nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases...

Nonradioactive methods for the assay of phosphoinositide3-kinases and phosphoinositide phosphatases and selectivedetection of signaling lipids in cell and tissue extractsq

Alexander Gray,a,*,1 Henric Olsson,b,1 Ian H. Batty,a Larisa Priganica,b

and C. Peter Downesa

a Division of Signal Transduction Therapy, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UKb Department of Molecular Sciences, AstraZeneca R&D, Scheeleva. 8, Lund, 221 87 Lund, Sweden

Received 9 July 2002

Abstract

We describe a novel approach to quantitation of phosphoinositides in cell extracts and in vitro enzyme-catalyzed reactions using

suitably tagged and/or labeled pleckstrin homology (PH) domains as probes. Stable complexes were formed between the biotiny-

lated target lipid and an appropriate PH domain, and phosphoinositides present in samples were detected by their ability to compete

for binding to the PH domain. Complexes were detected using AlphaScreen technology or time-resolved FRET. The assay pro-

cedure was validated using recombinant PI 3-kinase c with diC8PtdIns(4,5)P2 as substrate and general receptor for phosphoi-

nositides-1 (GRP1) PH domain as a PtdIns(3,4,5)P3-specific probe. This PI 3-kinase assay was robust, was suitable for high-

throughput screening platforms, and delivered expected IC50 values for reference compounds. The approach is adaptable to a wide

range of enzymes as demonstrated by assays of the tumor suppressor protein, PTEN, a phosphoinositide 3-phosphatase, which was

measured using the same reagents but with diC8PtdIns(3,4,5)P3 as substrate. PtdIns(3,4,5)P3 present in lipid extracts of Swiss 3T3

and HL60 cells stimulated with platelet-derived growth factor and fMLP, respectively, was also detectable at picomole sensitivity.

The versatility and general utility of this approach were demonstrated by exchanging the GRP1 PH domain for that of TAPP1

(which binds PtdIns(3,4)P2 and not PtdIns(3,4,5)P3). This system was used to monitor the accumulation of PtdIns(3,4)P2 in Swiss

3T3 cells exposed to an oxidative stress. It is therefore proposed that similar procedures should be capable of measuring any known

phosphoinositide present in cell and tissue extracts or produced in kinase and phosphatase assays by using one of several well-

characterized protein domains with appropriate phosphoinositide-binding specificity.

� 2003 Elsevier Science (USA). All rights reserved.

Phosphoinositide 3-kinases (PI 3-kinases)2 are ubiq-

uitously expressed lipid kinases that phosphorylatephosphoinositides at the 3-hydroxyl of the inositol ring.

The products of these enzymes serve as second mes-

sengers with key roles in fundamental cellular responses

such as proliferation, survival, adhesion, cell motility,and carbohydrate metabolism [1–3]. PI 3-kinases,

therefore, are increasingly attractive targets for drug

Analytical Biochemistry 313 (2003) 234–245

www.elsevier.com/locate/yabio

ANALYTICAL

BIOCHEMISTRY

qThis work was supported by the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit in Dundee

(AstraZeneca, Boehringer Ingelheime, Novo-Nordisk, Pfizer, SmithKline Beecham) and by MRC Program Grant G9823062.* Corresponding author. Fax: +44-0-1382-345893.

E-mail address: [email protected] (A. Gray).1 These authors contributed equally to this work.2 Abbreviations used: PI 3-kinase, phosphoinositide 3-kinase; HTS, high-throughput screening; PH, pleckstrin homology; BSA bovine serum

albumin; GST, glutathione S-transferase; DTT, dithiothreitol; Chaps, 3-[(3-cholamido)dimethylammonio]-1-propane sulfonate; TCA, tricholoro-

acetic acid; GRP1, general receptor for phosphoinositides 1; DMEM, Dulbecco�s modified Eagle�s medium; FCS, fetal calf serum; PDGF, platelet-derived growth factor; PBS, phosphate-buffered saline; PtdIns, phosphatidylinositol; TR-FRET, time resolved fluorescence resonance energy

transfer; TAPP1, tandem PH domain containing protein 1; PTEN, phosphatase and tensin homologue deleted on chromosome 10; FMLP, N-formyl

Methionyl-Leucyl Phenylalanyl peptide.

0003-2697/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0003-2697(02)00607-3

mail to: [email protected]

development, especially in the disease areas of inflam-mation and cancer. The recent demonstration that mice

deficient in PI 3-kinase c have an attenuated inflam-

matory response [4–6] indicates that PI 3-kinase iso-

forms are functionally specialized and that selective

inhibitors with acceptable toxicity profiles might be

therapeutically effective.

Several compounds which inhibit PI 3-kinase have

been identified, including wortmannin and the quercetinderivative LY294002. Although these are reasonably

specific for PI 3-kinases compared to other kinases they

display little selectivity within the PI 3-kinase family and

thus have no therapeutic potential. The recent crystal-

lographic structures of PI 3-kinase c bound to these

compounds and other broad-spectrum kinase inhibitors

[7] provide a way forward to the discovery of isoform-

specific PI 3-kinase inhibitors. However, a remainingbottleneck is the current PI 3-kinase assay technology,

which more or less excludes high-throughput screening

(HTS) as a tool to identify novel chemical starting

points. The detection of phosphoinositides in tissue ex-

tracts involves even more laborious procedures than

those required for enzyme assays and this limitation

prevents assessment of inhibitors and/or stimuli in vivo.

