A

aPsblss©

K

1

pcecautispan

oAf

0d

Cell Calcium 43 (2008) 515–519

Short communication

A fluorimetric method for determination of calcineurin activity

Brian Roberts a,b, Jan Pohl c, Jennifer L. Gooch a,b,∗a Department of Medicine, Division of Nephrology, Emory University School of Medicine, United States

b Atlanta Veterans Administration Medical Center, United Statesc Microchemical and Proteomics Facility, Emory University School of Medicine, United States

Received 14 June 2007; received in revised form 6 August 2007; accepted 7 August 2007Available online 29 September 2007

bstract

Calcineurin is a calcium-dependent, serine/threonine phosphatase that is involved in a variety of signaling pathways. Calcineurin is distinctmong phosphatases because its activity requires calcium and is not sensitive to inhibition by compounds that block the related phosphatasesP1A and PP2A. Therefore, the most common methods to measure calcineurin activity rely on calcium-dependent dephosphorylation of a sub-trate derived from the RII subunit of protein kinase A in the presence of PP1A/PP2A inhibitors. However, current techniques quantify activityy measurement of released radioactive phosphate or detection of free phosphate with malachite green. Both methods involve technical chal-enges and have undesirable features. We report a new calcineurin fluorimetric assay that utilizes a fluorescently labeled phosphopeptide sub-

trate and separation of dephosphorylated peptide product by titanium-oxide. The method is rapid, quantitative, involves no radioactivity and isuitable for high throughput assays. Furthermore, with the use of a standard curve, precise measurements of calcineurin activity can be obtained.

2007 Elsevier Ltd. All rights reserved.

eywords: Calcineurin; Assay; Fluorimetric

hfRposittrnf

. Introduction

Calcineurin is a calcium-dependent, serine/threoninehosphatase. The most common method for detection of cal-ineurin activity is based on earlier published work of Frumant al. [1] who established optimal reaction conditions for cal-ineurin activity. In brief, a peptide substrate is generatednd then incubated with protein kinase A and 32P�[ATP]nder appropriate conditions to result in phosphorylation ofhe peptide with a radioactive residue. The labeled substrates then purified and used within a short period of time as a sub-trate for calcineurin. To measure calcineurin activity, equal

arts of cell lysate, reaction mixture, and labeled substratere incubated at 30 ◦C for 10 min before the reaction is termi-ated. To determine how much of the phosphorylated peptide

∗ Corresponding author at: Department of Medicine, Division of Nephrol-gy, Emory University School of Medicine, 101 Woodruff Circle, WMB 338,tlanta, GA 30322, United States. Tel.: +1 404 727 2525;

ax: +1 404 727 3425.E-mail address: jgooch@emory.edu (J.L. Gooch).

it

ulpiam

143-4160/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2007.08.004

as been dephosphorylated, individual columns are preparedor each reaction containing pre-charged ion-exchange resin.eactions are loaded on the column and unincorporated phos-hate, which does not bind the resin, is eluted. The amountf radioactivity in the eluted fractions is then measured in acintillation counter and used to quantify calcineurin activ-ty. In general, the method has several drawbacks includinghe use of radioactive phosphate for labeling of the pep-ide substrate, background due to unincorporated phosphate,eliance upon ion exchange to separate phosphorylated fromon-phosphorylated peptide, and the final measurement ofree phosphate to represent calcineurin activity. These factorsncrease variability of the data and reduce reproducibility ofhe assay.

We report a new assay to determine calcineurin activitysing well-characterized reaction conditions, a fluorescentlyabeled phosphopeptide substrate, and separation of dephos-

horylated substrate by titanium-oxide. The method is fast,nvolves no radioactivity, and is suitable for high throughputssays. Furthermore, with the use of a standard curve, preciseeasurements of calcineurin activity can be obtained.

