A Homogeneous Phosphoinositide 3-Kinase Assay on Phospholipid FlashPlate Platforms. Busi Maswoswe, Hao Xie, Pat Kasila and Li-an Yeh

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1 A Homogeneous Phosphoinositide 3-Kinase Assay on Phospholipid FlashPlate Platforms Busi Maswoswe, Hao Xie, Pat Kasila and Li-an Yeh

2 Abstract Phosphoinositide 3-kinases (PI 3-kinase) consist of a family of lipid kinases that regulate major pathways by which biological signaling systems operate to control cell functions. The enzymes catalyze the addition of a phosphate molecule to the D-3 position of the inositol ring phophoinositides. Conventional PI 3-kinase assay involves organic solvent extractions and sometimes TLC separation. This study demonstrates a homogeneous method for measuring PI 3-kinase activity using the Phospholipid FlashPlate platform. In this method, a solid phase substrate is used by coating PIP 2 onto the Phospholipid FlashPlate. The enzyme kinetics and validation of this assay by inhibitor studies will be illustrated. This simple and robust assay for PI 3-kinase can be easily adapted into high throughput screening format for drug discovery. Introduction FlashPlate is a white, opaque, 96-well scintillant coated microplate. The Phospholipid FlashPlate uses a novel technology (U.S. Patent 5,972,595) which enables the attachment of lipid substrates to the FlashPlate surface through a hydrophobic interaction. Phosphoinositide 3-kinase (PI 3-kinase) is an enzyme which is involved in the induction of mitogenesis and cell transformation by growth factors and oncogenic agents. (1-5) The exact mechanism of the involvement of PI 3-kinase in the regulation of cell growth is still unknown, however, it was postulated that the PI 3-kinase generates a novel class of second messenger phospholipids by phosphorylating phosphatidylinositol at the D-3 position of the inositol ring. (6) The substrate for this enzyme (PIP 2 ) is a very hydrophobic molecule and is partitioned into a hydrophobic lipid layer created on the FlashPlate surface (Phospholipid FlashPlate). When the PIP 2 immobilized on the surface of the FlashPlate platform is phosphorylated with [ 33 P]ATP by the PI 3-kinase, the light generated by the close proximity of the scintillant can be measured using a Microplate Scintillation Counter. The validation of this solid phase substrate assay is demonstrated by comparing enzyme kinetics and inhibitor studies. 2

3 1 Experimental Procedures Materials: Phospholipid FlashPlate (NEN, Cat #SMP108); Substrate Coating Buffer (NEN, Cat #SMP900) [ 33 P]ATP (NEN, Cat #NEG302H) Purified recombinant PI 3-kinase was kindly provided to us by Lucia Rameh in Dr. L. Cantley s laboratory at Harvard Medical School, Boston, MA. Phosphatidylinositol 4,5-bisphosphate, PIP 2 (Cal Biochem); Phosphatidylserine (Sigma Chemicals). Methods: FlashPlate Assay Immobilization of substrate on FlashPlate PIP 2 was diluted in Substrate Coating Buffer and 200 µl pipetted into each well of a Phospholipid FlashPlate. The plates were incubated overnight at room temperature. After the incubation step, wells were washed 1X with 1 mm DOC in PBS, followed by 2X wash with PBS. Wells were aspirated prior to use in the kinase assay. PI 3-Kinase Assay The assay solution contained 20 mm Tris-HCl (ph 7.4), 5 mm MgCl 2, 10 µm ATP, 1 µci/reaction [ 33 P]ATP, 2 µl/well PI 3-kinase, final volume 200 µl/well. Plates were covered and the reaction mixture was shaken for 5 minutes before incubating at 30 o C for 1 hour. Following the incubation step, wells were aspirated and washed 2X with PBS. The bound radioactivity was determined by counting the FlashPlate. Solution Phase Assay Solution Phase lipid kinase assay was conducted as described by Carpenter (4). Briefly, the assay mixture (total volume 200 µl) contained 20 mm Tris-HCl (ph 7.4), 5 mm MgCl 2, 10 µm ATP, 1 µci [ 33 P]ATP, 0.1 mg/ml PIP 2, 0.1 mg/ml Phosphatidylserine. Wortmannin was added at varying concentrations. The enzyme reaction was initiated by adding 2 µl of PI 3-kinase. Reaction mixtures were incubated at 30 o C for 1 hour before stopping the reaction with an equal volume of 1M HCl. Lipids were extracted by the addition of chloroform/methanol (1:1). The amount of radioactivity in the chloroform phase (corresponding to PI 3,4,5 P 3 ) was measured by liquid scintillation counter. 2 Results and Discussion Studies were done to determine the optimum conditions for binding PIP 2, (PI 3-kinase substrate) onto the Phospholipid FlashPlate. The substrate, [ 3 H]PIP 2 (NEN, custom preparation) was diluted in Substrate Coating Buffer. Varying concentrations (up to 5000 pmol) were added at 0.2 ml/well and the plates were incubated overnight at room temperature. Following the incubation step, the FlashPlates were read on a Packard TopCount. Maximum capacity of the phospholipid coated well is greater than 100 pmol. This level of binding is achieved after adding 2000 pmol PIP 2. Doubling the amount of substrate added to each well (up to 4000 pmol) did not result in significant increase in bound material. 3