The methods currently available for the assay oflipid-metabolizing enzymes rely on either the incorpo-

ration or the release of radioactive isotopes with some

form of substrate/product separation step before quan-

titation. Estimation of cellular mass of phosphoinosi-

tides usually requires labeling of cells with [3H]inositol

or [32P]orthophosphate followed by deacylation and

HPLC analysis. Estimation of phosphoinositide mass in

tissues is at present restricted to PtdIns(3,4,5)P3 andrequires extraction of the lipids and removal of the

phosphoinositol head groups followed by a radiometric

displacement assay which also requires synthesis of ra-

diolabeled inositol(1,3,4,5)tetrakisphosphate. These as-

says also suffer from the drawback that the labeled

substrates or precursors required are expensive and

samples require extensive processing before analysis.

Here, we describe novel assays for phosphoinositidesbased on their ability to bind specifically to certain

pleckstrin homology (PH) domains. PH domains are the

major intracellular targets of PtdIns(3,4,5)P3,

PtdIns(3,4)P2, and several other phosphoinostides. Re-

cently the number of characterized PH domains binding

inositol lipids with a broad range of affinity and speci-

ficity has increased [8]. Other recently discovered phos-

phoinositide binding domains, like the phox homologydomain [9,10] and FYVE domains [11], could poten-

tially also be used in the assay formats described herein.

Other critical components of the assay concept are bi-

ologically active short acyl chain phosphoinositides that

recently have become commercially available. Important

structural features of these include short acyl side chains

(diC4 to diC8), rendering them water soluble, and the

addition of biotin to the terminus of the sn-1 acyl chainwhile still allowing selective recognition of the inositol

head group. Finally, sensitive nonradioactive detection

of lipid/PH domain complexes relies on either TR-

FRET using Lance reagents (Perkin–Elmer Wallac) or

the recently introduced AlphaScreen technology (Bio-

Signal Packard). In both cases, PtdIns(3,4)P2 or

PtdIns(3,4,5)P3 present in samples was detected in

competition assays by its ability to dissociate signal-generating complexes between PH domain, biotinylated

lipid, and detection reagents.

The assay systems presented here allow the assay of

many lipid-metabolizing enzymes in a homogeneous

format compatible with HTS and, in the case of the TR-

FRET format, capable of real-time kinetic measure-

ments. The assays presented also address the problem of

the determination of the mass of phosphoinositides incells and tissues.

Materials and methods

Detection of biotinylated phosphoinositide/PH domain

complexes. AlphaScreen detection was in 384-well mi-

croplates in 50mM Hepes, pH 7.4, 50mM NaCl, and0.1% BSA. Biotinylated, short-chain (diC6) phosphoi-

nositides and GRP1 PH–GST were added at 15 and

3.75 nM, respectively. Donor and acceptor AlphaScreen

beads (Perkin–Elmer) were added at 5 lg/ml to a final

volume of 50 ll. Plates were incubated in the dark for 5 hto ensure binding was complete and then read in an

AlphaQuest AD instrument (Perkin–Elmer) using stan-

dard settings. The TR-FRET sensor complex consistedof 50mM Hepes, pH 7.4, 150mM NaCl, 5mM DTT,

and 0.05% Chaps with APC–streptavidin (Prozyme

Ltd.) 32 nM, 120 nM biotinylated, short-chain (diC6)

phosphoinositides, and 35 nM Lance chelate labeled

GST-PH domain. Alternatively the TR-FRET sensor

complex contained 21 nM Lance chelate labeled anti-

GST antibody and unlabeled PH domain in a final

volume of 50 ll. All assays contained Chaps at lowconcentration to prevent loss of lipids by adsorption

onto plastic surfaces.

For all assays plates were read in an LJL Analyst

with the following settings: excitation 360–35 nm filter,

emission 665 nm filter, dichroic filter 505 nm, PMT

1000V set to digital sensitivity 2, 100 flashes per well, 10-

ms interval between flashes, read 50ms after flash, and

integration 1000ms.The GRP1 PH domain (amino acids 263 to 380) was

PCR cloned from a mouse brain cDNA library (Strat-

agene) as described previously [12]. The protein was

expressed from the pGEX 4T1 vector (Amersham

Pharmacia) in Escherichia coli and affinity purified on

glutathione–agarose using the manufacturer�s standardprotocols. The TAPP1 PH domain [13] was a gift from

A. Gray et al. / Analytical Biochemistry 313 (2003) 234–245 235

Dr. D. Alessi and W. Kimber. The purified PH domainswere labeled with Lance chelate according to the man-

ufacturer�s protocols.Lance chelate reagents were obtained from LKB

Wallac and biotinylated lipids were initially from Ech-

elon Biosciences, Inc., who no longer supply; subsequent

supplies of biotinylated lipids and all nonbiotinylated

lipids were obtained from Cell Signals, Inc. (Lexington,

KY, USA).PI 3-kinase assays. Standard enzyme reactions were

performed using the AlphaScreen in 50mM Hepes, pH

7.4, 50mM NaCl, 5mMMgCl2, 5mM DTT, and 0.05%

Chaps containing 40 lM ATP, 40 lM diC8 PtdIns

(4,5)P2, and 20 ng of PI 3-kinase c in a total volume of

20 ll. The reaction was stopped by the addition of 10 llof EDTA/diC6 PtdIns(3,4,5)P3–biotin followed by 20 llof GRP1 PH–GST/AlphaScreen beads both in 50mMHepes, pH 7.4, 50mM NaCl, and 0.1 % BSA. Final

