A direct fluorometric activity assay for lipid kinases and phosphatases

Journal of Lipid Research Volume 61, 2020 945 Copyright © 2020 Sun et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. kinases, lipid phosphatases, and phospholipases, which interconvert different lipid species and thereby control their cellular levels. For instance, the cellular levels of phosphoinositides, which play pivotal roles in cell signaling and membrane trafficking, are tightly regulated by a panel of kinases and phosphatases in a spatiotemporally specific and stimulus-dependent manner (4, 5). Due to their crucial roles in health and disease, lipid kinases and phosphatases have been extensively studied in terms of structure, physiological function, and cellular regulation (6, 7). However, detailed studies of the enzymatic properties of these proteins, which are necessary for full understanding of their biological functions and development of specific small molecule modulators for them, have been hampered by lack of direct and quantitative continuous enzyme activity assays. Enzymatic activity of lipid kinases and phosphatases is typically measured by a radioactivity-based assay (8, 9), which is suited for neither quantitative and mechanistic enzyme studies nor small molecule modulator screening. To overcome these technical limitations, we developed a fluorescence-based real-time activity assay for lipid kinases and phosphatases. This new assay allows quantitative analysis of enzyme kinetics for these enzymes and rapid screening of their small molecule modulators. Class I phosphoinositide 3-kinase (PI3K) converts phosphatidylinositol-4,5-bisphosphate (PI4,5P2) in the plasma membrane (PM) to phosphatidylinositol-3,4,5-trisphosphate (PIP3) (10). PIP3 is a potent signaling lipid that activates myriad of cellular processes (10, 11). PIP3 carries out its signaling functions primarily by facilitating PM recruitment Abstract Lipid kinases and phosphatases play key roles in cell signaling and regulation, are implicated in many human diseases, and are thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here, we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Because of its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided detailed mechanistic information about the product inhibition and substrate acyl-chain selectivity of PI3K and enabled rapid evaluation of small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.—Sun, J., I. Singaram, M. H. Soflaee, and W. Cho. A direct fluorometric activity assay for lipid kinases and phosphatases. J. Lipid Res. 2020. 61: 945–952.

of cellular proteins with PIP 3 -binding domains and motifs, most notably the pleckstrin homology (PH) domain (12). Dysregulated PI3K signaling has been linked to various human diseases, including cancer (13,14) and inflammatory diseases (15), and thus PI3K signaling pathways are major targets for drug development (16). Despite numerous studies on PI3K signaling pathways, the enzymatic properties of PI3K have not been fully characterized largely due to the lack of a direct and continuous assay that allows thorough and systematic enzyme kinetic studies (8). The action of PI3K is counterbalanced by phosphatase and tensin homolog (PTEN), which converts PIP 3 to PI4,5P 2 , thereby serving as a tumor suppressor (17,18). PTEN is frequently deleted in cancer. It has been recently reported that there are multiple isoforms of PTEN with different subcellular localization and function (19,20) and that PTEN may have promiscuous lipid phosphatase activity (21). As is the case with PI3K, the lack of an available direct activity assay has hampered full characterization of PTEN isoforms (9). Our new fluorescence-based activity assay, which enables direct quantitative analysis of enzyme kinetics for PI3K and PTEN through real-time quantification of their substrate and/or product, provides new mechanistic insight for these enzymes and also serves as a convenient tool for identification and characterization of enzyme modulators.

Vesicle preparation
Lipid solutions were mixed according to the final lipid composition of vesicles and the solvent was evaporated under a stream of nitrogen gas. Tris buffer (pH 7.4; 20 mM) containing 0.16 M NaCl was added to the lipid film and the mixture was shaken for 0.5 h and then sonicated for 1 min. Large unilamellar vesicles (LUVs) with a 100 nm diameter were then prepared by extrusion using the Avanti Mini-Extruder with a 100 nm polycarbonate filter (Whatman).
The cDNA for PTEN (OriGene) was subcloned into the pET-30 a (+) vector with a His 6 -tag, which was then transfected into Escherichia coli BL21 RIL codon plus (Stratagene) cells. After the optical density of cell suspension reached 0.6-0.8, protein expression was induced overnight at room temperature with 0.2 mM (final concentration) isopropyl -d-1-thiogalactopyranoside. Cell pellets were lysed by sonication and the supernatant was incubated with the Ni-NTA resin after centrifugation of the homogenate. The protein was purified as described above. The purity of the proteins was checked by sodium dodecylsulfate polyacrylamide gel electrophoresis.

