellular region has a large diversity. PTP belongs to subclass IIB, called "MAM-containing PTP" (2). Besides the MAM domain (meprin/A5-protein/PTPmu domain; Ref.3), their extracellular region contains a single immunoglobulin (Ig)-like domain and four fibronectin (FN) III repeats (4). This structural architecture of ectodomain is similar to members of the cell-adhesion molecule superfamily.PTP is strongly expressed in the endothelial cell layer of the arteries and continuous capillaries as well as in cardiac muscle, bronchial and lung epithelia, retina, and several brain areas (4 -6). At the subcellular level, it is localized at sites of cell-cell contact (7). In this regard, it has been demonstrated that PTP restores E-cadherin-mediated cellular adhesion, when it is expressed in LNCaP human prostate carcinoma cells (8). Physiologically, PTP has been shown to be involved in promotion and regulation of neurite outgrowth (5, 9).Numerous experiments have clearly demonstrated that the extracellular region of PTP promotes cell-cell aggregation in a Ca 2ϩ -independent manner (10, 11). The homophilic binding has been also evidenced in the ectodomains of PTP (12) and PTP (13), strongly suggesting that these RPTPs may be involved in signal transduction through cell-to-cell contact in vivo. Evidence concerning the physiological role of PTP-mediated homophilic binding has been reported in a recent article (14) showing that homophilic interactions trigger rearrangements of the axonal growth cone. However, the molecular mechanism of this interaction remains larg...

Which substrate will it be? Phosphotyrosine peptide microarrays have allowed the substrate specificity to be mapped for two prototypical protein‐tyrosine phosphatases (PTPs): PTP1B and PTPμ. The knowledge gained was used for molecular docking studies (see picture: the docking of a peptide in the binding pocket of PTPμ) and the design of an inhibitor for PTPμ.

Protein phosphorylation on tyrosine residues is tightly controlled by protein tyrosine phosphatases (PTPs) at multiple levels: spatio-temporal expression, subcellular localization and post-translational modification. Structural and functional analysis of the PTP domains has provided insight into catalysis and regulatory mechanisms that control the enzymatic activity. Understanding the molecular basis of PTP regulation is of fundamental importance to dissect the pleiotropic effect of these enzymes in both health and disease. Here, we review recent insights into the regulation of receptorlike PTPs by extracellular ligands and into regulation by reversible oxidation that impairs catalysis directly. The physiological roles of PTPs are essential in homeostasis in eukaryotic cells and pertubation of their functional attributes causes different disease states. As an example, we discuss recent findings indicating how inappropriate oxidation of PTPs in cancer cells may contribute to cell transformation. On the other hand, PTPs from many pathogens are key virulence factors and manipulate signalling pathways in the host cells to promote invasion and survival of the microorganisms. This research area has received relatively little attention but has advanced remarkably. We review the structural features of pathogenic PTPs, their similarities and differences with eukaryotic PTPs, and the possible exploitation of this knowledge for therapeutic intervention. Structures of PTPsAnalysis of protein tyrosine phosphatase (PTP) structures is not only important for understanding their function and regulation but also for identifying strategies for pharmacological modulation of PTP activity. Given the progress in establishing PTP deregulation in different pathologies such as cancer, PTPs deserve much attention as candidate drug targets. We focus on Cys-based PTPs [1] and discuss novel insights in PTP structure which may be important for pharmacological modulation, and emphasize novel findings and controversial issues for transmembrane (receptor-like) PTPs (RPTPs).Abbreviations AML, acute myeloid leukaemia; DUSP, dual specificity PTP; FLT3, Fms-like tyrosine kinase 3; FLT3 ⁄ ITD, internal tandem duplication of FLT3; LMWPTP, low molecular weight PTP; PDGF, platelet-defined growth factor; Prx, peroxiredoxin; PTP, protein tyrosine phosphatase; PTPLP, PTP-like phytase; ROS, reactive oxygen species; RPTP, transmembrane PTP; RTK, receptor tyrosine kinase; YopH, Yersinia outer protein H.

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In the present study, lecithin-cholesterol acyltransferase (LCAT) catalyzed esterification of oxysterols was investigated by using discoidal bilayer particles (DBP) containing various oxysterols, phosphatidylcholines, and apolipoprotein A-I. The esterified oxysterols were analyzed by high pressure liquid chromatography, gas chromatography, and mass spectrometry. LCAT esterified all oxysterols tested that are known to be present in human plasma. The esterification yields in almost all cases were relatively high, often as high as the yield of cholesterol esterification. When DBP preparations containing 27-hydroxycholesterol and various phosphatidylcholines were used for the LCAT reaction, both monoesters and diesters were produced. The mass spectrometry analysis showed that the monoester was produced by the esterification of the 3 beta-hydroxyl group and not the 27-hydroxyl group. The diesters were apparently produced by the esterification of the 27-hydroxyl group only after the esterification of the 3 beta-hydroxyl group. Phosphatidylcholine containing a saturated acyl group at sn-1 position and an unsaturated acyl group at sn-2 position gave generally high esterification yield. The esterification of various oxysterols was compared by using DBP containing dioleoyl-phosphatidylcholine and individual oxysterols. All oxysterols produced 3 beta-oleoyl monoesters. Unlike 27-hydroxycholesterol, 25-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, or cholestanetriol did not produce diesters. Various factors influencing the formation of the monoesters and diesters from 27-hydroxycholesterol were investigated. When dioleoyl-phosphatidylcholine was used as the acyl donor, prolonged dialysis of DBP preparations and increase in the ratio of the enzyme concentration to substrate particle concentration increased the diester formation. Significant amounts of diesters were also produced by using 1-palmitoyl-2-oleoyl-phosphatidylcholine and other phosphatidylcholines as the acyl donors. By analyzing the conditions of monoester and diester formation, a scheme for the LCAT reaction pathway was proposed.