concentrations were 50mM EDTA, 15 nM biotinylated

diC6PtdIns(3,4,5)P3, 3.75 nMGRP1 PH–GST, and 5 lg/ml AlphaScreen beads. Inhibitors were added to dry

wells in 0.5 ll Me2SO giving a finalMe2SO concentration

of 2.5% in the assay. For wortmannin, a 20-min prein-

cubation with enzyme before the start of the reaction was

included.The TR-FRET assays were conducted in Hepes assay

buffer (50mM Hepes, pH 7.4, 150mM NaCl, 5mM

MgCl2, 5mM DTT, 0.05% Chaps) using a two-compo-

nent reaction. The first component was a twofold-

concentrated sensor complex consisting of 32 nM

APC–Streptavidin, 120 nM biotinylated diC6PtdIns(3,4,

5)P3, and 35 nMLance chelate labeled GST–PH domain.

Alternatively the sensor complex contained 21 nM Lancechelate labeled anti-GST antibody and unlabeled PH

domain. The first component also contained 100 ng re-

combinant PI 3-kinase c in a final volume of 25 ll Hepesassay buffer. The second component contained the

diC8PtdIns(4,5)P2 at two times the required final con-

centrations and ATP at 100 lM again in 25 l l Hepes as-say buffer. The assays were started by mixing the two

components in a 96-well plate (White Lumitrac 200;Greiner Ltd) and reading in an LJL Analyst at the re-

quired time intervals to obtain rates of reaction.

PTEN assay. The PTEN assay was carried out as a

stopped assay system using diC8PtdIns(3,4,5)P3 as

substrate at a concentration of 100 lM in 50mM Hepes,

pH 7.4, 150mM NaCl, 10mM DTT. The assays were

started by the addition of enzyme in the required

amounts (20–300 ng/assay) in a final volume of 30 ll andincubated at 37 �C for 30min. The reaction was then

stopped by heating to 70 �C for 5min and 25 ll added to25 ll of sensor complex as described above before

measurement of TR-FRET.

Phosphoinositide mass assays. After indicated stimu-

lations the medium was aspirated from Swiss 3T3 cells

and cellular material precipitated by the immediate ad-

dition of 0.5ml of ice-cold 0.5M TCA. After standing onice for 5min the cells were scraped off and the wells rinsed

with additional TCA if required and the precipitate was

pelleted. The pellet was then washed two times with 1ml

of 5% TCA 1mM EDTA. Neutral lipids were extracted

from the pellet with 1ml of methanol:chloroform 2:1 by

vortexing three to four times over a 10-min period at

room temperature. This extraction was repeated and the

solvent supernatants were discarded. The acidic lipidswere then extracted as follows: 750 ll chloroform:meth-anol:12M HCl 40:80:1 was added to the pellet and vor-

texed occasionally over a 15-min period at room

temperature. A phase split was then carried out by the

addition of 250 ll chloroform and 450 ll 0.1M HCl fol-

lowed by centrifugation to separate the organic and

aqueous phases. The organic phase was collected into a

clean tube and dried in a Speed Vac centrifuge. The pelletat this stage was just visible. The lipids were then resus-

pended by sonication in a water bath in 60 ll of the assaybuffer as for the PI 3-kinase assay but withoutMgCl2 and

with Na cholate increased to 1.5%. Phosphoinositide

levels were estimated by mixing 25 ll of the lipid extract

with 25 ll of sensor complex as described above, but

containing 1.5% Na cholate and measuring displacement

after 30min. The mass of phosphoinositide present wasestimated by comparison to standard curves constructed

by addition of known amounts of phosphoinositide to the

sensor complex.

Comparative neutral/acid extraction of phosphoinosi-

tides. Cells (1321N1 astrocytoma) were labeled to equi-

librium with [3H]inositol as previously described [14].

The labeled cells were then extracted sequentially with

neutral and acidic solvent as described above for themass assay and the extracts dried and processed for

HPLC analysis [14].

Results and discussion

Signal detection

Studies in vitro and in vivo have shown that the PH

domain of GRP1 binds PtdIns(3,4,5)P3 with high af-

finity and selectivity [12,15,16]. It has also been reported

that short-chain, water-soluble analogues of phosphoi-

nositides retain biological activity when biotinylated at

the terminus of the sn-1 acyl chain [17–19]. In this study,

our approach to a homogeneous PI 3-kinase assay was

to form a complex between GST-tagged PH domainsand biotinylated (water-soluble) phosphoinositides. The

latter act as a bridge between streptavidin-coated donor

beads and anti-GST-conjugated acceptor beads, in the

case of the AlphaScreen, or in the case of the TR-FRET

assay, between the PH domain and the streptavidin–

APC complex. For TR-FRET detection either the PH

domain itself or an antibody directed against the GST

236 A. Gray et al. / Analytical Biochemistry 313 (2003) 234–245

tag is labeled with Eu chelate. In both assays this gen-erates a stable basal signal that can be quenched when

nonbiotinylated PtdIns(3,4,5)P3 is added or is generated

from PtdIns(4,5)P2 by PI 3-kinase. The principles of the

methods are illustrated in Fig. 1A (AlphaScreen) and 1B

(TR-FRET).

Using the TR-FRET approach, the addition of

biotinylated PtdIns(3,4,5)P3 to a sensor complex

comprising Eu chelate labeled GST–GRP1 PH andstreptavidin-coupled APC gave a strong, concentration-

dependent signal (Fig. 2A). The optimal signal was ob-

tained with 32 nM APC–streptavidin and 35 nM labeledGST–GRP1 PH at a biotinylated PtdIns(3,4,5)P3 con-

centration of 120 nM. This is consistent with the APC–

streptavidin binding capacity of 4mol of biotin per mole.

