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. Author manuscript; available in PMC: 2024 Apr 25.
Published in final edited form as: Methods Mol Biol. 2024;2743:285–300. doi: 10.1007/978-1-0716-3569-8_18

In vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase

Christopher Alderman 1, Aaron Krueger 1,2, John Rossi 1, Heide L Ford 3, Rui Zhao 1,*
PMCID: PMC11044973  NIHMSID: NIHMS1984888  PMID: 38147222

Abstract

Protein tyrosine phosphatases (PTP) such as the Eyes Absent (Eya) family of proteins play important roles in diverse biological processes. In vitro phosphatase assays are essential tools for characterizing the enzymatic activity as well as discovering inhibitors and regulators of these phosphatases. Two common types of in vitro phosphatase assays use either a small molecule substrate which produces a fluorescent or colored product, or a peptide substrate which produces a colorimetric product in a malachite green assay. Here we describe detailed protocols of a phosphatase assay using small molecule 3-O-methylfluorescein phosphate (OMFP) as a substrate and a malachite green assay using the pH2AX peptide as a substrate to evaluate the phosphatase activity of EYA2 and the effect of small molecule inhibitors of EYA2. These protocols can be easily adapted to study other protein tyrosine phosphatases.

Keywords: Eya2, protein tyrosine phosphatase, phosphatase assay, OMFP, Malachite Green assay

1. Introduction

The Eya family of genes, Eyas1-4, were first discovered as transcriptional co-activators of the Six family of homeoprotein transcription factors [1,2]. The Six1-Eya transcriptional complex is critical for development in species from Drosophila to mammals, where it plays a key role in organogenesis of the eye, thymus, thyroid, parathyroid, muscle, kidney, and ear [3-9]. (We will use the mouse protein nomenclature such as Six1 and Eya in the Introduction to represent these proteins from multiple species and will use the human protein nomenclature in all subsequent sections). Six1 and Eya are typically downregulated after organ development is complete, but they can be abnormally re-expressed and reinitiate developmental programs out of context, driving tumorigenesis and metastasis in many tumor types including breast cancer, leukemia, peripheral nerve-sheath tumors, Wilms’ tumor, cervical, ovarian, liver, kidney, lung, and colorectal cancers [10-32].

In addition to being transcriptional co-activators, Eya family members contain tyrosine phosphatase enzymatic activity and are members of the haloacid dehalogenase (HAD) family of tyrosine phosphatases [33,7,34]. Eya and other HAD phosphatases use an Asp as the active site residue instead of the Cys more commonly used in cellular tyrosine phosphatases [35]. The phosphatase activity of HAD family members requires Mg++ [35]. All other known HAD phosphatases target phosphorylated Ser/Thr, while Eya is the only HAD family protein tyrosine phosphatase [36].

The tyrosine phosphatase activity of Eyas plays important roles in multiple cellular processes. Eyas have been shown to regulate transcription of a subset of Six1 target genes, making Eya the first coactivator with transcription-modifying phosphatase activity [7]. The Eya1-3 tyrosine phosphatase activity was also found to be important for transformation, migration, invasion, and metastasis in breast cancer [24,37-40], though its precise mechanism of action is not known. In addition, Eyas1-3 direct cells to the repair instead of apoptotic pathway upon DNA damage by dephosphorylating pY142 on H2AX [36,41]. Eya2 can also inhibit the tumor suppressive activity of estrogen receptor β (ERβ) by dephosphorylating its pY36 residue [42]. Recently, we found that the Tyr phosphatase activity of Eya2 is critical for Glioblastoma stem cell maintenance, potentially through mitotic spindle regulation [43], although a direct target for the Eya2 tyrosine phosphatase in this context has yet to be identified.

Characterization of a protein tyrosine phosphatase such as Eya using an in vitro phosphatase assay is a critical first step in understanding the mechanism and regulation of these enzymes. There are two common types of in vitro phosphatase assays: one uses a small molecule and the other uses a phospho-peptide as a substrate. In a small molecule phosphatase assay, the phosphatase dephosphorylates the small molecule substrate and generates either a fluorescent or colored product that can be monitored by fluorescence or colorimetry. Small molecule substrates commonly used for phosphatase assays include 3-O-Methylfluorescein phosphate (OMFP), fluorescein diphosphate (FDP), and p-Nitrophenyl Phosphate (pNPP), which are dephosphorylated to fluorescent OMF, fluorescent fluorescein, and colorimetric pNP, respectively. Although these small molecules are generic substrates that do not differentiate Tyr or Ser/Thr phosphatases, this small molecule phosphatase assay is easy to set up, and is a simple and quantitative way to evaluate the effect of potential regulators on tyrosine phosphatase activity. Furthermore, this assay can be readily adapted to a high throughput screening format to identify potential inhibitors.

In a phospho-peptide based phosphatase assay, the free orthophosphate released from the phospho-peptide in the phosphatase reaction forms a green complex with Malachite Green and molybdate, which can be detected at 620 nm on a spectrometer. Unlike the small molecule phosphatase assays, in this assay, a protein tyrosine phosphatase only acts on a peptide containing a phosphor-tyrosine, while a Ser/Thr phosphatase only acts on a peptide containing a phosphor-serine or threonine.

