Surface phosphophilicity of aluminum-containing adjuvants probed by their efficiency for catalyzing the P--O bond cleavage with chromogenic and fluorogenic substrates.
AuthorsZhao Q, Sitrin R
JournalAnal Biochem
PubMed ID11476547
'Aluminum-containing adjuvants are widely used in a variety of vaccine products, such as recombinant proteins, virus-like particles, conjugated polysaccharides, and recently DNA vaccines. Aluminum-containing adjuvants are also known to have a high affinity to inorganic phosphate and its mono- or diesters. Since phosphate groups are present in many antigens as ... More
Negative regulation of EGFR signalling through integrin-alpha1beta1-mediated activation of protein tyrosine phosphatase TCPTP.
AuthorsMattila E, Pellinen T, Nevo J, Vuoriluoto K, Arjonen A, Ivaska J
JournalNat Cell Biol
PubMed ID15592458
'Integrin-mediated cell adhesion regulates a multitude of cellular responses, including proliferation, survival and cross-talk between different cellular signalling pathways. So far, integrins have been mainly shown to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signalling. Here we show that a collagen-binding integrin alpha(1)beta(1) functions as a negative regulator of ... More
Discovery of a novel shp2 protein tyrosine phosphatase inhibitor.
AuthorsChen L, Sung SS, Yip ML, Lawrence HR, Ren Y, Guida WC, Sebti SM, Lawrence NJ, Wu J
JournalMol Pharmacol
PubMed ID16717135
'Shp2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. It is involved in growth factorinduced activation of mitogen-activated protein (MAP) kinases Erk1 and Erk2 (Erk1/2) and has been implicated in the pathogenicity of the oncogenic bacterium Helicobacter pylori. Moreover, gain-of-function Shp2 mutations have been found in ... More
Loss of phosphatase activity in Ptp69D alleles supporting axon guidance defects.
AuthorsMarlo JE, Desai CJ
JournalJ Cell Biochem
PubMed ID16514605
'PTP69D is a receptor protein tyrosine phosphatase that was identified as a key regulator of neuromuscular axon guidance in Drosophila, and has subsequently been shown to play a similar role in the central nervous system and retina. Three Ptp69D alleles with mutations involving catalytically important residues exhibit a high degree ... More
High yield expression of serine/threonine protein phosphatase type 5, and a fluorescent assay suitable for use in the detection of catalytic inhibitors.
AuthorsNi L, Swingle MS, Bourgeois AC, Honkanen RE,
JournalAssay Drug Dev Technol
PubMed ID17939754
'Protein phosphatase type 5 (PP5) belongs to the PPP family of serine/threonine protein phosphatases and is expressed in most, if not all, human tissues. Although the physiological roles played by PP5 are not yet clear, PP5 is found in association with several proteins that influence intracellular signaling networks initiated by ... More
An automated fluorescence-based method for continuous assay of PP2A activity.
AuthorsWegner AM, McConnell JL, Blakely RD, Wadzinski BE,
JournalMethods Mol Biol
PubMed ID17200554
'Protein serine/threonine phosphatase (PP2A) is a major cellular enzyme implicated in the control of numerous signaling processes. The accurate measurement of PP2A activity in crude cell lysates, immune complexes, and purified preparations provides insight into the function and regulation of this essential enzyme, which, in turn, can lead to a ... More
Protein tyrosine phosphatase: enzymatic assays.
AuthorsMontalibet J, Skorey KI, Kennedy BP
JournalMethods
PubMed ID15588980
Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, ... More
A fluorescent microplate assay for microcystin-LR.
AuthorsFontal OI, Vieytes MR, Baptista de Sousa JM, Louzao MC, Botana LM
JournalAnal Biochem
PubMed ID10222000
A fluorescent enzyme inhibition assay for microcystin-LR was developed using a new fluorescent substrate of protein phosphatases 1 (PP1) and 2A (PP2A), 6,8-difluoro-4-methylumbelliferyl phosphate. The PP1 and PP2A inhibition assay for microcystin-LR was performed in a microtiter plate and the fluorescence yielded by the enzymatic hydrolysis of the substrate was ... More
6,8-Difluoro-4-methylumbiliferyl phosphate: a fluorogenic substrate for protein tyrosine phosphatases.