The complex of biotinylated lipid with APC–streptavidin

can be considered to be effectively irreversible due to the

very low Kd (<10�15 M) of the streptavidin/biotin inter-

action. The absolute concentrations of the complex

components can be varied two- to threefold with equiv-alent variation in the strength of signal obtained although

the molar ratios for the optimal signal remain constant.

Fig. 1. Diagrammatic representation showing the principles of detection of PI 3-kinase products by the AlphaScreen and TR-FRET, respectively. (A)

The use of the AlphaScreen, in which a sensor complex is formed between donor beads coated with anti-GST antibodies to which is bound GST-

tagged PH–GRP1 and streptavidin-coupled acceptor beads with bound biotinylated diC6PtdIns(3,4,5)P3. The interaction of donor and acceptor

through PtdIns(3,4,5)P3/PH–GRP1 binding generates a light signal that is detected in an Alpha Quest plate reader capable of reading a range of

multiwell plate formats. (B) A TR-FRET sensor complex is produced by the binding of Eu chelate labeled PH–GRP1 with biotinylated

diC6PtdIns(3,4,5)P3 attached to streptavidin-tagged APC. PI 3-kinase assays are performed in the presence of the appropriate sensor complex with a

source of enzyme, nonbiotinylated diC8PtdIns(4,5)P2, and ATP. Nonbiotinylated diC8PtdIns(3,4,5)P3 formed in such assays displaces PH–GRP1

from the sensor complex, causing a reduction in the AlphaScreen or TR-FRET signal.


The values shown above are considered optimal in that

there will be virtually no free biotinylated PtdIns(3,4,5)P3in the assay. If the concentration of biotinylated PtdIns

(3,4,5)P3 is increased such that the streptavidin–APC

binding is saturated, free biotinylated PtdIns(3,4,5)P3begins to compete theGRP1 PHdomain out of the sensor

complex, resulting in a severe drop in signal.Similar overall results were obtained using the Al-

phaScreen system except that an optimal signal was ob-

tained at 3.75 nM GST–GRP1 PH, 15 nM biotinylated

PtdIns(3,4,5)P3, and 5 lg/ml AlphaScreen donor and

acceptor beads (data not shown). The lower concentra-

tions of reagents used by this system reflect the greater

sensitivity of detection of the AlphaScreen system.

The selectivity of the detection system forPtdIns(3,4,5)P3 was tested using the AlphaScreen as

illustrated in Fig. 2B. Whereas biotinylated PtdIns-

(3,4,5)P3 gave a strong signal, biotinylated PtdIns,

PtdIns3P, and PtdIns(3,4)P2 were essentially undetect-

able. A small signal amounting to about 10% of that seen

with PtdIns(3,4,5)P3 was observed using biotinylated

PtdIns(4,5)P2. These results are compatible with the

previously characterized phosphoinositide binding spec-ificity of GRP1 PH [12].

We then examined whether nonbiotinylated phos-

phoinositides could displace biotinylated PtdIns(3,4,5)P3in the complex with GST–GRP1 PH. It was found that

diC8PtdIns(3,4,5)P3 effectively and dose-dependently

competed with the biotinylated analogue for binding to

GST–GRP1 PH in both assays as shown in Fig. 3. The

IC50�s for displacement by diC8PtdIns(3,4,5)P3 were

Fig. 2. Generation and specificity of sensor complexes. (A) Streptavi-

din-tagged APC was incubated with GRP1–PH and the indicated

concentrations of biotinylated diC6PtdIns(3,4,5)P3 in kinase assay

buffer (Materials and methods). Measurements were made in 96-well

plates and read in a fluorimeter with the settings described under

Materials and methods. The data presented are the means and stan-

dard deviations of triplicate determinations and are representative of

several experiments. (B) The signals obtained using 0.5lMPtdIns(3,4,5)P3 were compared with those obtained for the same

concentrations in the range of lipids indicated using the AlphaScreen

(black bar) or TR-FRET (gray bar). The symbols H and # represent a

signal of less than 1% and not determined respectively. The data are

means and standard deviations of quadruplicate determinations.

Fig. 3. Dissociation of sensor complexes by nonbiotinylated phos-

phoinositides. The effects of increasing concentrations of added non-

biotinylated diC8PtdIns(3,4,5)P3 (d) or diC8PtdIns(4,5)P2 (s) on the

sensor complex signals obtained using TR-FRET (A) or the Alpha-

Screen (B) were determined. TR-FRET or light emission was detected

as described under Materials and methods and the legends to Figs. 1

and 2. All data points are the means and standard deviations of trip-

licate determinations; again the data are representative of several

experiments.


0.1 lM and 1.0 lM for the AlphaScreen system andTR-FRET assays, respectively. The lower IC50 in the

AlphaScreen system is compatible with the lower con-

centration of biotinylated PtdIns(3,4,5)P3 required to

optimize the signal and confirms the competitive nature

of the displacements observed [20]. At high concentra-

tions PtdIns(4,5)P2 also decreased the signal with an IC50

of 7 lM in the AlphaScreen system, compared with

216 lM in the TR-FRET assay. Thus, GST–GRP1 PHexhibits between 70- and 200-fold lower affinity for

PtdIns(4,5)P2 compared with PtdIns(3,4,5)P3 under the

assay conditions used to generate Fig. 3. This degree of

selectivity was more than adequate to perform PI 3-ki-

nase assays in which the initial concentration of

PtdIns(4,5)P2 substrate was insufficient to cause signifi-

cant displacement of the signal.