In this chapter, we present detailed protocols of Eya2 Eya Domain (ED) expression and purification from E. coli, an OMFP based fluorescent phosphatase assay to analyze the enzyme kinetics of Eya2 phosphatase activity, use of the OMFP assay to evaluate the effect of small molecule inhibitors on the Eya2 phosphatase activity, and a pH2AX-based malachite green assay to evaluate the tyrosine phosphatase activity of Eya2 and the effect of small molecule inhibitors targeting the Eya2 tyrosine phosphatase. These protocols can be easily adapted to other Eya family members, as well as to other protein tyrosine phosphatases to evaluate their activities and identify potential regulators or inhibitors.

2. Materials

2.1. Express and Purify EYA2 ED from E. coli.

  1. Human EYA2 ED (residues 253-538) subcloned into the pGEX-6P1 plasmid (GE Healthcare) using the BamHI and XhoI sites.

  2. E. coli strain: XA90 cell obtained from colleagues (unpublished). Other E. coli strains such as BL21 or Rosetta should also work.

  3. E. coli culture media 1: autoclave LB media (10 g tryptone, 10 g NaCl, 5 g yeast extract, and q.s. 1L with H2O) and add Ampicillin to a final concentration of 100 μg/mL.

  4. E. coli culture media 2: autoclave 2xYT (16 g Tryptone, 10 g yeast extract, 5g NaCl, adjust pH to 7.0 with 5N NaOH, and q.s. 1L with H2O) media and add Ampicillin to a final concentration of 50 μg/mL.

  5. Isopropyl-beta-D-thiogalactoside (IPTG): dissolve 2.38 g into 10 mL H2O to make a 1000x stock solution at 1M and store in aliquots at −20 °C.

  6. Leupeptin: dissolve 10 mg/mL in DMSO and store in aliquots at −20 °C.

  7. Pepstatin A: dissolve 25 mg/mL in DMSO and store in aliquots at −20 °C.

  8. Phenylmethylsulfonyl Fluoride (PMSF): dissolve 17.42 g into 100 mL DMSO to make a 100x stock solution at 1M and store in aliquots at −20 °C.

  9. NaCl: dissolve 292.2 g/L in H2O to make a stock solution at 5M.

  10. Lysis buffer: 50 mM Tris, pH 7.5, 250 mM NaCl, 5% glycerol, and 1 mM dithiothreitol (DTT).

  11. Sonicator: Ultrasonic Processor purchased from Toption.

  12. Glutathione resin: Glutathione-Sepharose 4B resin purchased from GE Healthcare and stored at 4 °C.

  13. PreScission protease: PreScission Protease purchased from Cytiva and stored at −80 °C.

  14. Elution buffer: lysis buffer plus 30 mM DTT.

  15. Glass chromatography column: Glass Econo-Column purchased from Bio-Rad.

  16. Protein concentrator: Pierce™ Protein Concentrators PES, 10K MWCO, 0.5–100 mL purchased from Thermo Fisher.

  17. Size exclusion column: Superdex 200 purchased from GE Healthcare.

  18. Sodium dodecyl sulfate (SDS) gel: 4.1 mL H2O, 3.3 mL acrylamide/bis (30% 37.5:1; Bio-Rad), 2.5 mL Tris–HCl (1.5 M, pH 8.8), 100 μL 10% SDS, 10 μL N,N,N′,N′-tetramethylethylene-diamine (TEMED; Bio-Rad), and 32 μL 10% ammonium persulfate (APS).

  19. Coomassie Brilliant Blue stain: 1 g of Coomassie Brilliant Blue (Bio-Rad), 500 mL methanol, 100 mL glacial acetic acid, and bring the final volume to 1 L with H2O.

  20. 5x protein-loading buffer: 100 mg bromophenol blue, 2.5 mL 2 M Tris-HCL (pH 6.8), 10 mL glycerol, 2 g SDS, and 1.542 g DTT.

  21. Destain buffer: 500 mL methanol, 100 mL glacial acetic acid, and bring the final volume to 1 L with H2O.

2.2. Analyze the Eya2 phosphatase kinetics using an OMFP-based fluorescent phosphatase assay.

  1. 2X Assay buffer: 100 mM MES, pH 6.5, 100 mM NaCl, 5 mM MgCl2, 0.1% bovine serum albumin, 2 mM DTT.

  2. Black, half-volume 96-well flat bottom plate: purchased from Greiner Bio-one.

  3. Clear, 96-well round bottom plate: purchased from Greiner Bio-one or Corning.

  4. OMFP: 10 mM stock, dissolved in DMSO.

  5. Multichannel pipettor (8-channel).

  6. Solution reservoir for multichannel pipettor: purchased from VWR.

  7. Plate reader capable of recording time points automatically over the course of the assay, for example the Promega GloMax.