4 3 Phosphorylation of PIP 2 bound to the plate was studied by measuring incorporation of [ 33 P]ATP. Phosphate incorporation increased with increasing PIP 2 bound to the FlashPlate (up to 40 pmol). When higher amount of substrate was coated on the plate, the enzyme activity decreased as shown. This suggests that at higher concentrations, the substrate presentation on the plate may no longer be in a favorable conformation for the enzyme. 4 Regression Analysis to Determine the K m of the Enzymes for the Immobilized Substrate Nonradioactive substrate was added to each well as described above. PI 3-kinase assay was conducted as described in the methods section. The amount of phosphorylated substrate was determined by measuring the incorporation of [ 33 P]ATP into PIP 2. The K m of PI 3-kinase for PIP 2 in solution phase assay is 4 µm. (4) The K m obtained for this solid phase substrate is 25 nm. The decrease in K m for PIP 2 may indicate that the substrate presented on the Phospholipid FlashPlate surface is a better substrate compared with the PIP 2 in solution assay. The V max of the enzyme under FlashPlate assay conditions is µmoles/mg/min which is comparable to the literature value of 0.22 µmoles/mg/min. (4) 4

5 5 Time Course Showing PI 3-kinase Activity Kinase assay was performed on a Phospholipid FlashPlate using 1 and 2 µl of enzyme. Phosphorylation of substrate at 30 o C was followed for up to 1 hour. Doubling the enzyme concentration resulted in increased phosphorylation of the substrate. In the presence of 2 µl enzyme the rate of phosphorylation was linear for 1 hour. 6 Effect of Wortmannin on PI 3-kinase Activity (FlashPlate assay) Wortmannin inhibits PI 3-kinase in the FlashPlate assay. IC 50 for the FlashPlate assay is 1.64 nm, which is comparable to the literature value of 5 nm. 5

6 7 Effect of LY on PI 3-kinase Activity (FlashPlate Assay) LY inhibits PI 3-kinase in the FlashPlate assay. The IC 50 value is 0.44 µm, which is comparable to the literature value of 1.4 µm. 8 Effect of Wortmannin on PI 3-kinase Assay (Solution Phase Assay) This confirms the inhibition of PI 3-kinase by Wortmannin. In this assay the IC 50 value was 1.6 nm, which is comparable to the FlashPlate assay. 6

7 9 Summary Table Comparing PI 3-kinase Assay on FlashPlate to Solution Phase Assay IC 50 K m V max Wortmannin LY PIP2 PIP2 nm µm nm µmole/mg/min FlashPlate ` Assay Solution Phase 1.6 ND ND ND Assay Literature Value (Reference 7) (Reference 8) (Reference 4) (Reference 4) Conclusion Comparison of literature enzyme kinetic values shows that the enzyme performs equally well on the FlashPlate. This indicates that substrate bound to the FlashPlate is available for phosphorylation. Side-by-side comparisons of IC 50 values show that the FlashPlate assay is comparable to the conventional solution phase assay. IC 50 values are similar to the published literature values. Data shown validates the use of the FlashPlate method to measure PI 3-kinase activity. The FlashPlate method involves fewer steps and generates less radioactive waste than the conventional solution phase assay. The FlashPlate method is homogeneous and can be readily formatted for performance by robotics, and is amenable to high throughput screening. References 1. Auger, K.R., Serunian, L.A., Soltoff, S.O., and Cantley, L.C. (1989) Cell 57, Boyle, W.J., Van Der Geer, P., and Hunter, T. (1991) Methods Enzymology 201, Cantley, L.C., Auger, K.R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, Carpenter, L.C., Duckworth, B., Auger, K.R., Cohen, B., Schaffhausen, B.S., and Cantley, L.C. (1989) J. Biol. Chem. 265, Coughlin, S.R., Escobedo, J.A., and Williams, L.T. (1989) Science 243, Whitman, M., Downes, C.P., Keeler, T., and Cantley, L. (1988) Nature (London) 332, Duckworth, B., and Cantley, L.C. J. Biol. Chem. 272, Baumann, P., and West, S.C. Proc. Natl. Acad. Sci. USA 95, NEN is a registered trademark of NEN Life Science Products, Inc. Phospholipid FlashPlate is protected under US Patent 5,972,595 and foreign equivalents and patents pending. FlashPlate is a registered trademark of Packard Instrument Company, Inc. exclusively licensed to NEN Life Science Products. TopCount is a registered trademark of Packard Instrument Company. FlashPlate is protected under U.S. Patent 5,496,502 and foreign equivalents, to all of which NEN Life Science Products, Inc. holds an exclusive, worldwide license. 7

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