Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling.

Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this signalling pathway. Specifically, recent evidence has suggested that PTP1B might be a key regulator of leptin signalling, based on the resistance to diet-induced obesity and increased leptin signalling observed in PTP1B-deficient mice. The present study was undertaken to investigate the mechanism by which PTP1B mediates the cessation of the leptin signal transduction. Leptin-induced activation of a STAT3 responsive reporter was dose-dependently inhibited by co-transfection with PTP1B. No inhibition was observed when a catalytically inactive mutant of PTP1B was used or when other PTPs were co-transfected. PTP1B was able to dephosphorylate activated JAK2 and STAT3 in vitro, whereas either no or a minimal effect was observed with cluster of differentiation 45 (CD45), PTPalpha and leukocyte antigen-related (LAR). By utilisation of a selective PTP1B inhibitor, the leptin-induced STAT3 activation was enhanced in cells. In conclusion, these results suggested that the negative regulatory role of PTP1B on leptin signalling is mediated through a direct and selective dephosphorylation of the two signalling molecules, JAK2 and STAT3.

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.

Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function.

Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B.

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.

Residue 259 is a key determinant of substrate specificity of protein-tyrosine phosphatases 1B and alpha.

The aim of this study was to define the structural elements that determine the differences in substrate recognition capacity of two protein-tyrosine phosphatases (PTPs), PTP1B and PTPalpha, both suggested to be negative regulators of insulin signaling. Since the Ac-DADE(pY)L-NH(2) peptide is well recognized by PTP1B, but less efficiently by PTPalpha, it was chosen as a tool for these analyses. Calpha regiovariation analyses and primary sequence alignments indicate that residues 47, 48, 258, and 259 (PTP1B numbering) define a selectivity-determining region. By analyzing a set of DADE(pY)L analogs with a series of PTP mutants in which these four residues were exchanged between PTP1B and PTPalpha, either in combination or alone, we here demonstrate that the key selectivity-determining residue is 259. In PTPalpha, this residue is a glutamine causing steric hindrance and in PTP1B a glycine allowing broad substrate recognition. Significantly, replacing Gln(259) with a glycine almost turns PTPalpha into a PTP1B-like enzyme. By using a novel set of PTP inhibitors and x-ray crystallography, we further provide evidence that Gln(259) in PTPalpha plays a dual role leading to restricted substrate recognition (directly via steric hindrance) and reduced catalytic activity (indirectly via Gln(262)). Both effects may indicate that PTPalpha regulates highly selective signal transduction processes.

2-(oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases.

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp(181), Lys(120), and Tyr(46). PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.

Protein tyrosine phosphatases (PTPs) as drug targets: inhibitors of PTP-1B for the treatment of diabetes.

The phosphorylation of key proteins on tyrosine residues is an important part of many different intracellular signaling cascade mechanisms triggered by hormones and other agents. The deactivation of such signaling processes is catalyzed by protein-tyrosine phosphatases (PTPs), and therefore inhibition of these enzymes is being explored in different indications as a means whereby signaling may be prolonged or even initiated in the absence of the triggering agent. In the case of the signaling cascade initiated by the activation of the insulin receptor, an important gene knockout study in mice has identified PTP-1B as a potential target for anti-diabetes therapy, and has thus made it a focus of attention for several groups. Recent advances in the structure-based design of potent and selective inhibitors of this enzyme are described, as well as some preliminary data for such inhibitors in animal models which, together with more recently published data from further studies on PTP-1B knockout mice and from antisense studies, illustrate the potential of this approach for the treatment of both Type I and Type II diabetes.

Protein-tyrosine phosphatase alpha regulates Src family kinases and alters cell-substratum adhesion.

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.

Mutant forms of the protein tyrosine phosphatase alpha show differential activities towards intracellular substrates.

BHK cells overexpressing five million IR (BHK-IR) respond to insulin with reduced growth and detachment from the dish surface. We have recently identified protein tyrosine phosphatase (PTP) alpha as a negative regulator of the insulin receptor (IR) tyrosine kinase that is able to rescue BHK-IR cells from the insulin effect. In this report we describe the effect of several point mutations in PTP alpha on the phosphatase activity and regulation of insulin signaling in BHK-IR cells. Analysis of total cellular phosphotyrosine protein revealed several molecules that were dephosphorylated when PTP alpha or a phosphatase active mutant was overexpressed. By contrast, some proteins were tyrosine phosphorylated as strong or to an even higher extent as in the parental line when PTP alpha Y798F was present. We conclude that mutation of the carboxyterminal tyrosine in PTP alpha uncovers a dual function of this phosphatase in BHK cells: reduction of the IR signal and activation of an endogenous kinase.

