Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate

Huyer, Gregory; Liu, Susana; Kelly, John; Moffat, Jason; Payette, Paul; Kennedy, Brian P.; Tsaprailis, George; Gresser, Michael J.; Ramachandran, Chidambaram

doi:10.1074/jbc.272.2.843

Cited by 777 publications

(626 citation statements)

References 66 publications

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“…Sodium orthovanadate (Na 3 VO 4 ) is a commonly used general inhibitor of PTPs, most likely acting as a phosphate Tyrosine protein phosphatase of M. xanthus analogue (Huyer et al, 1997). Sodium orthovanadate, at 1 mM, inhibited phosphatase activity by 77±4 %, and similarly, ammonium metavanadate (NH 4 VO 3 ) at 1 mM inhibited phosphatase activity by 70±10 % against pNPP (Fig.…”

Section: Inhibitors For Phosphatase Activity Of Phpamentioning

confidence: 97%

PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide

et al. 2012

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Protein-tyrosine phosphorylation plays a significant role in multiple cellular functions in bacteria. Bacterial tyrosine phosphatases catalyse the dephosphorylation of tyrosyl-phosphorylated proteins. Myxococcus xanthus PhpA shares homology with DNA polymerase and histidinol phosphatase family members. Recombinant His-tagged PhpA requires Mn 2+ or Co 2+ for phosphatase activity, and shows strict specificity for phosphorylated tyrosine residues. The k m values of PhpA for p-nitrophenyl phosphate (pNPP) and phosphotyrosine peptide, RRLIEDAEpYAARG, were 803 and 139 mM, respectively. The phosphatase activity of PhpA was inhibited by sodium orthovanadate with a k i of 33 mM. phpA gene expression was observed under both vegetative and developmental conditions, but peaked during late fruiting body formation. A phpA mutant exhibited an elevated level of tyrosine phosphorylation of a 79 kDa protein and cytoplasmic tyrosine kinase, BtkA. In M. xanthus, exopolysaccharide (EPS) is essential for cellcell adhesion and fruiting body formation. phpA mutant cells exhibited enhanced capacity for cellcell agglutination in agglutination buffer. Under starvation conditions, phpA mutation caused early aggregation and sporulation. The EPS production assay showed that the phpA mutant produced an increased amount of EPS in comparison with the wild-type. These results indicate that PhpA may negatively regulate the production of EPS in M. xanthus.

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Section: Inhibitors For Phosphatase Activity Of Phpamentioning

confidence: 97%

PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide

et al. 2012

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“…EDTA has been routinely use in the literature to assay PTP activity as well as probing for reversibility of potent inhibitors [20]. Interestingly, if EDTA is included in the assay solution, the inhibition of all three tested enzymes by VO-OHpic is fully abolished.…”

Section: Pten Inhibition Assaymentioning

confidence: 99%

Targeting PTEN using small molecule inhibitors

Mak

Woscholski

2015

Methods

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PTEN (phosphatase and tensin homologue deleted on chromosome 10) is well known as a tumour suppressor. It's PI(3,4,5)P 3 lipid phosphatase activity is an important counteracting mechanism in PI3K (phosphoinositide 3-kinase) signalling. Furthermore, PTEN lies upstream of Akt kinase, a key enzyme in insulin signalling regulating glucose uptake and cell growth. Therefore, PTEN has recently gained attention as a valuable drug target for the treatment of diabetes, stroke, cardiac infarct and fertility. This review summarizes the use of small molecules as PTEN inhibitors. Currently available methodologies and techniques for accessing PTEN inhibition in vitro and in cellulo will be discussed. Keywords: PTEN, inhibition, Vanadium compounds, OMFP, VO-OHpic IntroductionThe phosphatidylinositol (PI) 3-phosphatase PTEN has been initially characterised as tumour suppressor gene with a putative tyrosine phosphatase domain [1], but has since then proven to be a dual specificity phosphatase having inositol lipids as substrates [2]. PTEN is dephosphorylating 3-phosphorylated inositol lipids, counteracting the action of PI3 kinases by removing the "second messengers" created by the action of growth factors, cytokines and cell survival stimulators [2][3][4]. Chemical intervention tools, such as wortmannin [5], LY294002 [6] or theophylline [7] facilitated the discovery of new chemical inhibitors, with all chemical intervention tools having a tremendous impact on the elucidation of the role of PI 3-kinases in disease and cancer development [8,9]. PTEN was discovered much later than the PI 3-kinases, and the first PTEN inhibitors were developed in due course a decade ago exploiting the dual specificity nature of the PTEN phosphatase. Bisperoxovanadium compounds were known tyrosine phosphatase inhibitors, which showed a remarkable potency for PTEN in vitro and in vivo [10]. However, these inhibitors are rather promiscuous targeting PTEN in the nanomolar dosage range as well as other phosphatases containing the CX5R active site motif, whereas the 2nd generation PTEN inhibitor VO-OHpic is fairly specific

