Development and optimization of high-throughput in vitro protein phosphatase screening assays

Tierno, Marni Brisson; Johnston, Paul A.; Foster, Caleb; Skoko, John; Shinde, Sunita; Shun, Tong Ying; Lazo, John S.

doi:10.1038/nprot.2007.155

Cited by 60 publications

(55 citation statements)

References 29 publications

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“…The activity of VHR phosphatase was measured using the substrate 3-Omethylfluorescein phosphate (OMFP; Sigma) in a 96-well microtiter plate assay based on methods described previously. 17 The NSC-87877 (Calbiochem) and OMFP were solubilized in H2O and DMSO, respectively. All reactions were performed at a final concentration of 1% DMSO.…”

Section: Notesmentioning

confidence: 99%

Regulation of Vaccinia H1-related (VHR) Phosphatase Activity by NSC-87877

Park¹,

Song²,

Cho

2009

Bulletin of the Korean Chemical Society

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Half maximal inhibitory constant of enzyme was determined to 7.83 ± 0.98 µM. VHR phosphatase (100 nM) was incubated with various concentrations of NSC-87877 at 37 o C for 30 min. Fluorescence emission from the product was measured with a multiwell plate reader as described in Experimental section.Based on amino-acid sequences of catalytic domains, protein tyrosine phosphatases (PTPs) can be classified into four groups 1 ; class I Cys-based PTPs, class II Cys-based PTPs, class III Cys-based PTPs, and Asp-based PTPs. Each PTP has a characteristic role in a variety of cellular processes including cellular metabolism, growth, proliferation, differentiation and also has been emerged as a therapeutic target in many diseases such as diabetes, leukemia, neurodegradation, and cancer. Class I PTPs comprise dual specificity phosphatases (DUSPs) including VHR that has diverse substrate specificities both or either on phospho-Tyr, phospho-Ser, and phospho-Thr residues. Class II PTPs that dephosphorylate only pTyr are encoded by a single gene ACP1 and related to diseases like Alzheimer's disease and asthma. Class III PTPs comprise CDC25 family proteins that regulate cell cycle by acting on Cdks. They dephosphorylate Thr-Tyr motifs of N-terminal Cdks, thus keep them from driving the cell progression.2 Asp-based PTPs encoded by only four Eya genes recently have been listed as a new family.VHR phosphatase, also called DUSP3, is a 20 kDa protein that might be one of the smallest known PTPs. VHR phosphatase can be elucidated by two particular characteristics in cellular physiology. First, VHR phosphatase shows more expression in the nucleus of cancer cells than that of normal cells. It is not due to the increase of transcription of VHR gene, but due to stabilization VHR phosphatase in cancer cell. Its expression was increased in cervix carcinoma and prostate cancer. 4 Another intriguing aspect of VHR phosphatase is that it fluctuates during the phases of cell cycle. VHR phosphatase during G1 phase is not detected due to rapid degradation. Gradually, it accumulates by means of its augmented transcription during late G1 and S phases. 5 The siRNA interference analyses also show that depletion of VHR phosphatase arrests cells at the G 1 /S, and G 2 /M phases. 5 Highly active extracellular signal regulated kinase (ERK) is a main reason of the cell cycle arrest. 6 Elevated VHR phosphatase expression results in cell cycle progression by dephosphorylating ERK. Due to its elevated protein level in cancers such as cervix carcinoma and prostate cancer and involvement in cell cycle progression, VHR phosphatase has been emerged as a therapeutic target. Therefore, it is important to identify novel inhibitor of VHR phosphatase for cancer treatment. Several VHR phosphatase inhibitors including stevastelins, 7 glucosamine-aminoehoxy triphenyltin (GATPT), 8 RK-682, 9 and 4-iA 10 have been identified so far. In search for more putative VHR phosphatase inhibitors, we investigated whether 8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid ...

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Section: Notesmentioning

confidence: 99%

Regulation of Vaccinia H1-related (VHR) Phosphatase Activity by NSC-87877

Park¹,

Song²,

Cho

2009

Bulletin of the Korean Chemical Society

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“…An initial drawback of OMFP was that the commercially available form is the cyclohexylammonium salt, which has very poor solubility in many organic solvents (including DMSO), requiring sonication to prepare stock solutions. 48 However, we found that this can be easily overcome by making stocks with an acidified organic solvent, thereby forming the, quite soluble, OMFP free acid (see ''Materials and Methods'' section). An examination of the reaction progress curves for an enzyme and substrate provides a basis for choosing appropriate conditions for the biochemical screening assay: concentrations of substrate and enzyme, and length of reaction.…”

Section: Resultsmentioning

confidence: 99%

Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

Swingle

Honkanen

2014

ASSAY and Drug Development Technologies

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Protein phosphatase types 1 a (PP1a/PPP1C) and 5 (PP5/PPP5C) are members of the PPP family of serine/threonine protein phosphatases. PP1 and PP5 share a common catalytic mechanism, and several natural compounds, including okadaic acid, microcystin, and cantharidin, act as strong inhibitors of both enzymes. However, to date there have been no reports of compounds that can selectively inhibit PP1 or PP5, and specific or highly selective inhibitors for either PP1 or PP5 are greatly desired by both the research and pharmaceutical communities. Here we describe the development and optimization of a sensitive and robust (representative PP5C assay data: Z 0 = 0.93; representative PP1Ca assay data: Z 0 = 0.90) fluorescent phosphatase assay that can be used to simultaneously screen chemical libraries and natural product extracts for the presence of catalytic inhibitors of PP1 and PP5.

