Protein tyrosine phosphatases: from structure to function

In the past few years, a diverse family of receptor-like and nontransmembrane protein tyrosine phosphatases (PTPases) have been identified and characterized at the level of primary structure. Progress is now being made towards defining physiological processes in which the activity of PTPases is important. One thing seems clear: the PTPases cannot be regarded simply as antagonists of the protein tyrosine kinases (PTKs)--rather, they have the potential to act both positively and negatively in mediating cellular signalling responses.     

Protein tyrosine phosphatases as negative regulators of mitogenic signaling

The regulation of tyrosine phosphorylation represents a key mechanism governing cell proliferation. In fibroblasts, inputs from both growth factor and extracellular matrix receptors are required for cell division. Triggering such receptors induces a wave of tyrosine phosphorylation on key signaling molecules, culminating in the activation of cyclin-dependent kinases and cell cycle progression. In general, protein tyrosine kinases stimulate, while protein tyrosine phosphatases inhibit, such cell proliferation pathways. The role of protein tyrosine kinases in mitogenesis has been extensively studied, but the identity and targets of the protein tyrosine phosphatases that regulate cell growth are not well described. In this review, I will survey recent advances in the identification and regulation of protein tyrosine phosphatases that downregulate cell proliferation. J. Cell. Physiol. 180:173–181, 1999. © 1999 Wiley-Liss, Inc.     

Structure and function of the extraembryonic membrane persisting around the larvae of the parasitoid Toxoneuron nigriceps

The embryo of Toxoneuron nigriceps (Hymenoptera, Braconidae) is surrounded by an extraembryonic membrane, which, at hatching, releases teratocytes and gives rise to a cell layer embedding the body of the 1st instar larva. This cell layer was studied at different developmental times, from soon after hatching up to the first larval moult, in order to elucidate its ultrastructural, immunocytochemical and physiological function. The persisting "larval serosa" shows a striking structural and functional complexity: it is a multifunctional barrier with protective properties, limits the passage of macromolecules and it is actively involved in the enzymatic processing and uptake of nutrients. The reported results emphasizes the important role that the embryo-derived host regulation factors may have in parasitism success in Hymenoptera koinobionts.     

Protein tyrosine phosphatases as novel targets in breast cancer therapy

Breast cancer is linked to hyperactivation of protein tyrosine kinases (PTKs), and recent studies have unveiled that selective tyrosine dephosphorylation by protein tyrosine phosphatases (PTPs) of specific substrates, including PTKs, may activate or inactivate oncogenic pathways in human breast cancer cell growth-related processes. Here, we review the current knowledge on the involvement of PTPs in breast cancer, as major regulators of breast cancer therapy-targeted PTKs, such as HER1/EGFR, HER2/Neu, and Src. The functional interplay between PTKs and PTK-activating or -inactivating PTPs, and its implications in novel breast cancer therapies based on targeting of specific PTPs, are discussed.     

Protein tyrosine phosphatases and neural development

During neural development, cells interact dynamically with each other and with the extracellular matrix, using cell signaling to control differentiation, axonogenesis, and survival. Enzymes that regulate protein tyrosine phosphorylation often lie at the core of such cell signaling. Protein tyrosine phosphatases (PTPases) are recognized as being of central importance here, and a growing family of PTPases are now known to be expressed in embryonic neurons and glia. Both receptor-like and cytoplasmic enzymes have been identified. The receptor family includes immunoglobulin superfamily members that influence cell–cell adhesion, proteoglycans that control neurite growth, and enzymes in Drosophila that regulate axon guidance and target cell recognition. Cytoplasmic PTPases are implicated in nerve cell commitment and potentially in the regulation of cell survival. This review outlines what we currently know about PTPases in the nervous system and presents concepts concerning their possible modes of action. BioEssays 20 :463–472, 1998. © 1998 John Wiley & Sons, Inc.     

Protein tyrosine phosphatases in cell transformation

The role of tyrosine phosphorylation in cell transformation has been well established. It has been proposed that protein tyrosine phosphatases (PTPases) may be capable of dephosphorylating critical substrates involved in the transformation process, suggesting that they represent a tumor suppressor family of enzymes. Indeed, recent work showed that overexpression of some PTPases in malignant cells counteracted the action of oncogenic tyrosine kinases although overexpression of other forms of these enzymes increased tumorigenicity. The work described herein has provided some insight into the action, both antagonistic and synergistic, of the kinases and phosphatases on cell growth and transformation.     