5 Calciu

2

2

bo(MVDscppTrb1c0M(po

2

rv1c(iTcpam5

2

mpw[iiGbewa[

2

HAntitEtcTTdfbtmover the m/z range of 50–1350. Precursors were determinedby each cycle’s MS spectrum from the m/z range of 375–1100.Straight MS was performed over the m/z range of 350–1350.For each sample, extracted ion chromatograms (XIC) were

Fig. 1. Diagram of fluorimetric calcineurin assay. The RII peptide substrateis synthesized with a phospho-serine 15 residue and an amino-terminus flu-orescein tag. In a 96-well plate, the labeled substrate is mixed in equal partswith reaction buffer and sample and allowed to incubate at 30 ◦C for 10 min.

16 B. Roberts et al. / Cell

. Methods

.1. Materials and reagents

Recombinant calcineurin was purchased from Cal-iochem (San Diego, CA) and all other chemicals werebtained from Sigma (St. Louis, MO). Titanium-oxideTiO4)-coated plates were obtained from Glygen (Colombus,

A). The RII peptide, fluoresceinyl-DLDVPIPGRFDRR-SVAAE, and its phosphorylated analog (Fluoresceinyl-LDVPIPGRFDRRVpSVAAE where pS = l-phospho-

erine) were synthesized at the Emory University Micro-hemical and Proteomics Facility by Fmoc-based solid-phaseeptide synthesis using model Liberty microwave-assistedeptide synthesizer (CEM Corporation, Matthews, NC).he peptides were purified to apparent homogeneity by

eversed-phase HPLC and their masses were confirmedy mass spectrometry. Peptide was diluted in Tris 50 mM,00 mM NaCl, 0.5 mM DTT, and 0.1 mg/ml BSA to a finaloncentration of 30 ng/ml. Reaction buffer consisted of.1 mg/ml BSA, 35 mM Tris pH 7.5, 25 mM NaCl, 2.0 mMgCl2, 270 �M DTT, 500 �M EDTA, 419 nM okadaic acid

in 0.63% ethanol), 25 mM CaCl2 or 500 uM EGTA. TiO4lates were pre-incubated with a binding buffer consistingf 0.1% acetic acid in 10% acetonitrile.

.2. Protocol

Twenty microliters diluted peptide substrate, 20 �l ofeaction buffer, and 20 �l of sample were loaded into indi-idual wells of a 96-well plate and incubated at 30 ◦C for0 min. During the reaction time, a 96-well plate with TiO4-oated wells was prepared by adding 80 �l of binding buffer0.1% acetic acid in 10% acetonitrile) per well. After thencubation period, reactions were transferred into preparediO4-coated wells followed by gentle shaking for 5 min. Theontents of each well were then removed to a white 96-welllate preloaded with 20 �l of 3 N ammonium hydroxide. Themount of fluorescent label that did not bind to the TiO4atrix was quantified by fluorimetry at 485 nm excitation

28 nm emission.

.3. Experimental models

Primary mouse renal fibroblasts were obtained frominced kidneys and propagated in culture using RPMI sup-

lemented with 5% serum and pen/strep antibiotics. Lysatesere prepared using a hypotonic lysis buffer as described

2,3]. Wildtype, adult mice are maintained in the animal facil-ty at the Atlanta Veterans Administration Medical Centern accordance with Institutional Care and Use Committeeuidelines. Where indicated, wildtype mice were treatedy sub-cutaneous injection with either vehicle alone (10%

thanol in Ringer’s lactate solution) or with 10 mg/kg bodyeight cyclosporin A daily for 3 days. Organs were harvested

nd calcineurin activity determined as previously described4].

E(spa

m 43 (2008) 515–519

.4. Mass spectrometry

The system used for the analysis was an Ultimate capillaryPLC system (LC Packings) with a FAMOS autosampler.n 0.5 mm × 150 mm C18SB-300 Zorbax (Agilent, Tech-ologies, Palo Alto, CA) reversed-phase column was used ashe analytical column. The LC eluent was directly sprayednto the 4000Qtrap mass spectrometer using a TurboV elec-rospray ion source (Applied Biosystems, Foster City, CA).lution from the column was accomplished with an acetoni-