2.3. Evaluate small molecule inhibitors of the Eya2 phosphatase using an OMFP-based fluorescent phosphatase assay.

  1. Assay buffer: 25 mM MES, pH 6.5, 50 mM NaCl, 5 mM MgCl2, 0.33% bovine serum albumin, 5 mM DTT.

  2. Eya protein: 300 nM in assay buffer.

  3. Dimethyl sulfoxide (DMSO): purchased from Sigma-Aldrich.

  4. OMFP: purchased from Scientific Resources Ptd Ltd.

  5. Disodium ethylenediaminetetraacetate (EDTA): 0.5 M, pH 8.0.

  6. Black, half-volume 96-well flat bottom plate: purchased from Greiner Bio-one.

  7. Plate reader with fluorometer: Promega GloMax.

2.4. Evaluate small molecule inhibitors of the EYA2 phosphatase using a pH2AX-based Malachite Green Assay.

  1. 2X Malachite green assay buffer containing EYA2: 200 mM MES, pH 6.0, 100 mM NaCl, 2 mM MgCl2, 100 μM DTT, 7.8 μM Eya2.

  2. pH2AX phospho-peptide: the KATQASQEpY phospho-H2AX peptide was purchased from Abgent and dissolved in water to make a 1mM stock solution.

  3. Microplate reader: Spectramax PLUS 384 plate reader (Molecular Devices). Malachite Green reagent: 0.34 mg/mL Malachite green in 8.3% HCl, 3.4% ethanol, 75 mM ammonium molybdate, 0.01% Tween-20 or purchased from Sigma-Aldrich.

  4. Data analysis software: Origin Pro 8.0 (OriginLab).

3. Methods

3.1. Express and purify EYA2 ED from E. coli.

Brief summary of the protocol: Human EYA2 ED is expressed and purified as a GST-fusion protein from E. coli. GST is then cleaved, and EYA2 ED is further purified using gel filtration for subsequent enzymatic assays.

  1. Grow overnight starter culture of E. coli containing the EYA2-ED plasmid by inoculating 10 ml of E. coli culture media 1 with a single colony or 10 μL glycerol stock (prepare 10 ml starter culture for each liter of large culture in step 2). The culture will be grown at 200 RPM, 37 °C, on a shaker for 12-16 h.

  2. Inoculate each liter of E. coli culture media 2 that has been prewarmed to 37 °C with 10 mL of the overnight starter culture.

  3. Grow the E. coli at 37 °C and 200 RPM on a shaker until OD600 reaches ~0.8-1.0.

  4. Add 300 μL of IPTG stock (1M) per liter of culture to induce EYA2 ED expression at 0.3 mM IPTG concentration.

  5. Reduce temperature to 25 °C and continue to grow the bacteria at 200 RPM overnight (18-20 hrs).

  6. Harvest bacteria culture by centrifugation at 5000 RPM, 6 °C for 10 min.

  7. Bacteria pellet can be lysed immediately as in step 8, or be frozen at −80 °C for later use. If frozen, thaw the pellet at room temperature before resuspension.

  8. Add approximately 10 mL of lysis buffer to pellet from 1 L of cells to resuspend the pellet (see Note 1 in section 4.1). Take 20 μL whole cell lysate sample for SDS PAGE in step 21 (see Note 2 in section 4.1).

  9. Add protease inhibitors to their respective final concentrations: Leupeptin: 1 μM, Pepstatin A: 1 μg/mL, PMSF: 1 mM.

  10. Lyse bacteria cells via sonication (45 seconds x 6 at 85% power) while manually rotating on ice (to keep the solution cool) with 2 min rests between each 45-second sonication.

  11. (Optional) Bring NaCl concentration up to 0.5 M for centrifugation (which helps making the pellet compact) by adding 5 M NaCl directly to the lysate.

  12. Collect the supernatant after centrifugation (~18,000g for 40 min). Take 20 μL supernatant and pellet samples for SDS PAGE in step 21.

  13. (Optional) Resuspend the pellet with 200 mL lysis buffer, sonicate, and spin down using the same condition in 12 and combine the supernatants (see Note 3 in section 4.1).

  14. (Optional) Centrifuge one or two more times (~18,000xg for 40 min) to generate clear supernatant.

  15. While centrifugation is underway, prewash glutathione resin with 2-3 bed volumes of lysis buffer.

  16. Load clear supernatant onto glutathione resin-packed column. Flow through the column via gravity at 4 °C (see Note 4 in section 4.1).

  17. Collect flowthrough and load onto column again using gravity flow.

  18. Wash column thoroughly by gravity-flow with 100-150 mL of the lysis buffer (>20x bed volume). Take 20 μL of 50% resin slurry as the pre-cut sample for SDS PAGE in step 21.

  19. After wash, add 10 mL lysis buffer and 2 units of PreScission protease per 100 μg of GST-fusion proteins to resin and incubate at 4 °C overnight on an orbital shaker.

  20. Elute in 5 individual 1 mL aliquots of lysis buffer with gravity flow. Take 20 μL from each elution and 50% resin slurry as the elution and post-cut samples for SDS PAGE in step 21.

  21. Boil samples mentioned in steps 9, 12, 18, and 20 with 5x protein-loading buffer for 5 min and run on SDS gel for 90 min at 100 V. Stain gel with Coomassie Brilliant Blue stain on an orbital shaker for 60 min. Destain with destain buffer on an orbital shaker for 90 min. Evaluate to ensure the EYA2 ED purification proceeded normally and to evaluate the EYA2 ED purity.

  22. Combine all eluates from step 20 and concentrate to 2-5 mL with a concentrator at 3,000g, 4 °C. Stop immediately if protein precipitation is observed.