Protein tyrosine phosphatases: from genes, to function, to disease

The protein tyrosine phosphatase (PTP) superfamily of enzymes functions in a coordinated manner with protein tyrosine kinases to control signalling pathways that underlie a broad spectrum of fundamental physiological processes. In this review, I describe recent breakthroughs in our understanding of the role of the PTPs in the regulation of signal transduction and the aetiology of human disease.     

Protein tyrosine phosphatases: prospects for therapeutics

Protein tyrosine phosphatases (PTPs) form a large family of enzymes that serve as key regulatory components in signal transduction pathways. Recent gene knockout studies in mice identify PTP1B as a promising target for anti-diabetes/obesity drug discovery. PTPs are also implicated in a wide variety of other disorders, including cancer. Significant progress has been made in identifying small molecules that simultaneously bind both the active site and a unique adjacent site that enables specific inhibition of individual PTP isoenzymes. As a consequence, there are compelling reasons to believe that PTP inhibitors may ultimately serve as powerful therapeutic weapons in our arsenal for battling human diseases.     

Protein phosphatases of the guinea-pig parotid gland

The nature of protein phosphatases of the guinea-pig parotid gland was investigated. The protein phosphatases were characterized by (a) the use of five different 32P-labelled substrate proteins (phosphorylase a, histone H2B, casein, and the alpha and beta subunits of phosphorylase kinase), (b) their behaviour during ion-exchange chromatography, (c) their relative molecular mass distribution during gel filtration, (d) their sensitivity towards inhibition by inhibitor 2, (e) their ability to be stimulated by protamine and (f) by their behaviour during freezing and thawing in the presence of 2-mercaptoethanol. The following results were obtained. 1. The 'cytosol' (100,000 X g supernatant) contains protein phosphatases of the types 1, 2A and 2B. 2. On the basis of inhibition with inhibitor 2 (1.2 micrograms/ml) the 'cytosolic' phosphorylase phosphatase activity consists to about 40% of protein phosphatase 1 and to about 60% of protein phosphatase 2A. 3. In the cytosol about 80-90% of the protein phosphatases 1 and 2A exist in an inactive state. 4. A 5-10-fold activation can be achieved by ethanol precipitation, which results in the generation of a mixture of forms of low apparent molecular mass of about 30 kDa. 5. Microsome-associated phosphorylase phosphatase activities can be extracted in a highly active state by detergent (1% Triton X-100) or by 0.8 M NaCl. 6. Activity measurements in the presence of inhibitor 2 (1.2 micrograms/ml) indicate that the microsomal activities consist to about 75% of protein phosphatase 1 and to about 25% of protein phosphatase 2A. Activities corresponding to protein phosphatases 2B and 2C could not be detected. 7. The 'microsomal' protein phosphatase activities exhibit lower apparent molecular masses (70 kDa and 30 kDa) than the 'cytosolic' protein phosphatases (about 260 kDa). 8. After ethanol treatment of the microsomal protein phosphatases only activities with apparent molecular masses of about 30 kDa can be detected. These share several similarities with the ethanol-treated cytosolic protein phosphatases. 9. Both cytosolic and microsomal protein phosphatases display activity towards histone H2B and casein.     

Ultrastructural localization of some phosphatases in the prothoracic gland of the insect Leucophaea Maderae