Expression of protein-tyrosine phosphatases in the major insulin target tissues.

Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semiquantitative measure of the expression levels of selected PTPs. Surprisingly, PTP-LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor-type PTP-sigma and PTP-alpha were expressed in relatively high levels in all four tissues. The low levels of LAR PTP mRNA in skeletal muscle were further confirmed by Northern blot analysis.

The transmembrane protein tyrosine phosphatase alpha dephosphorylates the insulin receptor in intact cells.

Protein tyrosine phosphatases (PTPs) are key regulators in a variety of signal transduction processes. However, substrates for most PTPs have not been determined. In a previous report, we demonstrated that in a transient expression system the intracellular phosphatases PTPs 1B and TC preferentially dephosphorylated the precursor form of several receptor tyrosine kinases. In this paper we show that the dephosphorylation of kinase precursors is a specific feature of PTPs 1B and TC that is not shared by two other intracellular PTPs, PTPH1 or SHP-1. By contrast, the receptor phosphatase PTP alpha preferentially dephosphorylated the beta-subunit of the insulin receptor localized on the cell surface. The insulin receptor was a better substrate for PTP alpha than for other receptor type PTPs. We conclude that the intracellular PTPs 1B and TC regulate the autophosphorylation of receptor tyrosine kinases during their posttranslational processing while receptor type PTPs regulate the mature, cell surface localized receptor tyrosine kinases.

ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation.

ERK6, a mitogen-activated protein (MAP) kinase-related serine/threonine kinase, is highly expressed in human skeletal muscle and appears to function as a signal transducer during differentiation of myoblasts to myotubes. In transfected 293 cells, activation of the 45-kDa enzyme results in tyrosine-phosphorylated 46- and 56-kDa forms, which phosphorylate myelin basic protein. Overexpression of wild-type ERK6 or the inactive mutant Y185F has no effect on fibroblast and myoblast proliferation, but it enhances or inhibits C2C12 cell differentiation to myotubes, respectively. Our findings suggest ERK6 to be a tissue-specific, differentiation signal-transducing factor that is connected to phosphotyrosine-mediated signaling pathways distinct from those activating other members of the MAP kinase family such as LRK1 and ERK2.

Selective down-regulation of the insulin receptor signal by protein-tyrosine phosphatases alpha and epsilon.

Binding of insulin to its receptor (IR) causes rapid autophosphorylation with concomitant activation of its tyrosine kinase which transmits the signal by phosphorylating cellular substrates. The IR activity is controlled by protein-tyrosine phosphatases, but those directly involved in regulating the insulin receptor and its signaling pathways have not yet been identified. Using baby hamster kidney cells overexpressing the IR and a novel insulin-based selection principle, we established stable cell lines with functionally coupled expression of the IR and protein-tyrosine phosphatases. The two closely related protein-tyrosine phosphatases alpha and epsilon were identified as negative regulators of IR tyrosine kinase.

Expression, purification and crystallization of human phosphotyrosine phosphatase 1B.

Protein phosphotyrosine phosphatases are believed to be involved in the regulation of the activity of cellular proteins, such as receptor tyrosine kinases, by controlling their phosphorylation status. One of the best described and characterized protein of this class of enzymes is the phosphotyrosine phosphatase 1B. To obtain sufficient quantities for structural investigations, truncated forms of PTP1B encompassing the catalytic domain were over-expressed in Escherichia coli and purified to apparent homogeneity by conventional chromatography. The activity of these purified enzymes has been compared with the wild-type enzyme expressed in mammalian cells. By measuring the activities against p-nitrophenyl phosphate, the pH dependence of this activity, and responses to different modulators, it could be demonstrated that the truncated forms of PTP1B retained the same characteristics as the full-length mammalian enzyme, but are not subject to inhibition of enzymic activity mediated by the C-terminus. Due to their improved solubility, it can be assumed that the catalytic domains are advantageous for crystallization studies in comparison to the natural enzyme. In a screening for crystallization conditions, we obtained protein crystals indicating that the quality of the purified protein is sufficient for crystallographic studies.

Src kinase associates with a member of a distinct subfamily of protein-tyrosine phosphatases containing an ezrin-like domain.