PI 3-kinase assay

To test whether detection of PtdIns(3,4,5)P3 gener-

ated in vitro by PI 3-kinase was feasible, short-chain

PtdIns(4,5)P2 (diC4 or diC8) was incubated with PI 3-

kinase under standard conditions. This and other ap-

proaches established that very short chain derivatives

are poor substrates for PI 3-kinase c. However,diC8PtdIns(4,5)P2 was efficiently phosphorylated by PI

3-kinase c and the production of diC8PtdIns(3,4,5)P3was readily detected as a marked decrease in the signal

obtained using the AlphaScreen system. It is not clear

whether the recognition of the diC8 substrate reflects the

presence of aggregated (micellar) structures in contrast

to true monomolecular species for diC4 substrate, but it

is clear that the diC8 compounds provide a convenientmeans of routinely assaying PI 3-kinases. A direct

comparison of the short-chain diC8 and long-chain

diC16 lipids as substrate for PI 3-kinase is not possible

since the reaction conditions required for the Alpha-

Screen and TR-FRET cannot be used for the diC16

lipids due to their insolubility in aqueous media. The

comparison could be made by using a vesicular sub-

strate containing both long- and short-chain lipids.However, such experiments are out of the scope of this

study, which is concerned with characterizing a homo-

geneous water-soluble assay system.

Using the decrease inAlphaScreen counts (Dcounts) asreadout, phosphorylation of diC8PtdIns(4,5)P2 was both

time (Fig. 4A) and enzyme concentration-dependent

(Fig. 4B). The assay system delivered expected enzyme

characteristics for PI 3-kinase c, reproducing inhibitionby low concentrations of the nonionic detergent Triton

X-100 at 0.01% [21] and dependence onMg2þ as divalent

cation with an optimal concentration of 5mM and a Km

for ATP around 35 lM (not shown). Saturation kinetics

were also obtained for diC8PtdIns(4,5)P2 with a Km

around 50 lM (Fig. 4C) and a Vmax comparable to that

obtained with vesicular diC8PtdIns(4,5)P2 (not shown).

The assay was robust, typically with Z 0 factors around 0.8(not shown), and sensitive to inhibition by the specific PI

3-kinase inhibitors wortmannin and LY294002 and the

broad-spectrum kinase inhibitor staurosporine (Fig. 4D),

in a manner similar to that previously reported for type I

PI 3-kinases (Table 1) [7,22,23]. The data presented in

Table 1 show IC50 values obtained from the literature.

There are to our knowledge no publishedKi values for the

inhibitors used obtained using a radiometric assay sys-tem. Unfortunately, from the information given in the

literature regarding the assay conditions, it is not possible

to accurately calculate Ki values from the given IC50

values.However, due to the lowATP concentrations used

in radiometric assays (well below the Km) these IC50

values will approximate the Ki�s. Furthermore wort-

mannin, although initially ATP competitive, is an irre-

versible inhibitor which covalently binds in the enzymeactive site and gives mixed inhibition kinetics and is not

therefore amenable to calculation of a Ki. These results

establish the feasibility of the assay for compound

screening.

PTEN assay

To demonstrate that the detection systems describedabove can be applied to the problem of assaying other

phosphoinositide-metabolizing enzymes, TR-FRET was

used to detect the conversion of diC8PtdIns(3,4,5)P3 to

PtdIns(4,5)P2 by the tumor suppressor phosphatase

PTEN. The experiment reported in Fig. 5 used a sensor

complex similar to that described for PI 3-kinase c but

with a modified buffer system (Materials and methods).

The data are derived from a stopped assay since in thepresence of nonbiotinylated PtdIns(3,4,5)P3 substrate

the sensor complex would be dissociated at the start of

the assay, exposing the biotinylated PtdIns(3,4,5)P3 to

digestion by the PTEN. The action of PTEN reduced

the amount of PtdIns(3,4,5)P3 present, which was de-

tected as a restoration of the TR-FRET signal. This

effect was clearly dependent on enzyme concentration

and was destroyed by prior heat treatment of the PTENpreparation. The maximum increase in signal shown

corresponds to >90% conversion of the PtdIns(3,4,5)P3to PtdIns(4,5)P2 and is comparable with the activity

observed in radiometric PTEN assays in that initial rate

measurements across the range of protein concentra-

tions shown were indistinguishable [24].

Generic reagents

The TR-FRET assay makes use of commercially

available APC, but requires the preparation of Eu che-

late labeled PH domain. As described below, the avail-

ability of a range of PH domains with differing

phosphoinositide specificities suggests it should be pos-

sible to develop assay procedures for the detection of all


the known phosphoinositide species and hence the en-

zymes that synthesize or metabolize them. Diversifica-

tion of the procedure would be facilitated by the use of a

generic fluorescence donor species. Since the PH do-mains used in the assay are expressed as GST fusion

proteins the ability of an Eu chelate labeled anti-GST

antibody to substitute for directly labeled PH domain

was assessed.