Summary The cytochemical localization of acid phosphatase and thiamine pyrophosphatase activity was studied by light and electron microscopy in prothoracic gland cells of the cockroach Leucophaea moderae. Nymphal and young adult animals were used.Prominent sites of acid phosphatase activity included large membrane-bounded dense bodies or lysosomes, and certain cisternae of the Golgi apparatus. The results suggest a possible difference in the enzymatic activity toward glycerophosphate and aromatic phosphates as substrates.Thiamine pyrophosphatase activity was localized in elements of the Golgi apparatus and endoplasmic reticulum, and in lysosome-like dense bodies. This latter activity was abolished by sodium fluoride treatment, whereas the phosphatase activity in the Golgi apparatus and endoplasmic reticulum is unaffected by such inhibition.The cytochemical results confirm through direct evidence the suggestions of Scharrer (1964), that the large dense bodies present in the prothoracic gland cells are lysosomes, and that their activity may be related to stages in the life history of the glands. Furthermore, the lysosomes or their derivative structures may play an essential role in the autolysis of the prothoracic glands toward the end of their active period.The enzymatic activity of the endoplasmic reticulum may indicate the involvement of this organelle in the metabolism of steroid-like precursor materials necessary for the synthesis of ecdysone.This study was supported by U.S.P.H.S. grants 5 T1-MH-6418 and NB-05219, and grant RO 1-AM-3984 to Dr. Berta Scharrer. I would like to express my appreciation to Dr. Scharrer for her encouragement and assistance during this study. I also wish to thank Mrs. Sarah Wurzelmann for her competent technical aid.     

In vitro stimulation of the prothoracic gland of the insect as a test system (prothoracic gland test)

In vitro cultures of prothoracic glands of larvae of Periplaneta americana and of some Lepidoptera as biological tests are described. Incorporation of ³H-5-uridine in the RNA of the prothoracic glands represented the measure of the cellular activity of the glands.Activation factor I separated from extracts of corpora cardiaca of the cockroach Periplaneta americana by means of gel filtration techniques caused significant stimulation of RNA synthesis of the glands.     

Protein tyrosine kinases as new potential targets against human schistosomiasis

In spite of the numerous efforts made to control their transmission, parasite schistosomes still represent a serious public health concern and a major economic problem in many developing countries. Praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis and the only one that is available for mass chemotherapy. However, its widespread use and its inefficacy on juvenile parasites raise fears that schistosomes will develop drug resistance, and make the development of alternative drugs highly desirable. Protein tyrosine kinases (PTKs) are key molecules that control cell differentiation and proliferation and they already represent important targets for molecular cancer therapy. The recent characterization in Schistosoma mansoni of several cytosolic and receptor PTKs, with properties similar but also divergent from their vertebrate counterparts, opens new perspectives for the development of novel strategies in chemotherapy of schistosomiasis, which could be based on the use of parasite-specific tyrosine phosphorylation inhibitors.     

trans-Beta-nitrostyrene derivatives as slow-binding inhibitors of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphotyrosyl (pY) proteins to produce tyrosyl proteins and inorganic phosphate. Specific PTPs inhibitors provide useful tools for studying PTP function in signal transduction processes and potential treatment for human diseases such as diabetes, inflammation, and cancer. In this work, trans-beta-nitrostyrene (TBNS) and its derivatives are found to be slow-binding inhibitors against protein tyrosine phosphatases PTP1B, SHP-1, and Yop with moderate potencies (K(I*) = 1-10 microM). Competition experiments with a substrate (pNPP) and iodoacetate indicate that TBNS is active site-directed. The mechanism of inhibition was investigated by UV-vis absorption spectroscopy, (1)H-(13)C heteronuclear single-quantum correlation NMR spectroscopy, and site-directed mutagenesis. These studies suggested a mechanism in which TBNS acts a pY mimetic and binds to the PTP active site to form an initial noncovalent E.I complex, followed by nucleophilic attack on the TBNS nitro group by Cys-215 of PTP1B to form a reversible, covalent adduct as the tighter E.I* complex. TBNS derivatives represent a new class of neutral pY mimetic inhibitors of PTPs.     

Calixarene-based phosphinic acids as inhibitors of protein tyrosine phosphatases

In the present work, the derivatives of calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene bearing four methylene(phenyl)phosphinic acid groups on the upper rim of the macrocycle were synthesized and studied as inhibitors of human protein tyrosine phosphatases. The inhibitory capacities of the three compounds towards PTP1B were higher than those for protein tyrosine phosphatases TC–PTP, MEG1, MEG2, and SHP2. The most potent sulfonylcalix[4]arene phosphinic acid displayed K_i value of 32?nM. The thiacalix[4]arene phosphinic acid was found to be a low micromolar inhibitor of PTP1B with selectivity over the other PTPs. The kinetic experiments showed that the inhibitors compete with the substrate for the active site of the enzyme. Molecular docking was performed to explain possible binding modes of the calixarene-based phosphinic inhibitors of PTP1B.     