  23. Spin tubes for 5 min at 4 °C to remove any protein precipitation.

  24. Load supernatant onto pre-washed Superdex 200 column.

  25. Analyze Superdex 200 fractions under the elution peak corresponding to the correct molecular weight using SDS PAGE. If concentration of the fractions with pure EYA2 ED is sufficiently high, aliquot and freeze at −80 °C. If not, concentrate to desired concentration, aliquot and freeze at −80 °C.

3.2. Analyze the EYA2 phosphatase kinetics using an OMFP-based fluorescent phosphatase assay.

Brief summary of the protocol: 150 nM EYA2 ED is incubated with 16.6 to 1000 μM OMFP substrate. The amount of fluorescent product OMF is monitored in a time course to determine the initial velocity. Plotting of the initial velocity versus OMFP concentration generates Vmax and Km by fitting to a Michaelis-Menton equation.

  1. Prepare a working stock of 300 nM EYA2 ED in 2X assay buffer in enough volume to add 25 μL for every replicate of the reaction. These assays are typically performed in triplicate.

  2. Using a multichannel pipette, transfer 25 μL from this EYA2 ED stock to each well to be used in the black 96-well plate. Deposit solution down the side of the well to avoid introducing bubbles, which will interfere with the fluorescence readings. If bubbles occur, gently swirl or tap the plate to dissipate.

  3. Transfer 25 μL of assay buffer to one column of the black 96-well plate to determine the background rate of background OMFP hydrolysis at each substrate concentration, serving as a no-enzyme control.

  4. Prepare 2X working stock solutions of OMFP in H2O at concentrations of 2000 μM, 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM, and 31.25 μM (see Note 1 in section 4.2). Transfer 150 μL of each prepared dilution, in descending concentration, to a single column of the clear 96-well plate.

  5. Set up plate reader to read fluorescence with excitation/emission wavelengths of 485 nm/515 nm, reading each well once every 2 minutes (see Note 2 in section 4.2). The reaction will begin upon addition of OMFP to the reaction plate, so be sure the plate reader will be ready to take readings immediately following OMFP addition.

  6. Initiate reaction by adding 25 μL of prepared OMFP from the clear plate to the corresponding wells of the black reaction plate using multichannel pipette. Gently pipette to mix and avoid introduction of bubbles. Note time. At a known time interval, repeat this addition for all replicates of the reaction, then place assay plate in plate reader and begin measurement. Allow reaction to proceed for one hour.

  7. Upon reaction completion, export data from plate reader as a table of RFU measurements versus time, in seconds. Correct the time measurement for the different replicates to account for the time interval during substrate addition in the previous step.

  8. Graph RFU values versus time to generate a saturation plot for each substrate concentration (Fig 1). Review the plots to determine which measurements were taken during the initial velocity phase of the reaction, then fit these measurements using a linear regression. The slope of this linear regression is the initial reaction velocity for the corresponding substrate concentration in RFU/sec.

  9. Determine the rate of background OMFP hydrolysis at each substrate concentration by fitting time points with linear regression. Subtract the rate of background OMFP hydrolysis at each substrate concentration from the initial velocities of Eya2 at each substrate concentration to account for hydrolysis of OMFP in assay buffer.

  10. Plot initial velocity versus substrate concentration to obtain a curve that can be fit to the Michaelis-Menton equation to determine Km and Vmax values (Fig 2, see Note 3 and 4 in section 4.2).

Fig. 1.

Fig. 1.

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Determining the initial velocity of Eya2 ED at different substrate concentrations using an OMFP assay.

Fig. 2.

Fig. 2.

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Determining the Vmax and Km of Eya2 ED using the Michaelis-Menton equation.

3.3. Evaluate small molecule inhibitors of the EYA2 phosphatase using an OMFP-based fluorescent phosphatase assay.

Brief summary of the protocol: 150 nM EYA2 ED is incubated with 25 μM OMFP substrate and 45.7 nM to 100 μM inhibitor for an hour. Plotting of the percentage activity versus inhibitor concentration on a log scale allows for fitting of a dose-response curve and determination of IC50.

  1. Dilute EYA2 ED protein to 300 nM in assay buffer (see Note 1 in section 4.3).

  2. Prepare serial dilutions of inhibitors at 100X of the desired concentrations using DMSO (see Note 2 in section 4.3).

  3. Dilute OMFP to 50 μM in H2O to be used as substrate.

  4. Dilute EDTA to 0.45 M in H2O to be used to stop the reaction.

  5. Load 49 μl of buffer without protein per well as a blank control (see Note 3 in section 4.3).

  6. Load 49 μL of 300 nM EYA2 ED per well as an uninhibited control.

  7. Load 49 μL of 300 nM EYA2 ED per well for all inhibitor concentrations.

  8. Add 1 μL DMSO to the blank control (buffer only), 1 μL of DMSO to uninhibited control (protein only), and 1 μL of prepared inhibitor to each respective well.

  9. Incubate 10 minutes at room temperature on an orbital rotator.

  10. Start the reaction by adding 50 μL of 50 μM OMFP to each well (see Note 4 in section 4.3). Final reaction conditions are 150 nM EYA2 ED and 25 μM OMFP.