The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor constants for DIFMUP ... More
Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase.
AuthorsMattila E, Marttila H, Sahlberg N, Kohonen P, Tähtinen S, Halonen P, Perälä M, Ivaska J,
JournalBMC Cancer
PubMed ID20055993
BACKGROUND: T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the alpha-cytoplasmic tail of alpha1beta1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond ... More
Characterization of the histidine-containing phosphotransfer protein B-mediated multistep phosphorelay system in Pseudomonas aeruginosa PAO1.
AuthorsHsu JL, Chen HC, Peng HL, Chang HY,
JournalJ Biol Chem
PubMed ID18256026
Certain bacterial two-component sensor kinases possess a histidine-containing phosphotransfer (Hpt) domain to carry out a multistep phosphotransferring reaction to a cognate response regulator. Pseudomonas aeruginosa PAO1 contains three genes that encode proteins with an Hpt domain but lack a kinase domain. To identify the sensor kinase coupled to these Hpt ... More
Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases.
AuthorsPastula C, Johnson I, Beechem JM, Patton WF
JournalComb Chem High Throughput Screen
PubMed ID12769677
A number of aromatic substrates were evaluated for their ability to detect tyrosine phosphatase and serine/threonine phosphatase activity. Results demonstrated that the fluorinated coumarin DiFMUP is the most sensitive substrate for detecting LAR and PP-2A activity. Using this substrate, selective high-throughput screening assays for serine/threonine and tyrosine phosphatases were developed. ... More
A fluorogenic substrate for detection of organophosphatase activity.
AuthorsSoukharev S, Hammond DJ
JournalAnal Biochem
PubMed ID15033522
A new fluorogenic substrate for the specific detection of organophosphatase (OPase) activity has been designed and evaluated. Our results indicate that 7-diethylphospho-6,8-difluor-4-methylumbelliferyl (DEPFMU) is hydrolyzed specifically by the OPases, mammalian serum paraoxonase and bacterial organophosphorus hydrolase (OPH). The apparent K(m) of DEPFMU is 29 microM for OPH and 91 and ... More
A microfluidics-based mobility shift assay to discover new tyrosine phosphatase inhibitors.
AuthorsPerrin D, Frémaux C, Besson D, Sauer WH, Scheer A
JournalJ Biomol Screen
PubMed ID17092920
Protein tyrosine phosphatases (PTPs) play key roles in regulating tyrosine phosphorylation levels in cells. Since the discovery of PTP1B as a major drug target for diabetes and obesity, PTPs have emerged as a new and promising class of signaling targets for drug development in a variety of therapeutic areas. The ... More
Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion.
AuthorsSun JP, Wang WQ, Yang H, Liu S, Liang F, Fedorov AA, Almo SC, Zhang ZY
JournalBiochemistry
PubMed ID16142898
The PRL (phosphatase of regenerating liver) phosphatases constitute a novel class of small, prenylated phosphatases that are implicated in promoting cell growth, differentiation, and tumor invasion, and represent attractive targets for anticancer therapy. Here we describe the crystal structures of native PRL-1 as well as the catalytically inactive mutant PRL-1/C104S ... More
A fluorogenic assay using pressure-driven flow on a microchip.
AuthorsKerby M, Chien RL
JournalElectrophoresis
PubMed ID11700721
A fluorogenic assay for human T-cell phosphatase (TCPTP) was conducted on an etched glass microchip using pressure driven flow. The TCPTP enzyme catalyzes the removal of a phosphate group from 6,8-difluoro-4-methylumbelliferyl/phosphate (DiFMUP) to produce the fluorogenic product 6,8-difluoro-4-methylumbelliferone (DiFMU). Enzyme assays with real-time on-chip dilution were performed in both low-viscosity ... More
Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase, phosphatase, and protease reactions.