Protein tyrosine phosphatases: the quest for negative regulators of insulin action

Type 2 diabetes is increasing at an alarming rate worldwide, and there has been a considerable effort in several laboratories to identify suitable targets for the design of drugs against the disease. To this end, the protein tyrosine phosphatases that attenuate insulin signaling by dephosphorylating the insulin receptor (IR) have been actively pursued. This is because inhibiting the phosphatases would be expected to prolong insulin signaling and thereby facilitate glucose uptake and, presumably, result in a lowering of blood glucose. Targeting the IR protein tyrosine phosphatase, therefore, has the potential to be a significant disease-modifying strategy. Several protein tyrosine phosphatases (PTPs) have been implicated in the dephosphorylation of the IR. These phosphatases include PTPalpha, LAR, CD45, PTPepsilon, SHP2, and PTP1B. In most cases, there is evidence for and against the involvement of the phosphatases in insulin signaling. The most convincing data, however, support a critical role for PTP1B in insulin action. PTP1B knockout mice are not only insulin sensitive but also maintain euglycemia (in the fed state), with one-half the level of insulin observed in wild-type littermates. Interestingly, these mice are also resistant to diet-induced obesity when fed a high-fat diet. The insulin-sensitive phenotype of the PTP1B knockout mouse is reproduced when the phosphatase is also knocked down with an antisense oligonucleotide in obese mice. Thus PTP1B appears to be a very attractive candidate for the design of drugs for type 2 diabetes and obesity.     

Peptidyl aldehydes as reversible covalent inhibitors of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. PTP inhibitors provide potential treatment of human diseases/conditions such as diabetes and obesity as well as useful tools for studying the function of PTPs in signaling pathways. In this work, we have shown that certain aryl-substituted aldehydes act as reversible, slow-binding inhibitors of modest potency against PTP1B, SHP-1, and a dual-specificity phosphatase, VHR. Attachment of the tripeptide Gly-Glu-Glu to the para position of cinnamaldehyde resulted in an inhibitor (Cinn-GEE) of substantially increased potency against all three enzymes (e.g., K(I) = 5.4 microM against PTP1B). The mechanism of inhibition was investigated using Cinn-GEE specifically labeled with (13)C at the aldehyde carbon and (1)H-(13)C heteronuclear single-quantum coherence spectroscopy. While Cinn-GEE alone showed a single cross-peak at delta 9.64 ((1)H) and delta 201 ((13)C), the PTP1B/Cinn-GEE complex showed three distinct cross-peaks at delta 7.6-7.8 ((1)H) and 130-137 ((13)C). Mutation of the catalytic cysteine (Cys-215 in PTP1B) into alanine had no effect on the cross-peaks, whereas mutation of a conserved active-site arginine (Arg-221 in PTP1B) to alanine abolished all three cross-peaks. Similar experiments with Cinn-GEE that had been labeled with (13)C at the benzylic position revealed a change in the hybridization state (from sp(2) to sp(3)) for the benzylic carbon as a result of binding to PTP1B. These results rule out the possibility of a free aldehyde, aldehyde hydrate, or hemithioacetal as the enzyme-bound inhibitor form. Instead, the data are consistent with the formation of an enamine between the aldehyde group of the inhibitor and the guanidine group of Arg-221 in the PTP1B active site. These aldehydes may provide a general core structure that can be further developed into highly potent and specific PTP inhibitors.     

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.     

Fullerene derivatives as a new class of inhibitors of protein tyrosine phosphatases

In this study, we identified water-soluble C₆₀ and C₇₀ fullerene derivatives as a novel class of protein tyrosine phosphatase inhibitors. The evaluated compounds were found to inhibit CD45, PTP1B, TC-PTP, SHP2, and PTPβ with IC₅₀ values in the low micromolar to high nanomolar range. These results demonstrate a new strategy for designing effective nanoscale protein tyrosine phosphatase inhibitors.