  11. Incubate in dark, covered, for 1 hour.

  12. Stop the reaction by adding 20 μL of 0.45 M EDTA, pH 8.0 to each well (see Note 4 in section 4.3). Final concentration of EDTA is 75 mM.

  13. Read fluorescence on the plate reader, with excitation at 485 nm and emission at 515 nm. An example of the dose response curve for one small molecule inhibitor is shown in Fig. 3.

Fig. 3.

Fig. 3.

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Evaluating the inhibition of Eya2 phosphatase activity by a small molecule using the OMFP assay.

3.4. Evaluate small molecule inhibitor of the EYA2 phosphatase using a pH2AX-based malachite green assay.

Brief summary of the protocol: 3.9 μM EYA2 ED is incubated with 50 μM pH2AX peptide substrate, the Malachite Green reagent, and 45.7 nM to 100 μM inhibitor for 1 hour. Plotting of the percentage activity versus inhibitor concentration on a log scale allows for fitting of a dose-response curve and determination of IC50.

  1. Make a 2X Malachite Green assay buffer containing 7.8 μM EYA2. Keep everything on ice until it is added to the plate.

  2. Add 25 μL of the above 2X Malachite green assay buffer and EYA protein to wells of clear, 96-well, half-area plates (see Notes 1-3 in section 4.4).

  3. Add 22 μL H2O to each well to bring the final volume to 50 μL when all components are added.

  4. Add 0.5 μL inhibitors to each well.

  5. Incubate at room temperature for 10 min.

  6. Add 2.5 μL pH2AX (1mM stock) to wells to achieve a final concentration of 50 μM and to start the reaction.

  7. Incubate at 25 °C for 1 h.

  8. Add 100 μL Malachite Green reagent to each well.

  9. Incubate 20 min at room temperature to develop the color complex.

  10. Read plate at 620 nm.

  11. Dose response curves were generated using Origin Pro 8.0. An example of the dose response curve for one small molecule inhibitor is shown in Fig. 4.

Fig. 4.

Fig. 4.

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Evaluating the inhibition of Eya2 phosphatase activity by a small molecule using the Malachite Green assay with the pH2AX peptide as a substrate.

4. Notes

4.1. Express and purify EYA2 ED from E. coli.

  1. EYA2 ED tolerates a wide variety of buffers for purification fairly well.

  2. Several samples will be taken throughout the protocol to be analyzed on a Coomassie-stained SDS gel to monitor the purification process and identify any potential issues.

  3. Resuspension of the pellet after initial sonication is only required if the expression level of the protein is low. This is not typically needed with the EYA2 ED construct.

  4. Protein binding to glutathione resin can also be done in batch mode. Resuspend pre-washed resin in 1-2 mL of lysis buffer and add to the lysate which is then incubated on an orbital shaker at 4°C for 2 hrs. Centrifuge the lysate and resin mixture at 4000 rpm, 4°C for 10 minutes. Resuspend the resin in 2 mL lysis buffer and add to a column. Continue with step 18 in section 3.1.

4.2. Analyze the EYA2 phosphatase kinetics using an OMFP-based fluorescent phosphatase assay.

  1. OMFP dilutions of 2000 μM, 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM, and 31.25 μM can be prepared by serial dilution at 1:2 ratio beginning with 2000 μM OMFP. Substrate range may need to be adjusted for other PTPs to cover from 0.2 to 5x of the Km.

  2. Rate of measurement will depend on the specifications of the plate reader. A time interval should be chosen such that each well is measured once during that interval; a faster plate reader may allow for a shorter interval between measurements.

  3. Due to the low solubility of OMFP, we cannot reach Vmax. Other more soluble small molecule substates such as DFP or pNPP can be used to overcome this problem.

  4. Vmax can be used to calculate Kcat using the equation Kcat=Vmax/[Eya2] after converting RFU to molar quantity of OMF using a standard curve and expressing the initial velocity in moles per unit time.

4.3. Evaluate small molecule inhibitors of the EYA2 phosphatase using OMFP-based fluorescent phosphatase assay

  1. It is ideal to evaluate inhibitor effect at an enzyme concentration that produces >5-fold signal/noise ratio at selected time point and a substrate concentration below Km.

  2. Ideal inhibitor concentrations will vary for different enzymes and inhibitors. For EYA2 ED, prepare 8 serial dilutions at a ratio of 1:3 beginning with 10 mM inhibitor. This will generate inhibitor concentrations (100x) of 10 mM, 3.33 mM, 1.11 mM, 370 μM, 123 μM, 41.2 μM, 13.7 μM, and 4.57 μM. The final inhibitor concentrations in each well are: 100, 33.3, 11.1, 3.7, 1.23, 0.412, 0.137, and 0.0457 μM. Make stocks of inhibitors in DMSO for each concentration to keep the DMSO concentration in each inhibitor condition the same.

  3. Quantities listed are for one replicate. Prepare triplicate reactions for all conditions (blank control, uninhibited control, and each concentration of inhibitor).

  4. OMFP and EDTA can be added to each replicate of different inhibitor concentrations simultaneously using a multi-channel pipette. Addition of OMFP to different replicates should be done sequentially in 20 or 30 second intervals to ensure consistent timing for all reactions. Quenching with EDTA should then follow the same intervals, ensuring each reaction has proceeded exactly 1 hour.

