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Sodium orthovanadate
The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of colocalized NADPH oxidases and is mediated by locally produced reactive oxygen species, in particular H2 O2 . How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of thioredoxin 1(TRX1) slows PTEN reactivation in intact living cells. Finally, using a mechanism-based trapping approach, we demonstrate direct thiol disulphide exchange between the active sites of thioredoxin and either phosphatase. The application of thioredoxin trapping mutants represents a complementary approach to direct assays of PTP oxidation in elucidating the significance of redox regulation of PTP function in the control of cell signaling.TRX1 physically interacts with PTP1B by anti tag coimmunoprecipitation (1, 2).
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ABSTRACT The secreted phosphatase activities of two trypanosomatid parasites were characterized and compared with supernatants of living cells. The plant parasite Phytomonas françai and the phytophagous hemipteran parasite Herpetomonas sp. hydrolyzed p-nitrophenylphosphate at a rate of 15.54 and 6.51 nmol Pi/mg of protein per min, respectively. Sodium orthovanadate (N(a)VO(3)) and sodium fluoride (NaF) decreased the phosphatase activities. The phosphatase activity of P. françai was drastically diminished (73% inhibition) in the presence of sodium tartrate, whereas the phosphatase activity of Herpetomonas sp. was inhibited by 23%. Cytochemical analysis showed the localization of these enzymes on the external surface and in the flagellar pocket of the two trypanosomatids. Sodium tartrate inhibited this reaction, confirming the biochemical data. Platelet-activating factor modulated the phosphatase activities, inhibiting P. françai activity and stimulating Herpetomonas sp. phosphatase activity.
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Phosphatases are remarkably diverse, having evolved up to 10 distinct protein folds to mediate the removal of phosphate from substrates. Their functions are correspondingly wide ranging and a number are important clinical targets. As for kinases, several phosphatase classes comprise catalytically dead members. The classical protein tyrosine phosphatases (PTPs) are a subclass that utilise a cysteine nucleophile for catalysis and are critical for the control of cellular phosphotyrosine levels. Of the 49 human PTP domains, 19 possess sequence variants in key catalytic motifs defining them as pseudophosphatase domains. Strikingly, in all but two cases the catalytic cysteine remains intact. This is in contrast to other pseudophosphatases such as the pseudo-dual specificity phosphatase (DUSP) MK-Styx, which is inactivated by loss of its catalytic cysteine. We find that, like many pseudokinases, pseudoPTPs can mediate protein-protein interactions, providing scaffolding functions, but also substrate recruitment to active PTP domains. Finally, given the striking conservation of cysteine residues in the pseudoPTPs, we have also explored a potential role for redox regulation in their signalling mechanisms.
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Normal human melanocytes, and some human melanoma cell lines, contain c-SRC which is constitutively activated by hypophosphorylation of tyrosine 530. We investigated the possibility that the activation of c-SRC in melanocytes might be attributable to elevated levels of tyrosine 530-directed protein tyrosine phosphatase activity in these cells. We found three times more of this phosphatase activity in cell extracts from melanocytes compared to fibroblasts. The majority of the tyrosine 530-dephosphorylating activity was present in the particulate fraction of cell homogenates, where c-SRC is also located. Treatment of melanocytes with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, caused inactivation of c-SRC. From these results, we conclude that activation of c-SRC in human melanocytes may be attributed to an elevated level of protein tyrosine phosphatase activity directed against tyrosine 530.
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In the present work we have partially characterized an ecto-phosphatase activity in Crithidia deanei, using viable parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 3.55 ± 0.47 nmol Pi/h x 10 8 cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this phosphatase activity and the value of the apparent K m for p-NPP was 5.35 ± 0.89 mᴍ. This phosphatase activity was inhibited by the product of the reaction, the inorganic phosphate. Experiments using classical inhibitors of acid phosphatases, such as ZnCl 2 and sodiumfluoride , as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and ammonium molybdate, showed a decrease in this phosphatase activity, with different patterns of inhibition.
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Read the full review for this Faculty Opinions recommended article: Preferential oxidation of the second phosphatase domain of receptor-like PTP-alpha revealed by an antibody against oxidized protein tyrosine phosphatases.
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Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.
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