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“…Phosphatase activity was measured using the substrate OMFP (Sigma-Aldrich, St. Louis, MO) in a 96-well microtiter plate assay based on methods described previously. 17 PTP inhibitor V and OMFP were solubilized in dimethyl sulfoxide (DMSO). All reactions were performed at the final concentration of Figure 2.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of PTPN2 by PTP inhibitor V

Kim¹,

Cho

2013

Bulletin of the Korean Chemical Society

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Protein phosphorylation is closely linked to critical pathways in numerous human diseases, which is regulated by the coordinated activities of kinases and phosphatases.1 Protein phosphorylation occurs predominantly on serine, threonine, and tyrosine residues of proteins and plays important roles in the regulation of physiological processes including gene expression, proliferation, differentiation, cell cycle arrest, and apoptosis.2 About 30% of proteins are phosphorylated in the mammalian cell. In case of eukaryotic cells, tyrosine phosphorylation occupies only 0.01-0.05% of total protein phosphorylation, while most of protein phosphorylation occurs on serine or threonine residues. However, growth factor stimulation or tumorigenesis enhances the level of tyrosine phosphorylation up to 1-2% of total protein phosphorylation.3 Protein phosphorylation is a reversible process that is controlled by protein kinases and protein phosphatases. In particular, protein tyrosine phosphatases (PTPs) are involved in most signaling pathways and thus play important roles in eukaryotes.4 PTPs superfamily comprises 107 enzymes in human genome. Based on the amino acid sequences of their catalytic domains, the PTPs can be grouped into four main families.1 Since PTPs play critical roles for cell homeostasis and thus diseases, chemical inhibitors that regulate PTPs have been extensively investigated to be used as therapeutic reagents.PTP inhibitor V, also known as phenyl hydrazono pyrazolone sulfate 1 (PHPS1), is 4-{N'-[3-(4-nitrophenyl)-5-oxo-1-phenyl-1,5-dihydro-pyrazol-(4Z)-ylidene]-hydrazino}-benzenesulfonic acid ( Fig. 1(a)). PTP inhibitor V was originally identified as a potent cell-permeable inhibitor that acts as active-site targeting, reversible, competitive inhibitor of SHP-2 with the IC 50 value of 2.1 μM.5 PTP inhibitor V inhibits ECPTP, PTP1B and SHP-1 (IC 50 = 5.4, 19, 30 μM, respectively).5 To investigate the effect of PTP inhibitor V on other PTPs, we performed in vitro phosphatase assays with purified recombinant PTPs (Table 1). We found that protein tyrosine phosphatase non-receptor type 2 (PTPN2) was inhibited by PTP inhibitor V while other PTPs were not.PTPN2 that is also called T cell protein tyrosine phosphatase (TC-PTP) belongs to class I Cys-based PTPs as the intracellular non-receptor PTPs with a high degree of sequence and structural homology within the catalytic domain.6,7 The mRNA levels of PTPN2 increase at G 1 phase and return to basal level after G 1 phase during cell cycle progression. 8PTPN2 is positively involved in cell proliferation.9 Abnormal expression of PTPN2 is correlated with several diseases such as diabetes, obesity, and cancer.10 PTPN2 has been reported to modulate cytokine receptor signaling, including IFN-γ signaling.11,12 In another case, PTPN2 negatively regulates and inactivates Src tyrosine kinases to suppress Protein tyrosine phosphatase IC 50 (µM) DUSP18 > 10 (n = 3) DUSP23 > 10 (n = 3) DUSP26 > 10 (n = 3) ACP1 >10 (n = 3) PTPN2 3.79 ± 0.59 (n = 3)

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Development and optimization of high-throughput in vitro protein phosphatase screening assays

Cited by 60 publications

References 29 publications

Regulation of Vaccinia H1-related (VHR) Phosphatase Activity by NSC-87877

Regulation of Vaccinia H1-related (VHR) Phosphatase Activity by NSC-87877

Development and Validation of a Robust and Sensitive Assay for the Discovery of Selective Inhibitors for Serine/Threonine Protein Phosphatases PP1α (PPP1C) and PP5 (PPP5C)

Inhibition of PTPN2 by PTP inhibitor V

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