Ratiometric lipid sensor preparation and characterization
The engineered epsin1 ENTH domain (eENTH) (23), tandem PH domains of myoxin X (eMyoX-tPH) (24), and C-terminal PH domain of Tapp2 (eTapp2-cPH), which have been employed as specific sensors for PI4,5P 2 (23), PIP 3 (24), and PI3,4P 2 (25), respectively, were expressed as glutathione-S-transferase-tagged proteins in BL21 RIL codon plus cells and purified as described previously. Protein expression was induced overnight at room temperature with 0.5 mM (final concentration) isopropyl -d-1thiogalactopyranoside when the optical density of the media reached 0.6-0.8. Cells were harvested and cell pellets were suspended in 20 mM Tris buffer (pH 7.4) with 160 mM NaCl, 1 mM TCEP, and 1 mM PMSF and then lysed by sonication. The supernatant was incubated with glutathione resin (GenScript) for 2 h. The resin mixture was then poured into a small column and washed with 20 mM Tris buffer (pH 7.4) containing 0.16 M NaCl. After the resin became clear, the buffer solution was replaced by 5 ml of labeling buffer [50 mM Tris-HCl, 300 mM NaCl, 1 mM TCEP, 50 mM arginine, 50 mM glutamic acid (pH 8.0)]. After adding 100 l of a cysteine-reactive solvatochromic fluorophore (10 mg/ml in DMSO), acrylodan (Thermo Fisher), or a Nile Red derivative, NR3 (24), the mixture was gently shaken at room temperature for 2 h. The resin was then washed with 20 mM Tris buffer (pH 7.4) containing 0.16 M NaCl and 5% DMSO until the free dye was completely removed. The resin was then suspended in the digest buffer [20 mM Tris-HCl, 160 mM NaCl, 20 mM CaCl 2 , 0.5 mM TCEP, 50 mM arginine, 50 mM glutamic acid (pH 7.4)] containing 40 U of bovine -thrombin (Haematologic Technologies). After the overnight incubation at 4°C to remove the glutathione-S-transferase tag, the sensor was eluted from the column and any insoluble matter was removed by centrifugation at 4°C. The purity of the sensor was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis and the protein concentration was determined by the Bradford assay. The activity of the purified sensor was routinely checked by a quick three-point fluorometric measurement. For DAN-eENTH, for example, its fluorescence emission intensity at 470 nm (F 470 ) and at 530 nm (F 530 ) was measured with three LUVs, e.g., 10, 50, and 100 M of POPC/POPS/PI4,5P 2 (77:20:3). The ratio (F 470 /F 530 ) values from these measurements should lie within the 10% range of the standard calibration curves (see Fig. 1A) for the sensor to be qualified for the enzyme assay.

Spectrofluorometric activity assay
All cuvette-based continuous activity assays were performed with the FluoroLog3 spectrofluorometer at 37°C in a 1 ml quartz cuvette (Hellma Analytics). For the PI3K activity assay, 874 l of 20 mM Tris buffer (pH 7.4) containing 0.16 M NaCl were mixed with 100 l of POPC/POPS/PI4,5P 2 (77:20:3; 400 g/ml) LUVs at the indicated concentration, 5 l of 50 M lipid senor (final concentration, 500 nM), 10 l of 0.1 mM pY2 peptide (final concentration,: 500 nM), and 10 l of 0.1 M ATP (final concentration: 0.1 mM). The reaction was initiated by adding 1 l of enzyme solution (0.5-12.5 M) to the mixture to a final concentration of 1-25 nM and continuously monitored by measuring the blue-shifted emission of the sensor at 470 nm with the excitation wavelength set at 380 nm. Alternatively, the reaction was triggered by adding ATP or pY2 peptide to the mixture containing all other components. The reaction by PTEN was monitored in a similar manner except for the absence of ATP and the pY2 peptide in the reaction mixture. For NR3-eTapp2-cPH, the reaction was monitored at 675 nm with the excitation wavelength set at 560 nm.