After the amount of antibody required to form a

sensor complex was optimized, as described for the la-

beled PH domain (results not shown) the ability of

nonbiotinylated PtdIns(3,4,5)P3 to displace GRP1 fromthe sensor complex containing Eu chelate labeled anti-

GST antibody was assessed and found to give results

similar to those obtained for the directly labeled PH

domain (Fig. 6A). PI 3-kinase assays were performed in

parallel using both configurations and the results are

shown in Fig. 6B as a plot of initial rate of activity

against PtdIns(4,5)P2 concentration. Rates can be esti-

mated since the TR-FRET readings can be taken duringthe assay, unlike the AlphaScreen, which is an end-point

determination. The data show that there is no significant

difference in the assay results using a generic anti-GST

antibody or directly labeled PH domain.

We were especially interested in developing an assay

for PtdIns(3,4)P2, which is produced in stimulated cells

via the dephosphorylation of PtdIns(3,4,5)P3, as well asthrough PtdIns(3,4,5)P3-independent pathways. In or-

der to confirm the generic nature of the assay using the

Eu chelate labeled anti-GST antibody the utility of the

TAPP1 PH domain for the detection of PtdIns(3,4)P2 in

a system identical to that described for the GRP1 PH

domain was assessed. Using the Eu chelate labeled anti-

GST antibody and GST–TAPP1 PH domain a sensor

Fig. 4. Analysis of PI 3-kinase c using the AlphaScreen sensor complex. (A) Time course. 20 ng of PI 3-kinase c was incubated with 40 lM ATP,

40lM diC8PtdIns(4,5)P2 (s) or diC4PtdIns(4,5)P2 (d), and the components of the sensor complex. Reactions were terminated by the addition of

EDTA and light emission was detected in an Alpha Quest plate reader. PI 3-kinase activity was detected as a decrease in light emission compared

with zero time. (B) Dependency on protein concentration. Incubations were carried out as in A for 10min with 40lM diC8PtdIns(4,5)P2 and the

indicated amounts of PI 3-kinase. (C) Lipid substrate dependency. Incubations were carried out as in B using the indicated concentrations of

diC8PtdIns(4,5)P2. (D) Inhibition of PI 3-kinase c (20 ng) under the conditions given for B by wortmannin (d), LY294002 (j), and quercetin (N) at

the concentrations shown; details of inhibitor presentation are given under Materials and methods.

Table 1

Comparison of some known inhibitors of PI-3 kinase estimated using

the AlphaScreen with previously published values

Compound Reported

IC50

Ki for PtdIns

3-kinase c

Wortmannin 4.2 nM [22] 12 nMa

LY294002 1.4 lM [21] 5.6 lMStaurosporine 9 lM [7] 3.8 lM

A comparison of published IC50 values for some common PI 3-

kinase inhibitors determined by radiometric assays as referenced and

Ki values derived from the data in Fig. 4d is shown.a Since wortmannin is an irreversible inhibitor the IC50 value is

given.


complex was formed upon the addition of increasing

amounts of biotinylated PtdIns(3,4)P2 with character-

istics very similar to those of the GRP1 PH domain–

PtdIns(3,4,5)P3–biotin complex (Fig. 7A). The system

also worked in a displacement assay allowing the de-tection of exogenously added PtdIns(3,4)P2 with an IC50

of 0.9 lM for diC8PtdIns(3,4)P2 (Fig. 7B), giving a de-

tection limit of about 2 pmol in a 50-ll assay volume.

This indicates that the system is applicable to other PH

domains produced as GST-fusion proteins and removes

the requirement for labeling the PH domains directly.

Mass assays

Since the assay system described depends on dis-

placement of a PH domain from the sensor complex, it

should be possible to adapt the procedure to determine

the concentration of phosphoinositides derived from

any source from which they can be extracted in quan-

tities compatible with the sensitivity of the detection

system. In practice, however, several problems had to beovercome in order to use the detection system in this

way. First, tissue extracts contain large quantities of

lipids other than the phosphoinositides of interest and

which might be expected to interfere nonspecifically.

This was dealt with by using a two-step procedure with

the majority of noninositol lipids being extracted with

neutral solvents before quantitative extraction of rele-

vant polyphosphoinositides using acidified solvents. The

efficacy of this approach was verified by HPLC analysis

of lipids extracted by both the neutral and the acidicsolvents. The data shown in Table 2 clearly demonstrate

that no significant depletion of the polyphosphoinosi-

tides occurs during extraction with neutral solvent.

Second both the sensitivity and the selectivity of the

displacement curves shown in Fig. 3 are problematic

when assaying cell and tissue samples. Our previous

analysis of phosphoinositide levels in cells labeled to

Fig. 6. Alternative TR-FRET assay using Eu chelate tagged anti-GST

antibodies. The TR-FRET sensor complex was modified to contain

unlabeled PH GRP1–GST fusion protein and Eu chelate labeled anti-

GST as described in detail under Materials and methods. (A) Modified

sensor complex signal and its displacement by the indicated concen-

trations of diC8PtdIns(3,4,5)P3. The data are means and standard

deviations of triplicate determinations. (B) PI 3-kinase assays using the

indicated concentrations of diC8PtdIns(4,5)P2 and either standard

sensor complex (s) or the modified sensor complex (j). The assays

were carried out by incubating the diC8PtdIns(4,5)P2 with 20 ng re-

combinant PI 3-kinase c and 100lM ATP in standard kinase assay

buffer (Materials and methods) in a final volume of 50 ll containing25 ll of the relevant sensor complex. The assay components were

mixed in the wells of a 96-well plate and the reaction was started by

addition of the ATP. Readings of TR-FRET were taken every minute

and initial rates of reaction were determined and plotted against

diC8PtdIns(4,5)P2 concentration.