4.4. Evaluate small molecule inhibitors of the EYA2 phosphatase using a pH2AX-based Malachite Green assay.

  1. A kinetic assay similar to what is described in section 3.2 (but using the pH2AX peptide and Malachite Green assay instead) can be performed first to determine the optimal enzyme and substrate concentration to be used in the endpoint assay for evaluating the effect of inhibitors.

  2. Use caution when pipetting. Try not to create bubbles in the wells as they will interfere with the reading.

  3. Always do the following controls to check for possible free phosphate contamination: protein + peptide without inhibitor (uninhibited), protein + peptide + EDTA (fully inhibited). If the fully inhibited sample has significant absorbance reading, it indicates potential free phosphate contamination in either the protein, peptide, or reagents (buffer, DMSO, small molecule). Malachite Green assay with each of these individual components alone can be carried out to identify the source of contamination and replace the reagent with one from a different source to remove the contamination.

Acknowledgement

Research reported in this paper was supported by the National Cancer Institute of the National Institute of Health under award number R03DA030559 (R.Z. and H.L.F.), R01CA221282 (H.L.F. and R.Z.), R01CA224867 (H.L.F.), R01NS108396 (H.L.F.), as well as National Institute of General Medical Sciences of the National Institute of Health under award number R35GM145289 (R.Z.). C.A. is supported by the NIH NRSA T32CA174648 Training in Translational Research of Lung, Head and Neck Cancer. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References