Fluorescence plate reader assay
Enzyme reactions were also monitored with the BioTek Synergy Neo HTS multi-mode plate reader using nontreated black polystyrene 96-well plates (Corning Inc.). For the PI3K activity assay, 200 l of buffer solution [20 mM Tris buffer (pH 7.4) with 0.16 M NaCl] containing PI3K and lipid LUVs with the indicated concentrations, 500 nM lipid sensor and 10 M pY2 peptide were added to each well. After a 5 min incubation, the reaction was triggered by adding ATP (final concentration: 0.1 mM) to the mixture and the fluorescence emission intensity was simultaneously measured at two wavelengths (470 nm and 530 nm for DANbased sensors with the excitation set at 380 nm). The PTEN assay was performed in a similar manner except that ATP and pY2 were absent in the reaction mixture. For NR3-eTapp2-cPH, the emission intensity was measured at 600 nm and 675 nm with the excitation set at 560 nm.

PI3K inhibition assay
Two hundred microliters of buffer solution [20 mM Tris buffer (pH 7.4) with 0.16 M NaCl] containing 10 nM PI3K and 50 M lipid LUVs, 500 nM DAN-eENTH, 10 M pY2 peptide, and varying concentrations of inhibitor (0-1 M for GDC-0941 and wortmannin and 0-1 mM for LY294002) were added to each well. After a 10 min incubation, the reaction was triggered by adding ATP (final concentration: 0.1 mM) to the mixture and the fluorescence ] were added to each sensor (500 nM) and fluorescence emission spectra were monitored with the excitation wavelength set at 380 nm for DAN-labeled sensors and 560 nm for NR3-labeled sensors, respectively. The spectra of the sensors without lipid vesicles are indicated by arrows. The change in fluorescence emission intensity (F) for each vesicle was normalized against the maximal fluorescence increase value (F max ) observed for each sensor. Each spectrum is from a single representative scan selected from triplicate measurements (n = 3) that showed essentially the same patterns. D-F: From the emission spectra, the ratio of fluorescence intensity at 470 nm versus that at 530 nm (F 470 /F 530 ) for DAN-eENTH (D) and DAN-eMyoX-tPH (E) and F 600 /F 675 for NR3-eTapp2-cPH (F), respectively, were calculated. Nonlinear least-squares analysis of the plots using the equation (e.g., for DAN-eMyoX-tPH): F 470 /F 530 = (F 470 /F 530 ) min + (F 470 /F 530 ) max /(1 + K d / [PIP 3 ]) yielded K d , (F 470 /F 530 ) max , and (F 470 /F 530 ) min values and the calibration curves were constructed using these parameters. K d , (F 470 /F 530 ) max , and (F 470 /F 530 ) min values are the equilibrium dissociation constant, the maximal F 470 /F 530 value, and the minimal F 470 /F 530 value, respectively. Data in D-F indicate mean ± SD from the triplicate measurements. emission intensity was simultaneously measured at 470 nm and 530 nm with the excitation set at 380 nm.

Kinetic data analysis
All fluorescence intensity ratios (F 470 /F 530 for DAN-eENTH and DAN-eMyoX-tPH and F 600 /F 675 for NR3-eTapp2-cPH) at different time points were converted into total PI4,5P 2 (PIP 3 or PI3,4P 2 ) concentrations by Excel using respective ratiometric calibration curves ( Fig. 1D-F) to yield full enzyme reaction curves. The initial rate (V o ) of enzyme reaction was then calculated from the initial linear part of the reaction curves. Apparent Michaelis-Menten kinetic parameters were calculated by nonlinear least-squares analysis using the Michaelis-Menten equation, where E o and S o are the bulk molar concentrations of enzyme and substrate, respectively, and k cat and K m are the turnover number and Michaelis constant, respectively. The enzyme inhibition data were analyzed by nonlinear least-squares analysis using a simple competitive inhibition equation, and IC 50 are maximal V o , the initial inhibitor concentration, and the inhibitor concentration yielding half-maximal inhibition. All kinetics parameters were expressed as average ± SD from minimum of triplicate measurements.