Fig. 5. Analysis of PTEN lipid phosphatase activity using TR-FRET

detection. The protein dependence of PTEN activity was determined

by incubating diC8PtdIns(3,4,5)P3 (100lM) with the indicated

amounts of recombinant PTEN (d) or boiled PTEN (j) in phos-

phatase assay buffer (Materials and methods). The reactions were

terminated by heat denaturation, and 25 ll of the assay mixtures was

removed and added to the TR-FRET sensor complex (this stopped

assay format avoids hydrolysis of diC4PtdIns(3,4,5)P3 in the sensor

complex). Hydrolysis of PtdIns(3,4,5)P3 was detected as an increase in

TR-FRET due to the shift in binding of PH GRP1 from nonbiotiny-

lated substrate to biotinylated diC4PtdIns(3,4,5)P3 bound to strepta-

vidin-coupled APC. The data shown are means and standard

deviations of triplicate determinations from two independent experi-

ments.


isotopic equilibrium with [3H]inositol show that PtdIns

(3,4,5)P3 is present at levels ranging from 100- to 1000-

fold lower than those of PtdIns(4,5)P2 [12] (agonist-stimulated and basal levels, respectively), while Fig. 3

shows a selectivity ratio between these species of about

200-fold. These problems were overcome by including

the anionic detergent sodium cholate (1.5%) in samples

for analysis. Fig. 8A shows that increasing concentra-

tions of cholate progressively shift the PtdIns(3,4,5)P3displacement curve to the left. This displacement de-

pendence on detergent concentration is also demon-strated by the mass assay for PtdIns(3,4)P2 (data not

shown). Plotting apparent IC50 against increasing de-

tergent concentration (Fig. 8B) gives a smooth curve

with no sharp transitions, suggesting that the effect is

independent of the critical micellar concentration for the

detergent. We attribute this effect to the presentation of

Table 2

Distribution of phosphoinositides in neutral and acid solvent extrac-

tions

Extraction Inositol lipids

PtdIns PtdInsP PtdInsP2

Neutral 53� 15% 25� 9% 6� 4%

Acid 46� 10% 74� 11% 94� 3%

1321N1 astrocytoma cells were grown to confluence in six-well

plates and labeled with ½3H�inositol as described. Cells were precipi-

tated with TCA and the washed pellets subjected to extraction first

using neutral solvents and then with acidified solvents as described

(Materials and methods). Phospholipids present in the extracts were

then deacylated and subjected to analysis by anion exchange HPLC.

The peaks of radiolabeled compounds were assigned by the elution

characteristics of defined standards. The data shown are the means and

range of two independent experiments, each lipid being assayed in

triplicate.

Fig. 8. Effect of Na cholate on the sensitivity of dissociation of the

TR-FRET sensor complex by nonbiotinylated PtdIns(3,4,5)P3. (A)

Dissociation of the TR-FRET sensor complex by the indicated

concentrations of diC8PtdIns(3,4,5)P3 as described for Fig. 3. Disso-

ciation curves were obtained using buffer containing 0.05% (d), 0.1%

(s), 0.5% (N), 1.0% (O), 1.5% (j), and 2.0% (�) Na cholate. The data

shown are the means of triplicate determinations and are representa-

tive of several independent experiments. Error bars have been omitted

for clarity. (B) IC50 values derived from the data in A are plotted

against log detergent concentration.

Fig. 7. Use of the TAPP1 PH domain to generate a PtdIns(3,4)P2-

specific sensor complex. The sensor complex comprised biotinylated

diC6PtdIns(3,4)P2, TAPP1 PH domain as a GST-fusion protein, Eu-

labeled anti-GST antibodies, and streptavidin-coupled APC as detailed

under Materials and methods. (A) TR-FRET signals obtained with the

indicated concentrations of biotinylated diC4PtdIns(3,4)P2. (B) Dis-

sociation of the sensor complex with increasing concentrations of

nonbiotinylated diC8PtdIns(3,4)P2. TR-FRET was measured as de-

scribed for Fig. 2. The data are means and standard deviations of

triplicate determinations.


lipids as mixed micelles in a large excess of detergent(micellar number for cholate under these conditions is

estimated at 4.8 [25]) over lipid. Thus the PtdIns(3,4,5)

P3 will be distributed at no more than one molecule per

micelle. Therefore addition of detergent to the extracted

lipids for mass assay is essential for accurate and sen-

sitive estimations of lipid mass. This simplifies the pre-

sentation of displacing ligand and avoids the formation

of vesicles in which some of the phosphoinositideswould be inaccessible to the PH domain. Exclusion of

divalent cations from the assay is also essential to pre-

vent the aggregation of the polyphosphoinositides re-

sulting in removal of lipid from the pool available for

binding.

To test the assay procedures Swiss 3T3 cells (grown

to 2� 106 cells per well of a six-well plate in DMEM

with 10% FCS) were serum starved for 6 h in DMEMwith 0.5% BSA and then stimulated with a range of

concentrations of PDGF. Samples were prepared as

described under Materials and methods and, after ad-

dition of detection mix containing appropriate PH do-

mains and biotinylated lipid, the mass of PtdIns(3,4,5)P3was estimated by comparison to a standard curve con-

structed by adding known amounts of the lipid of in-

terest to the sensor complex mix. In a 50-ll final assayvolume, the detection limit of this procedure is ap-

proximately 0.5 pmol. The data shown in Fig. 9A are

compatible with the mass of PtdIns(3,4,5)P3 determined

for these cells by other methods [26] and show typical

responses to PDGF. The effects of the latter were

completely blocked by 100 nM wortmannin (not

shown), confirming that the assay detects specifically the

products of a PI 3-kinase. In order to obtain these databy traditional labeling and HPLC analysis would have

required 22 HPLC runs, taking almost 2 weeks, and

substantial amounts of [3½H]inositol. The data presentedwere obtained in one and a half days including the tissue

culture preparation of the cells.