rile gradient from 2% to 80% with 0.1% formic acid as aounter ion for HPLC. The flow rate was set at 15 �l/min.he total LC run time was 60 min including equilibration.he 4000Qtrap was operated both in the information depen-ent acquisition (IDA) mode and straight MS mode. In IDA,or each cycle, a single MS spectrum was acquired followedy up to two MS/MS spectra based upon observed ions inhe MS spectrum. The MS spectrum was acquired over the/z range of 350–1350. Each MS/MS spectrum was acquired

ach well is then transferred to a 96-well plate coated with titanium-oxideTiO4) followed by gentle shaking to allow binding of phosphorylated sub-trate. Finally, the total contents of each well are then moved to a new 96-welllate and the amount of dephosphorylated peptide determined by fluorimetryt 485 nm excitation and 528 nm emission.

Calciu

gv1rp

3

dIptik

pmoro

loefllc

Fpawd

Fodos

B. Roberts et al. / Cell

enerated for the phosphorylated and non-phosphorylatedersions of the RII peptide. The width used for the XIC wasDa. Based on the areas of the peaks from each XIC, the

elative quantity of phosphorylated to non-phosphorylatedeptide was determined.

. Results and discussion

Calcineurin is an important enzyme but available assays toetect calcineurin activity are hampered by several problems.n the most commonly used assay, a radioactively labeled

eptide substrate is mixed with desired sample and reac-ion buffer. The most common drawbacks to this method arenconsistent labeling of the peptide with recombinant proteininase A, increased background due to incorporated 32P-

ctt

ig. 2. Validation of fluorescein-labeled RII peptide by mass spectrometry. (A) Reer reaction and then the relative amount of dephosphorylated to phosphorylated perea under the curve for dephosphorylated RII and phosphorylated RII with each coith TiO4 matrix in a 96-well plate. After binding, the samples were removed anetermined by mass spectrometry. Data shown is the ratio of the area under the cur

ig. 3. Dose–response with recombinant calcineurin. (A) Calcineurin assays were pr absence of calcium. Recombinant calcineurin resulted in increased amounts ofependent upon calcium. Data shown are the mean ± S.E.M. of triplicate reactionsut along with standard controls to verify calcineurin activity. Heat inactivation of tignificantly reduced activity (ANOVA). Data shown are the mean ± S.E.M. of trip

m 43 (2008) 515–519 517

hosphate, and quantification of “released” phosphate as aarker of calcineurin activity. An improved method would be

ne where the peptide substrate is uniformly labeled, does notequire radioactivity, and quantitatively measures the amountf dephosphorylated peptide.

Fig. 1 illustrates the steps we propose to solve these prob-ems. First, a peptide is synthesized that is phosphorylatedn Ser-15 during synthesis, thus eliminating the need fornzymatic labeling. The peptide was also generated with auorescein moiety at its amino-terminus. Next, the tagged,

abeled peptide is incubated with the desired lysate and well-haracterized reaction conditions [1] for 10 min at 30 ◦C.

Recently, TiO4 has been demonstrated to be highly spe-ific for binding of phosphorylated peptides [5,6]. Therefore,o separate phosphorylated from non-phosphorylated pep-ide, plates coated with titanium oxide are utilized. Reactions

actions were carried out with 0, 0.2, or 0.3 ng of recombinant calcineurinptide was determined by mass spectrometry. Data shown is the ratio of thendition. (B) Reactions were carried identically as in (A) and then incubatedd the relative amount of dephosphorylated to phosphorylated peptide wasve for dephosphorylated RII and phosphorylated RII with each condition.

erforms with increasing amounts of recombinant calcineurin in the presencedephosphorylated peptide in a dose-dependent manner. This activity was. (B) Reactions containing 0.2 ng of recombinant calcineurin were carriedhe enzyme, addition of an autoinhibitory peptide, or absence of calcium alllicate samples compared to a standard curve.