Assay strategy
We recently developed a fluorescence-based ratiometric imaging analysis that allows accurate in situ quantification of cellular lipids in live cells (23)(24)(25)(26)(27). This method utilizes a ratiometric fluorescence sensor prepared from a genetically engineered lipid binding domain that is chemically labeled on a single site with a solvatochromic fluorophore that exhibits a large change in fluorescence emission upon lipid binding. After in vitro calibration of the lipid sensor using lipid vesicles with the varying lipid composition, the sensor is delivered to cells for in situ lipid quantification with high spatiotemporal resolution and accuracy. In this work, we applied the same lipid quantification technology to the in vitro real-time activity measurement for lipid kinases and phosphatases. For instance, we directly measured the enzymatic kinetics of PI3K through real-time spectrofluorometric quantification of either its substrate, PI4,5P 2 , or its product, PIP 3 . Likewise, we monitored the enzyme activity of its counterbalancing enzyme, PTEN, by following the kinetics of the PIP 3 decrease or the PI4,5P 2 increase. As sensors for PI4,5P 2 and PIP 3 , we selected DAN-eENTH (23) and DAN-eMyoX-tPH (25), respectively, which have been fully characterized and successfully used for in situ quantification of cellular PI4,5P 2 and PIP 3 . Spectrofluorometric properties of these sensors and their ratiometric lipid titration curves are shown in Fig. 1. Briefly, these solvatochromic sensors displayed a hypsochromic shift (or blue shift) of the fluorescence emission peak from 530 to 470 nm upon membrane lipid binding, and the intensity at 470 nm was increased proportionally to the increase in the concentration of their cognate lipid (Fig. 1A, B). Data in Fig. 1 were collected by varying the total lipid concentration of vesicles with a fixed PI4,5P 2 (PIP 3 or PI3,4P 2 ) composition [e.g., POPC/POPS/PI4,5P 2 (77:20:3 in mole percent)], but similar results were obtained when the PI4,5P 2 (PIP 3 or PI3,4P 2 ) content in the vesicles was varied (e.g., POPC/ POPS/PI4,5P 2 = 80-x:20:x, x = 0-10 mol%) with the fixed total lipid concentration (not shown). The ratio of fluorescence intensity at 470 nm versus that at 530 nm (F 470 /F 530 ) showed hyperbolic dependence of the lipid concentration (Fig. 1D, E). These ratiometric calibration curves allowed direct conversion of F 470 /F 530 values into lipid concentrations, thereby enabling quantitative real-time monitoring of changes in the substrate or product concentration and thus robust kinetic analysis of the reaction.

Conditions and efficiency of the PI3K activity assay
The cellular activation PI3K, which is composed of two subunits, p110 (catalytic subunit) and p85 (regulatory subunit), involves binding of two SH2 domains in the p85 to phosphotyrosines (pY) in an activating protein, such as a receptor tyrosine kinase, which relieves p110 from its inhibitory tethering by p85 (22). It has been shown that PI3K can be activated in vitro by a pY-containing peptide derived from PDGF (pY2) (22). Addition of PI3K and cofactors to the mixture of POPC/POPS/PI4,5P 2 (77:20:3 in mole percent) LUVs and DAN-eENTH resulted in a rapid decrease in F 470 /F 530 (data not shown). Conversion of F 470 /F 530 into the total PI4,5P 2 concentration by the calibration curve (see Fig. 1D) yielded a kinetic curve of PI4,5P 2 disappearance ( Fig. 2A). The order of addition of different reagents did not affect the kinetic curve ( Fig. 2A). When the PIP 3 sensor (DAN-eMyoX-tPH) was employed in place of the PI4,5P 2 sensor, the reaction led to a rapid increase in F 470 /F 530 , which was converted into the total PIP 3 concentration, yielding the kinetic curve of PIP 3 appearance (Fig. 2B). Throughout the reaction, the sum of the PI4,5P 2 and PIP 3 concentrations remained constant ( Fig. 2A, B), verifying that our assay faithfully monitors the conversion of PI4,5P 2 to PIP 3 by PI3K. The reaction could be monitored with either a cuvette-based spectrofluorometer or a plate reader.  1D). B: Kinetics of PI3K-catalyzed PIP 3 formation. The same as in A except that DAN-eMyoX-tPH was employed in place of DAN-eENTH. Notice that the order of addition of reagents did not affect the kinetic curves. The data are representative sets from quadruple independent measurements (n = 4).