  • 1.Zimmerman JE, Bui QT, Liu H, Bonini NM (2000) Molecular genetic analysis of Drosophila eyes absent mutants reveals an eye enhancer element. Genetics 154 (1):237–246. doi: 10.1093/genetics/154.1.237 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Xu PX, Cheng J, Epstein JA, Maas RL (1997) Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function. Proc Natl Acad Sci U S A 94 (22):11974–11979. doi: 10.1073/pnas.94.22.11974 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Xu PX, Zheng W, Laclef C, Maire P, Maas RL, Peters H, Xu X (2002) Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid. Development 129 (13):3033–3044. doi: 10.1242/dev.129.13.3033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Zheng W, Huang L, Wei ZB, Silvius D, Tang B, Xu PX (2003) The role of Six1 in mammalian auditory system development. Development 130 (17):3989–4000. doi: 10.1242/dev.00628 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ikeda K, Ookawara S, Sato S, Ando Z, Kageyama R, Kawakami K (2007) Six1 is essential for early neurogenesis in the development of olfactory epithelium. Dev Biol 311 (1):53–68. doi: 10.1016/j.ydbio.2007.08.020 [DOI] [PubMed] [Google Scholar]
  • 6.Grifone R, Demignon J, Houbron C, Souil E, Niro C, Seller MJ, Hamard G, Maire P (2005) Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo. Development 132 (9):2235–2249. doi: 10.1242/dev.01773 [DOI] [PubMed] [Google Scholar]
  • 7.Li X, Oghi KA, Zhang J, Krones A, Bush KT, Glass CK, Nigam SK, Aggarwal AK, Maas R, Rose DW, Rosenfeld MG (2003) Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426 (6964):247–254. doi: 10.1038/nature02083 [DOI] [PubMed] [Google Scholar]
  • 8.Ozaki H, Nakamura K, Funahashi J, Ikeda K, Yamada G, Tokano H, Okamura HO, Kitamura K, Muto S, Kotaki H, Sudo K, Horai R, Iwakura Y, Kawakami K (2004) Six1 controls patterning of the mouse otic vesicle. Development 131 (3):551–562. doi: 10.1242/dev.00943 [DOI] [PubMed] [Google Scholar]
  • 9.Pignoni F, Hu B, Zavitz KH, Xiao J, Garrity PA, Zipursky SL (1997) The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91 (7):881–891. doi: 10.1016/s0092-8674(00)80480-8 [DOI] [PubMed] [Google Scholar]
  • 10.Ford HL, Kabingu EN, Bump EA, Mutter GL, Pardee AB (1998) Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis. Proc Natl Acad Sci U S A 95 (21):12608–12613. doi: 10.1073/pnas.95.21.12608 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Reichenberger KJ, Coletta RD, Schulte AP, Varella-Garcia M, Ford HL (2005) Gene amplification is a mechanism of Six1 overexpression in breast cancer. Cancer Res 65 (7):2668–2675. doi: 10.1158/0008-5472.CAN-04-4286 [DOI] [PubMed] [Google Scholar]
  • 12.Coletta RD, Christensen KL, Micalizzi DS, Jedlicka P, Varella-Garcia M, Ford HL (2008) Six1 overexpression in mammary cells induces genomic instability and is sufficient for malignant transformation. Cancer Res 68 (7):2204–2213. doi: 10.1158/0008-5472.CAN-07-3141 [DOI] [PubMed] [Google Scholar]
  • 13.Micalizzi DS, Wang CA, Farabaugh SM, Schiemann WP, Ford HL (2010) Homeoprotein Six1 increases TGF-beta type I receptor and converts TGF-beta signaling from suppressive to supportive for tumor growth. Cancer Res 70 (24):10371–10380. doi: 10.1158/0008-5472.CAN-10-1354 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wang CA, Jedlicka P, Patrick AN, Micalizzi DS, Lemmer KC, Deitsch E, Casas-Selves M, Harrell JC, Ford HL (2012) SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer. J Clin Invest 122 (5):1895–1906. doi: 10.1172/JCI59858 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Tan J, Zhang C, Qian J (2011) Expression and significance of Six1 and Ezrin in cervical cancer tissue. Tumour Biol 32 (6):1241–1247. doi: 10.1007/s13277-011-0228-8 [DOI] [PubMed] [Google Scholar]
  • 16.Wan F, Miao X, Quraishi I, Kennedy V, Creek KE, Pirisi L (2008) Gene expression changes during HPV-mediated carcinogenesis: a comparison between an in vitro cell model and cervical cancer. Int J Cancer 123 (1):32–40. doi: 10.1002/ijc.23463 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Behbakht K, Qamar L, Aldridge CS, Coletta RD, Davidson SA, Thorburn A, Ford HL (2007) Six1 overexpression in ovarian carcinoma causes resistance to TRAIL-mediated apoptosis and is associated with poor survival. Cancer Res 67 (7):3036–3042. doi: 10.1158/0008-5472.CAN-06-3755 [DOI] [PubMed] [Google Scholar]
  • 18.Ng KT, Lee TK, Cheng Q, Wo JY, Sun CK, Guo DY, Lim ZX, Lo CM, Poon RT, Fan ST, Man K (2010) Suppression of tumorigenesis and metastasis of hepatocellular carcinoma by shRNA interference targeting on homeoprotein Six1. Int J Cancer 127 (4):859–872. doi: 10.1002/ijc.25105 [DOI] [PubMed] [Google Scholar]
  • 19.Ng KT, Man K, Sun CK, Lee TK, Poon RT, Lo CM, Fan ST (2006) Clinicopathological significance of homeoprotein Six1 in hepatocellular carcinoma. Br J Cancer 95 (8):1050–1055. doi: 10.1038/sj.bjc.6603399 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Yu Y, Khan J, Khanna C, Helman L, Meltzer PS, Merlino G (2004) Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med 10 (2):175–181. doi: 10.1038/nm966 [DOI] [PubMed] [Google Scholar]
  • 21.Sehic D, Karlsson J, Sandstedt B, Gisselsson D (2012) SIX1 protein expression selectively identifies blastemal elements in Wilms tumor. Pediatr Blood Cancer 59 (1):62–68. doi: 10.1002/pbc.24025 [DOI] [PubMed] [Google Scholar]
  • 22.Ono H, Imoto I, Kozaki K, Tsuda H, Matsui T, Kurasawa Y, Muramatsu T, Sugihara K, Inazawa J (2012) SIX1 promotes epithelial-mesenchymal transition in colorectal cancer through ZEB1 activation. Oncogene 31 (47):4923–4934. doi: 10.1038/onc.2011.646 [DOI] [PubMed] [Google Scholar]
  • 23.Robin TP, Smith A, McKinsey E, Reaves L, Jedlicka P, Ford HL (2012) EWS/FLI1 regulates EYA3 in Ewing sarcoma via modulation of miRNA-708, resulting in increased cell survival and chemoresistance. Mol Cancer Res 10 (8):1098–1108. doi: 10.1158/1541-7786.MCR-12-0086 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Pandey RN, Rani R, Yeo EJ, Spencer M, Hu S, Lang RA, Hegde RS (2010) The Eyes Absent phosphatase-transactivator proteins promote proliferation, transformation, migration, and invasion of tumor cells. Oncogene 29 (25):3715–3722. doi: 10.1038/onc.2010.122 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Wang Y, Klijn JG, Zhang Y, Sieuwerts AM, Look MP, Yang F, Talantov D, Timmermans M, Meijer-van Gelder ME, Yu J, Jatkoe T, Berns EM, Atkins D, Foekens JA (2005) Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 365 (9460):671–679. doi: 10.1016/S0140-6736(05)17947-1 [DOI] [PubMed] [Google Scholar]