5 Calcium 43 (2008) 515–519

aipdnt

trpl0lctNdwtfuspo

pfacibptducopEn(

isfipsedswPmdpt

Fig. 4. Detection of calcineurin-mediated dephosphorylation. (A) Culturedrenal fibroblasts were treated with a variety of stimuli including argininevasopressin (AVP), dexamethasone, and phorbol myristate acetate (PMA)to induce calcineurin activity. In addition, some cells were pre-treated withcalcineurin inhibitors prior to addition of PMA. Cells were lysed accordingto previously established methods [2,3] and then calcineurin activity wasdetermined. Data shown is the mean ± S.E.M. of triplicate samples com-pared to a standard curve. (B) Mice were treated with cyclosporin A for 3dh[o

ts(

rdtoihrbp

18 B. Roberts et al. / Cell

re transferred to the TiO4 plate followed by gentle shak-ng at room temperature for 5 min to allow binding of thehosphorylated peptide. Dephosphorylated peptide (whichoes not bind to the TiO4 matrix) is then transferred to aew plate and quantified by fluorimetry of the fluoresceinag.

To validate this new method, we first verified by mass spec-rometry that the labeled, tagged peptide can be dephospho-ylated by calcineurin. Phosphorylated, fluorescein-taggedeptide was used as a substrate for calcineurin under estab-ished reaction conditions. After stopping the reaction with.1% acetic acid in 10% acetonitrile, the samples were ana-yzed by mass spectrometry. Fig. 2A shows that recombinantalcineurin stimulated a dose–responsive increase in the rela-ive amount of dephosphorylated to phosphorylated peptide.ext, we verified that the TiO4 matrix effectively separatedephosphorylated from phosphorylated peptide. Reactionsere performed identically as in panel (A), but with the addi-

ional step of incubating the reactions in TiO4-coated wellsor 5 min. The amount of dephosphorylated peptide in thenbound fraction was analyzed by mass spectrometry. Fig. 2Bhows that there is a 30-fold increase in the amount of dephos-horylated peptide in the unbound fraction with the additionf recombinant calcineurin.

After confirming that the labeled peptide could be dephos-horylated and the TiO4 effectively separated phosphorylatedrom non-phosphorylated peptide, we proceeded with char-cterizing the new assay. First, we analyzed reactionsontaining increasing amounts of recombinant calcineurinn either normal reaction buffer or calcium-free reactionuffer. Calcineurin in normal buffer resulted in dephos-horylation of the peptide in a dose-dependent manner. Inhe absence of calcium, however, there was no increase inephosphorylation (Fig. 3A). Next, we performed reactionssing 0.2 ng of recombinant calcineurin along with standardontrols for calcineurin activity including heat inactivationf the recombinant enzyme, addition of an autoinhibitoryeptide, absence of calcium, and chelation of calcium withGTA. Dephosphorylation by 0.2 ng calcineurin was sig-ificantly reduced (ANOVA) by each of these conditionsFig. 3B).

Finally, we explored the use of the new calcineurin assayn two model systems. Fig. 4A shows the result of calcineurintimulation by a variety of agents in cultured primary renalbroblasts. When the absorbance values obtained were com-ared to a standard curve of recombinant calcineurin runimultaneously, a determination of calcineurin activity inach condition can be made. Arginine vasopressin (AVP),examethasone, and phorbol myristate acetate (PMA) eachignificantly stimulated calcineurin activity. Pre-treatmentith the calcineurin inhibitor FK506 blocked stimulation byMA. Next, protein lysates were collected from liver and

uscle samples of mice treated with vehicle alone or treated

aily with 20 mg/kg cyclosporin A. Calcineurin assays wereerformed on 1 �g of total protein. Results indicated thathere is significantly more calcineurin activity per �g pro-

aali

ays to inhibit calcineurin activity and then liver and muscle samples werearvested. Tissues were lysed according to previously established methods4,7] and calcineurin activity determined. Data shown are the mean ± S.E.M.f triplicate samples compared to a standard curve.

ein in the liver compared to muscle. Cyclosporin treatmentignificantly decreased calcineurin activity in both tissuesFig. 4B).