Kinetic analysis of PI3K reaction
It has been shown that the reaction catalyzed by interfacial enzymes, most notably phospholipases, follows complex mechanisms involving interfacial binding/unbinding of the enzyme, which often makes it difficult to analyze interfacial enzyme kinetics by the conventional Michaelis-Menten kinetics (28,29). To determine whether the reaction catalyzed by PI3K could be analyzed by the Michaelis-Menten kinetics, we measured the initial rate (V o ) as a function of total enzyme concentration (E o ) and substrate concentration (S o 3C, D). The V o versus [PI4,5P 2 ] o plot was successfully fit by nonlinear least-squares analysis using the Michaelis-Menten equation (Fig. 3D) and the analysis yielded k cat (= 50 ± 5 s 1 ) and K m (36 ± 6 M) values. These results indicate that although the PI3K-catalyzed reaction might involve more steps than the conventional homogenous enzyme catalysis, our activity assay allows robust determination of (apparent) kinetic parameters by the straightforward Michaelis-Menten analysis and that these parameters can be used to quantitatively assess the effects of diverse factors, including PI3K mutations and variation of the substrate structure, on the PI3K enzyme activity.
Interestingly, the concentration of PIP 3 reached only 60% of PI4,5P 2 , even with the saturating concentration of PI3K (i.e., >50 nM; see also Fig. 3A). To explore the possibility that this was due to product inhibition, we carried out the PI3K reaction in the presence of varying concentrations of PIP 3 in the PI4,5P 2 -containing vesicles [i.e., POPC/ POPS/PI4,5P 2 /PIP 3 (77-x/20/3/x; x = 0-3 mol%)]. As shown in Fig. 3E, the initial rate decreased as a function of pre-added PIP 3 and essentially reached an undetectable . The k cat and K m for these PI4,5P 2 molecules were determined from the respective V o versus [PI4,5P 2 ] o plots. The data in A-C are representative sets from quadruple independent measurements (n = 4). Data in B-D indicate mean ± SD from the measurements. The data in E are a representative set from triplicate independent measurements, whereas the data in F indicate the mean ± SD from triplicate measurements. level when the equimolar PIP 3 and PI4,5P 2 were present in the same vesicles. These results support the notion that PIP 3 inhibits the PI3K reaction. This feedback inhibition mechanism might also contribute to the regulation of cellular PI3K activity under physiological conditions. In fact, our recent in situ quantification showed that stimulation of PI3K by growth factors converts only about 60% of PI4,5P 2 into PIP 3 at the PM of PTEN-null mammalian cells (25).

PI3K inhibition assay
PI3K is one of the most frequently mutated proteins in cancer and has thus been an attractive cancer drug target (16). Having established the conditions for the rapid plate reader-based PI3K assay, we tested to determine whether the assay could be used to screen molecules for PI3K-modulating activity. As a proof of principle, we measured the inhibitory activity of three well-characterized PI3K inhibitors, GDC-0941, LY294002, and wortmannin. GDC-0941 is a potent class I-selective PI3K inhibitor targeting their ATPbinding pocket with a reported IC 50 of 33 nM for PI3K (31). LY294002 is a nonselective inhibitor of PI3K with a reported IC 50 of 1.4 M (32), whereas wortmannin is an irreversible inhibitor of PI3K with the reported IC 50 value of 1.9 nM (33). Increasing concentrations (0-500 nM) of each of these molecules was added to each row of a 96-well plate and incubated with a fixed concentration (i.e., 10 nM) of PI3K for 10 min. After addition of lipid vesicles [POPC/ POPS/PI4,5P 2 (77:20:3)], the PI4,5P 2 sensor, ATP, and pY2 to the mixture, the reaction was monitored for 3 min and the V o was calculated. As shown in Fig. 4, the analysis of the plot of V o versus inhibitor concentration gave IC 50 values of 12 ± 2 nM, 5.2 ± 0.5 M, and, 4.2 ± 0.9 nM for GDC-0941, LY294002, and wortmannin, respectively. Importantly, when the enzyme activity was rapidly estimated by a single time point fluorescence measurement after 1 min (or 2 min) of incubation instead of continuous monitoring and V o determination, essentially the same IC 50 values were ob-tained (data not shown). These values compare well with reported IC 50 values for these compounds taking into account that the conditions for the inhibition assays, including the preparation and the concentration of PI3K isoforms, the composition, the physical state, and the concentration of the lipid substrate, and the assay method, vary widely among ours and other reports (31)(32)(33). These results thus demonstrate the feasibility of high-throughput screening for PI3K inhibitors.