The AlphaScreen assay also proved suitable for de-

tecting PtdIns(3,4,5)P3 in cell extracts as illustrated by

the data in Fig. 9B, which show a typical time course forthe accumulation and rapid decay of this lipid in dif-

ferentiated HL60 cells stimulated with fMLP. Cells were

differentiated for 48 h in the presence of 10mM dibutyl

cAMP in RPMI medium supplemented with 10% FCS.

This results in a more neutrophil-like phenotype. Cells

were then washed in PBS and suspended in Hepes-buf-

fered saline at a density of 5� 106/ml. One milliliter of

cell suspension was added to each well of a six-well plateand incubated at 37 �C for 15min before addition of

1 lM fMLP. The incubations were terminated by the

addition of TCA and the cell pellets processed as de-

scribed above for the PDGF stimulation of Swiss 3T3

cells. The response shown is known to result largely

from the activation of PI 3-kinase c by the G-protein-

coupled fMLP receptor and illustrates the combined

power of the methods described in this report; namely

the opportunity to carry out high-throughput inhibitor

screens coupled to the ability to test the effects of in-

hibitors in cell preparations and animal tissues.

When cells are subjected to an oxidative stress they

produce high levels of PtdIns(3,4)P2 which are sustained

for up to 30min [12,26,27]. The origin of this lipid is as

Fig. 9. Analysis of PtdIns(3,4,5)P3 in extracts of stimulated cells. (A)

Swiss 3T3 cells were grown to approximately 60% confluence in six-

well plates and stimulated with the indicated concentrations of PDGF

for 10min as described under Materials and methods. The incubations

were terminated by the addition of TCA and polyphosphoinositides

were selectively extracted from the precipitated material as described.

The dried extracts were dissolved in buffer containing 1.5% Na cholate

and the amount of PtdIns(3,4,5)P3 present in these samples was de-

termined by the displacement of the TR-FRET sensor complex by

comparison with a standard curve of known PtdIns(3,4,5)P3 concen-

trations (see Fig. 6A). (B) Time course of PtdInsP3 production in

differentiated HL60 cells stimulated with fMLP. Cells were cultured

and differentiated to a neutrophil-like phenotype as described. They

were suspended in HBSS at 5� 106 cells/ml. 1ml of cell suspension was

added to each well of a six-well plate and incubated for 15min before

stimulation with 1 lM fMLP for the times indicated. Polyphosphoi-

nositides were extracted as described for A and determined by dis-

placement of the AlphaScreen sensor complex by comparison with a

standard curve. The data are means and standard deviations of trip-

licate determinations and are representative of several similar experi-

ments.


yet unclear. Evidence exists for the direct production ofPtdIns(3,4)P2 from PtdIns(3)P by the action of an un-

characterized 4-kinase in platelets in response to integrin

activation [28,29]. Alternatively it could be produced

from PtdIns(3,4,5)P3 by the action of a 5-phosphatase

such as SHIP2. The latter data as well as the existence of

PtdIns(3,4)P2-selective binding proteins [28] imply that

this lipid is likely to have signaling functions. We

therefore exploited the previously characterized TAPP1PH domain, which binds PtdIns(3,4)P2 selectively, to

develop an assay for this lipid in cell and tissue extracts.

Swiss 3T3 cells were grown as described above and

treated in a manner identical to that described for the

simulation with PDGF with the exception that in this

case the cells were stimulated by the addition of 1mM

H2O2. This methodology is validated by the data in Fig.

10, showing increasing levels of PtdIns(3,4,)P2 and atransient increase in PtdIns(3,4,5)P3 in Swiss 3T3 cells

when treated with H2O2. These data are consistent with

results showing that PtdIns(3,4,5)P3 is also produced in

response to oxidative stress and could serve as a pre-

cursor for PtdIns(3,4)P2 [12,26,27]. The time course and

wortmannin sensitivity (not shown) of this response

were similar to previously reported results. This system

is currently the subject of further investigation usingthese assays

Summary

In this report we describe procedures for the rapid,

sensitive, and reliable assay of bioactive inositol lipid

species formed during enzyme-catalyzed reactions and

present in cell and tissue extracts. These open up op-portunities for high-throughput approaches to the

identification of agents which modify the activity of

therapeutically important enzymes, such as the PI

3-kinases and PTEN, and the means to evaluate the

efficacy of such compounds in cells and animals. In

principle it should now be possible to devise comparable

assays for all the known phosphoinositide species for

which a selective protein binding module has beenidentified and characterized. The mass assay has already

been used successfully in the analysis of PtdIns(3,4,5)P3levels in the B cells of PI 3-kinase delta knockout mice

[30]. This analysis of PtdIns(3,4,5)P3 in primary cells

would have been technically very difficult if not impos-

sible by traditional methods. The use of TAPP1 PH to

detect PtdIns(3,4)P2 production in Swiss 3T3 cells is the

first reliable, nonradioactive assay for this putative lipidsignal and should herald more detailed studies of its

metabolism and responsiveness to stimuli than was

hitherto possible.

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nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases...

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