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“…It is likely that the effects of exposing cells to H 2 O 2 are mediated by global inhibition of tyrosine phosphatases (27)(28)(29)(30)(31) 7 Jurkat cells (10 6 cells/ml) to a final concentration of 5 mM. After incubation at 37°C for 15 min, the cells were washed once in ice-cold, isotonic, Tris-buffered saline and then lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.2, 1% w/v sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 200 M Na 3 VO 4 , 50 mM NaF, 2 mM EDTA, 100 kallikrein-inactivating units/ml aprotinin) for 20 min at 4°C at a concentration of 10 7 cells/ml.…”

Section: From the ‡Molecular Biology And Virology Laboratory The Salmentioning

confidence: 99%

The Activated Form of the Lck Tyrosine Protein Kinase in Cells Exposed to Hydrogen Peroxide Is Phosphorylated at Both Tyr-394 and Tyr-505

Hardwick

Sefton

1997

Journal of Biological Chemistry

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Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H 2 O 2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H 2 O 2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members.p56 lck , a member of the Src family of non-receptor tyrosine protein kinases (1, 2), is expressed predominantly in T cells. Lck function is critical both for T-cell development in the thymus (3, 4) and activation of mature T cells in the periphery by antigen (5, 6). Lck stably associates with the inner surface of the plasma membrane as a result of myristoylation of Gly-2 and palmitoylation of Ser-3 and Ser-5 (7-10). There it binds to the T-cell receptor-associated glycoproteins CD4 and CD8 as well as other proteins through its unique amino terminus (11-15). The activity of Lck is regulated by phosphorylation of two conserved tyrosine residues. Tyr-505 (equivalent to Tyr-527 in c-Src) is located near the carboxyl terminus of Lck and, when phosphorylated, associates intramolecularly with the SH2 domain in the amino-terminal half of the protein. This helps stabilize Lck in a conformation that, biologically, is relatively inactive (16 -20). In the absence of phosphorylation at Tyr-505, intramolecular binding of the carboxyl terminus to the SH2 domain does not occur, and Lck exhibits increased activity in vivo. In contrast, phosphorylation of Tyr-394 (equivalent to Tyr-416 in c-Src) stimulates the catalytic activity of Lck (21-23). Phosphorylation of Tyr-394 allows the formation of hydrogen bonds between the phosphate of Tyr(P) 1 -394 and amino acid residues in the catalytic cleft. These interactions allow the enzyme to assume a conformation resembling that of activated cyclic AMP-dependent protein kinase A (19, 20, 24 -26).We have previously demonstrated that hydrogen peroxide, a potent activator of Lck, acts by inducing phosphorylation of Lck on 37). It is likely that the effects of exposing cells to H 2 O 2 are mediated by global inhibition of tyrosine phosphatases (27-31). The increase in phosphorylation of Lck at Tyr-394 that we observe in the presence of H 2 O 2 may therefore result largely from reduced dephosphorylation of this site. Our previous work did not address the question of whether or not activation of Lck by H 2 O 2 -induced phosphorylation of Tyr-394...

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Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate

Cited by 777 publications

References 66 publications

PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide

PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide

Targeting PTEN using small molecule inhibitors

The Activated Form of the Lck Tyrosine Protein Kinase in Cells Exposed to Hydrogen Peroxide Is Phosphorylated at Both Tyr-394 and Tyr-505

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