PTEN activity assay
A PTEN-catalyzed reaction was followed by monitoring the time-dependent decrease of PIP 3 or the time-dependent increase of PI4,5P 2 . The assay condition for PTEN was simpler than that for PI3K because PTEN is not known to require cofactors for activity as long as the reaction medium is kept under reducing conditions (9). Addition of recombinant PTEN to the mixture of POPC/POPS/PIP 3 (77:20:3) LUVs and DAN-eMyoX-tPH resulted in a rapid decrease in F 470 /F 530 (data not shown), which was converted to a kinetic curve of PIP 3 disappearance (Fig. 5A). The use of PI4,5P 2 sensor (DAN-eENTH) in place of the PIP 3 sensor yielded the kinetic curve of PI4,5P 2 formation (Fig. 5B). As was the case with the PI3K activity assay, V o was linearly proportional to E o in the range of 0-40 nM when [PIP 3 ] o was kept at 50 M (data not shown). Unlike the case with PI3K, however, the PTEN-catalyzed reaction reached near full conversion of PIP 3 to PI4,5P 2 and did not exhibit product PIP 3 generated by PI3K is subsequently converted to PI3,4P 2 by lipid phosphatases, including SHIP (25) and INPP5 (10), and PI3,4P 2 plays unique signaling roles (34,35). It has been reported that PTEN regulates PI3,4P 2 signaling by converting it to phosphatidylinositol-4-phosphate (21). To investigate the enzymatic basis of this finding, we rigorously determined the relative activity of PTEN toward PIP 3 and PI3,4P 2 by simultaneously measuring the PIP 3 and PI3,4P 2 dephosphorylation. To this end, we employed a well-established PI3,4P 2 sensor, NR3-eTapp2-cPH (25), which is spectrally orthogonal to the DAN-eMyoX-tPH (see Fig. 1C, F), thereby enabling direct simultaneous monitor-ing of PIP 3 and PI3,4P 2 dephosphorylation. As a negative control, we separately checked the activity of PTEN against POPC/POPS/PI4,5P 2 (77:20:3) LUVs (Fig. 5E). Addition of PTEN to the mixture containing POPC/POPS/PIP 3 (77:20:3) LUVs, POPC/POPS/PI3,4P 2 (77:20:3) LUVs, DAN-eMyoX-tPH, and NR3-eTapp2-cPH resulted in a rapid decrease in F 470 /F 530 (data not shown), which was converted to a kinetic curve of PIP 3 disappearance (Fig. 5E); however, the decrease in F 600 /F 675 , which reflects the dephosphorylation of PI3,4P 2 , was only slightly above the negative control under the same conditions (Fig. 5E). These results show that PTEN has much lower intrinsic enzymatic activity toward PI3,4P 2 than toward PIP 3 and suggest that the reported activity of PTEN to regulate PI3,4P 2 signaling might not derive from its catalytic action on PI3,4P 2 . It should be noted that our study was performed with the bacterially expressed PTEN and that one cannot preclude the possibility that posttranslational modification in mammalian cells might confer enhanced activity for PI3,4P 2 on PTEN. (77/20/3) LUVs for 1 min, and then 10 nM PTEN were added to the mixture to initiate the reaction. F 470 /F 530 and F 600 /F 675 were monitored to simultaneously track the dephosphorylation of PIP 3 and PI3,4P 2 , respectively. PTEN activity against POPC/POPS/PI4,5P 2 (77:20:3) LUVs was separately measured using DAN-eENTH. The data in A-C and E are representative sets from quadruple independent measurements (n = 4). The data in D represent the mean ± SD from triplicate measurements.

CONCLUSIONS
We have developed a new fluorescence-based real-time assay for PI3K and PTEN. The main advantages of this direct quantitative assay are high sensitivity, accuracy, speed, and a high degree of flexibility in assay design. Although the present study was confined to a single form of PI3K and PTEN, respectively, the assay is universally applicable to the kinetic analysis of any lipid kinase and phosphatase, as long as a sensor specific for its lipid substrate or product can be prepared. Our straightforward kinetic analysis of PI3K and PTEN produced the new mechanistic information about these enzymes, and our pilot study demonstrates feasibility for high-throughput screening of small molecules for their PI3K-modulating activity. The new method will facilitate further mechanistic studies on other lipid kinases and phosphatases as well as rapid screening and testing of small molecule modulators of pharmacologically important lipid kinases and phosphatases.

Data availability
All data are contained within the article. The raw data will be shared upon request: contact Wonhwa Cho (University of Illinois at Chicago, e-mail: wcho@uic.edu).