  • 26.Li CM, Guo M, Borczuk A, Powell CA, Wei M, Thaker HM, Friedman R, Klein U, Tycko B (2002) Gene expression in Wilms' tumor mimics the earliest committed stage in the metanephric mesenchymal-epithelial transition. Am J Pathol 160 (6):2181–2190. doi: 10.1016/S0002-9440(10)61166-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Farabaugh SM, Micalizzi DS, Jedlicka P, Zhao R, Ford HL (2012) Eya2 is required to mediate the pro-metastatic functions of Six1 via the induction of TGF-beta signaling, epithelial-mesenchymal transition, and cancer stem cell properties. Oncogene 31 (5):552–562. doi: 10.1038/onc.2011.259 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wang QF, Wu G, Mi S, He F, Wu J, Dong J, Luo RT, Mattison R, Kaberlein JJ, Prabhakar S, Ji H, Thirman MJ (2011) MLL fusion proteins preferentially regulate a subset of wild-type MLL target genes in the leukemic genome. Blood 117 (25):6895–6905. doi: 10.1182/blood-2010-12-324699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Miller SJ, Lan ZD, Hardiman A, Wu J, Kordich JJ, Patmore DM, Hegde RS, Cripe TP, Cancelas JA, Collins MH, Ratner N (2010) Inhibition of Eyes Absent Homolog 4 expression induces malignant peripheral nerve sheath tumor necrosis. Oncogene 29 (3):368–379. doi: 10.1038/onc.2009.360 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Auvergne RM, Sim FJ, Wang S, Chandler-Militello D, Burch J, Al Fanek Y, Davis D, Benraiss A, Walter K, Achanta P, Johnson M, Quinones-Hinojosa A, Natesan S, Ford HL, Goldman SA (2013) Transcriptional differences between normal and glioma-derived glial progenitor cells identify a core set of dysregulated genes. Cell Rep 3 (6):2127–2141. doi: 10.1016/j.celrep.2013.04.035 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Li Z, Qiu R, Qiu X, Tian T (2017) EYA2 promotes lung cancer cell proliferation by downregulating the expression of PTEN. Oncotarget 8 (67):110837–110848. doi: 10.18632/oncotarget.22860 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Anantharajan J, Zhou H, Zhang L, Hotz T, Vincent MY, Blevins MA, Jansson AE, Kuan JWL, Ng EY, Yeo YK, Baburajendran N, Lin G, Hung AW, Joy J, Patnaik S, Marugan J, Rudra P, Ghosh D, Hill J, Keller TH, Zhao R, Ford HL, Kang C (2019) Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells. Mol Cancer Ther 18 (9):1484–1496. doi: 10.1158/1535-7163.MCT-18-1239 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Rayapureddi JP, Kattamuri C, Steinmetz BD, Frankfort BJ, Ostrin EJ, Mardon G, Hegde RS (2003) Eyes absent represents a class of protein tyrosine phosphatases. Nature 426 (6964):295–298. doi: 10.1038/nature02093 [DOI] [PubMed] [Google Scholar]
  • 34.Tootle TL, Silver SJ, Davies EL, Newman V, Latek RR, Mills IA, Selengut JD, Parlikar BE, Rebay I (2003) The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature 426 (6964):299–302. doi: 10.1038/nature02097 [DOI] [PubMed] [Google Scholar]
  • 35.Jung SK, Jeong DG, Chung SJ, Kim JH, Park BC, Tonks NK, Ryu SE, Kim SJ (2010) Crystal structure of ED-Eya2: insight into dual roles as a protein tyrosine phosphatase and a transcription factor. FASEB J 24 (2):560–569. doi: 10.1096/fj.09-143891 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Krishnan N, Jeong DG, Jung SK, Ryu SE, Xiao A, Allis CD, Kim SJ, Tonks NK (2009) Dephosphorylation of the C-terminal tyrosyl residue of the DNA damage-related histone H2A.X is mediated by the protein phosphatase eyes absent. J Biol Chem 284 (24):16066–16070. doi: 10.1074/jbc.C900032200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Blevins MA, Towers CG, Patrick AN, Zhao R, Ford HL (2015) The SIX1-EYA transcriptional complex as a therapeutic target in cancer. Expert Opin Ther Targets 19 (2):213–225. doi: 10.1517/14728222.2014.978860 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Wu K, Li Z, Cai S, Tian L, Chen K, Wang J, Hu J, Sun Y, Li X, Ertel A, Pestell RG (2013) EYA1 phosphatase function is essential to drive breast cancer cell proliferation through cyclin D1. Cancer Res 73 (14):4488–4499. doi: 10.1158/0008-5472.CAN-12-4078 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Tadjuidje E, Wang TS, Pandey RN, Sumanas S, Lang RA, Hegde RS (2012) The EYA tyrosine phosphatase activity is pro-angiogenic and is inhibited by benzbromarone. PLoS One 7 (4):e34806. doi: 10.1371/journal.pone.0034806 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Krueger AB, Dehdashti SJ, Southall N, Marugan JJ, Ferrer M, Li X, Ford HL, Zheng W, Zhao R (2013) Identification of a selective small-molecule inhibitor series targeting the eyes absent 2 (Eya2) phosphatase activity. J Biomol Screen 18 (1):85–96. doi: 10.1177/1087057112453936 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Cook PJ, Ju BG, Telese F, Wang X, Glass CK, Rosenfeld MG (2009) Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions. Nature 458 (7238):591–596. doi: 10.1038/nature07849 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Yuan B, Cheng L, Chiang HC, Xu X, Han Y, Su H, Wang L, Zhang B, Lin J, Li X, Xie X, Wang T, Tekmal RR, Curiel TJ, Yuan ZM, Elledge R, Hu Y, Ye Q, Li R (2014) A phosphotyrosine switch determines the antitumor activity of ERbeta. J Clin Invest 124 (8):3378–3390. doi: 10.1172/JCI74085 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Zhang G, Dong Z, Gimple RC, Wolin A, Wu Q, Qiu Z, Wood LM, Shen JZ, Jiang L, Zhao L, Lv D, Prager BC, Kim LJY, Wang X, Zhang L, Anderson RL, Moore JK, Bao S, Keller TH, Lin G, Kang C, Hamerlik P, Zhao R, Ford HL, Rich JN (2021) Targeting EYA2 tyrosine phosphatase activity in glioblastoma stem cells induces mitotic catastrophe. J Exp Med 218 (11). doi: 10.1084/jem.20202669 [DOI] [PMC free article] [PubMed] [Google Scholar]

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