A 6.2-kb full-length clone encoding a distinct protein-tyrosine phosphatase (PTP; EC 3.1.3.48), PTPD1, was isolated from a human skeletal muscle cDNA library. The cDNA encodes a protein of 1174 amino acids with N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family, which also includes the two PTPs H1 and MEG1. The PTP domain is positioned in the extreme C-terminal part of PTPD1, and there is an intervening sequence of about 580 residues without any apparent homology to known proteins separating the ezrin-like and the PTP domains. Thus, PTPD1 and the closely related, partially characterized, PTPD2 belong to the same family as PTPH1 and PTPMEG1, but because of distinct features constitute a different PTP subfamily. Northern blot analyses indicate that PTPD1 and PTPD2 are expressed in a variety of tissues. In transient coexpression experiments PTPD1 was found to be efficiently phosphorylated by and associated with the src kinase pp60src.

Amino acid sequence of human pregnancy-associated plasma protein-A derived from cloned cDNA.

The amino acid sequence of human pregnancy-associated plasma protein-A (PAPP-A), a component of the circulating complex with the proform of eosinophil major basic protein (proMBP), has been determined from partial protein sequencing and from sequencing of cloned cDNA. The PAPP-A monomer contains 1547 amino acid residues, but is derived from a larger precursor of placental origin. PAPP-A contains 82 Cys residues, which are all bridged, 14 putative sites for N-glycosylation, and 7 putative sites for attachment of glycosaminoglycan groups. The C-terminal part of PAPP-A contains 5 approximately 60-residue motifs related to the short consensus repeats of complement proteins and selectins. The SCRs presently known can be grouped into three classes: complement-type, class I; selectin-type, class II; PAPP-A-type, class III. PAPP-A further contains three approximately 26-residue motifs, related to the lin-notch motifs of proteins regulating early tissue differentiation, and, in addition, a putative Zn2+ binding site similar to that found in many metalloproteinases has been identified. Apart from these features, the PAPP-A sequence is not related to other known protein sequences.

High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.

Examination of flk-1 receptor tyrosine kinase mRNA expression by in situ hybridization analysis revealed specific association with endothelial cells at all stages of mouse development, including the blood islands in the yolk sac of day 8.5-10.5 embryos, in which the early progenitors of this lineage originate. flk-1 transcripts were abundant in proliferating endothelial cells of vascular sprouts and branching vessels of embryonic and early postnatal brain, but were drastically reduced in adult brain, where proliferation has ceased. Identification of the angiogenic mitogen, vascular endothelial growth factor (VEGF), as the high affinity ligand of Flk-1 and correlation of the temporal and spatial expression pattern of Flk-1 and VEGF suggest a major role of this ligand-receptor signaling system in vasculogenesis and angiogenesis.

Identification of determinants that confer ligand specificity on the insulin receptor.

We have previously shown, using truncated soluble recombinant receptors, that substituting the 62 N-terminal amino acids of the alpha subunit from the insulin-like growth factor I receptor (IGFIR) with the corresponding 68 amino acids from the insulin receptor (IR) results in a chimeric receptor with an approximately 200-fold increase in affinity for insulin and only a 5-fold decrease in insulin-like growth factor I (IGFI) affinity (Kjeldsen, T., Andersen, A. S., Wiberg, F. C., Rasmussen, J. S., Schäffer, L., Balschmidt, P., Møller, K. B., and Møller, N. P. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408). We demonstrate that these 68 N-terminal amino acids of the IR also confer insulin affinity on the intact IGFI holoreceptor both in the membrane-bound state and when solubilized by Triton X-100. Furthermore, this domain can be subdivided into two regions (amino acids 1-27 and 28-68 of the IR alpha subunit) that, when replacing the corresponding IGFIR sequences, increases the insulin affinity of truncated soluble receptor chimeras 8- and 20-fold, respectively, with only minor effects on the IGFI affinity. Within the latter of these two regions, we found that amino acids 38-68 of the IR, representing 13 amino acid differences from IGFIR, confer the same 20-fold increase in insulin affinity on the IGFIR. Finally, the amino acids from position 42 to 50 are not responsible for this increase in insulin affinity. We thus propose that at least two determinants within the 68 N-terminal amino acids of the insulin receptor are involved in defining the ligand specificity of the insulin receptor, and that one or a combination of the remaining seven amino acid differences between position 38 and 68 are involved in conferring insulin affinity on the insulin receptor.

Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA.

Human beta 2-glycoprotein (beta 2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature beta 2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the beta 2gpI cDNA. Upon isoelectric focusing, recombinant beta 2gpI obtained from expression of beta 2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as beta 2gpI isolated from plasma, and at least 5 polypeptides were visible.