While our method offers several improvements over cur-ent methods, there are a several points that are worth briefiscussion. First, acidic peptides (other than phosphopep-ides) also show affinity for TiO4 as per literature and ourwn experience. The RII peptide is, in fact, rather acidic notncluding pSer-15 (five negative and three positive charges);owever this does not seem to negatively influence theuggedness and reproducibility of the assay. Second, 32P-ased assays are very specific as they measure inorganichosphate released upon the action of the phosphatase. This

ssay, on the other hand, measures fluorescence of the probettached to unbound peptide released to solution due to theoss of phosphate (loss of binding affinity to TiO4). However,t is possible that the fluorescence probe may be released in

Calciu

sptmssmitFiatrl

aTam

mbipwtnebn

A

atDR

R

[

[

[

[

[

[

B. Roberts et al. / Cell

olution by the action of a protease, e.g. a tryptic-like enzymeresent in the biological material, cleaving the substrate athe C-terminal side of arginines. In such case, peptide frag-

ents such as fluoresceinyl-DLDVPIPGR will be releasedimulating phosphatase activity (false positive). Hence, inclu-ion of protease inhibitors is highly advisable in order toinimize such artifacts. Finally, the fluorimetric method

n fact is not limited to the incorporation of a fluoresceinag and may be modified with other fluorescent moieties.luorescein can be quenched by common reagents includ-

ng dithiothreitol and its excitation can be altered with pHnd light exposure. Use of fluorescein requires the reactiono be protected from light and neutralized prior to fluo-imetry. Other tags such as Tamra may be less pH- andight-sensitive.

To further evaluate the reproducibility of the method, wenalyzed 14 separate samples with six replicate reactions.he intra-assay variability was 9.35%. In our hands, this isfar better variability rate than we achieved with previousethodologies.In conclusion, we present a novel method for the deter-

ination of calcineurin activity that has several importantenefits compared to standard methods. First, the techniques non-radioactive and utilizes a peptide substrate that is phos-horylated during synthesis. Next, the peptide is synthesizedith a fluorescein moiety at peptide’s NH2-terminus to facili-

ate measurement by fluorimetry. Finally, phosphorylated and

on-phosphorylated peptides are separated by TiO4 which ismbedded in coating at the base of a 96-well plate. Com-ined, these innovations results in an assay that is rapid,on-radioactive, quantitative, and reproducible.

[

m 43 (2008) 515–519 519

cknowledgements

The authors wish to acknowledge Ashalk Shulka for hisdvice and input and Matt Reed for his technical exper-ise. Funding for this work was provided by NIH/NIDDKK066422-02 and DK074854 (JLG) and by NIH-NCRRR022440 (JP).

eferences

1] D.A. Fruman, S.-Y. Pai, C.N. Klee, S.J. Burakoff, B.E. Bierer, Mea-surement of calcineurin phosphatase activity in cell extracts, MethodEnzymol. 9 (1996) 146–154.

2] J.L. Gooch, Y. Tang, J.M. Ricono, H.E. Abboud, Insulin-like growthfactor-I induces renal cell hypertrophy via a calcineurin-dependentmechanism, J. Biol. Chem. 276 (2001) 42492–42500.

3] J.L. Gooch, Y. Gorin, B.X. Zhang, H.E. Abboud, Involvement ofcalcineurin in transforming growth factor-beta-mediated regulation ofextracellular matrix accumulation, J. Biol. Chem. 279 (2004) 15561–15570.

4] J.L. Gooch, J.J. Toro, R.L. Guler, J.L. Barnes, Calcineurin A-alpha butnot A-beta is required for normal kidney development and function, Am.J. Pathol. 165 (2004) 1755–1765.

5] I. Kuroda, Y. Shintani, M. Motokawa, S. Abe, M. Furuno,Phosphopeptide-selective column-switching RP-HPLC with a titaniaprecolumn, Anal. Sci. 20 (2004) 1313–1319.

6] M.W. Pinkse, P.M. Uitto, M.J. Hilhorst, B. Ooms, A.J. Heck, Selectiveisolation at the femtomole level of phosphopeptides from proteolyticdigests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns,

Anal. Chem. 76 (2004) 3935–3943.

7] J.L. Gooch, J.L. Barnes, S. Garcia, H.E. Abboud, Calcineurin is acti-vated in diabetes and is required for glomerular hypertrophy andECM accumulation, Am. J. Physiol. Renal. Physiol. 284 (2003) F144–F154.