AuthorsSimeonov A, Bi X, Nikiforov TT
JournalAnal Biochem
PubMed ID12009695
We have previously reported that the kinase catalyzed conversion of fluorescently labeled phosphate acceptor peptides to the corresponding phosphopeptides can be conveniently followed by measuring the fluorescence polarization signal in the presence of polyarginine. In the present work, we demonstrate that the method can be used for other enzymes besides ... More
Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.
AuthorsGee KR, Sun WC, Bhalgat MK, Upson RH, Klaubert DH, Latham KA, Haugland RP
JournalAnal Biochem
PubMed ID10452797
Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase ... More
Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14.
The Cdc14 family of protein phosphatases is conserved within eukaryotes and antagonizes the action of cyclin-dependent kinases, thereby promoting mitotic exit and cytokinesis. We performed a detailed kinetic and mechanistic study of the Cdc14 phosphatases with both small molecule aryl phosphates and a physiological protein substrate hCdh1. We found that ... More
Kinetic analysis of human serine/threonine protein phosphatase 2Calpha.
AuthorsFjeld CC, Denu JM
JournalJ Biol Chem
PubMed ID10400656
The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes. Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family. Although ... More
Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding.
AuthorsMontalibet J, Skorey K, McKay D, Scapin G, Asante-Appiah E, Kennedy BP
JournalJ Biol Chem
PubMed ID16332678
Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, ... More
Transition state analysis and requirement of Asp-262 general acid/base catalyst for full activation of dual-specificity phosphatase MKP3 by extracellular regulated kinase.
AuthorsRigas JD, Hoff RH, Rice AE, Hengge AC, Denu JM
JournalBiochemistry
PubMed ID11284696
Dual-specificity phosphatase MKP3 down-regulates mitogenic signaling through dephosphorylation of extracellular regulated kinase (ERK). Unlike a simple substrate-enzyme interaction, the noncatalytic, amino-terminal domain of MKP3 can bind efficiently to ERK, leading to activation of the phosphatase catalytic domain by as much as 100-fold toward exogenous substrates. It has been suggested that ... More
Mechanistic basis for catalytic activation of mitogen-activated protein kinase phosphatase 3 by extracellular signal-regulated kinase.
AuthorsFjeld CC, Rice AE, Kim Y, Gee KR, Denu JM
JournalJ Biol Chem
PubMed ID10702230
The dual specificity mitogen-activated protein kinase phosphatase MKP3 has been shown to down-regulate mitogenic signaling through dephosphorylation of extracellular signal-regulated kinase (ERK). Camps et al. (Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., and Arkinstall, S. (1998) Science 280, 1262-1265) had demonstrated that ERK ... More
Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.
Authors
JournalMar Drugs
PubMed ID27827901
The Highly Recurrent PP2A Aα-Subunit Mutation P179R Alters Protein Structure and Impairs PP2A Enzyme Function to Promote Endometrial Tumorigenesis.
Authors
JournalCancer Res
PubMed ID31142515
Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature.
Authors
JournalJ Clin Invest
PubMed ID25180601
Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes.
Authors
JournalElife
PubMed ID34342579
Optical control of protein phosphatase function.
Authors
JournalNat Commun
PubMed ID31558717
Optimization of Fused Bicyclic Allosteric SHP2 Inhibitors.
Authors
JournalJ Med Chem
PubMed ID30688462
Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-βCTF (C99).
Authors
JournalJ Neurosci
PubMed ID31043483
Inactivation of PP2A by a recurrent mutation drives resistance to MEK inhibitors.
Authors
JournalOncogene
PubMed ID31541192
Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth.
Authors
JournalJ Clin Invest
PubMed ID28504649
6-Amino-3-methylpyrimidinones as Potent, Selective, and Orally Efficacious SHP2 Inhibitors.