Anti-FLAG monoclonal antibody (clone M2), wortmannin and bovine liver phosphatidyinositol were obtained from Sigma-Aldrich (St. Louis, MO). PIK-93 [9
] was purchased from Symansis (Washdyke, New Zealand).
293T (GenHunter, Nashville, TN) and COS-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin.
PI 4-kinase expression constructs
The constructs encoding full-length human PI4KA and PI4KB cDNAs tagged with an N-terminal 3XFLAG epitope have been previously reported [4
]. FLAG-PI4KB was generated by cloning the Flag sequence between EcoRV and Xho I sites at the N-terminus of human PI4KB in the pcDNA3.1 plasmid (generously provided by Gordon Polevoy and Julie Brill, The Hospital for Sick Children, Toronto, Canada). The kinase-inactive PI4KA mutant D1899A contains a mutation in the conserved lipid kinase catalytic domain corresponding to the kinase-inactivating mutation D656A for PI4KB reported by Godi et al.
] and was introduced into PI4KA by overlap extension PCR. The amplified region was completely sequenced to confirm that the D1899A mutation had been introduced without any unwanted mutations. Constructs encoding full-length human PI4K2A and PI4K2B cDNAs tagged with an N-terminal 3XFLAG epitope were constructed by PCR amplification from full-length cDNAs obtained from Open Biosystems (Huntsville, AL) and subcloning into the pFB retroviral vector (Stratagene; La Jolla, CA). Primer sequences and a detailed cloning strategy will be provided upon request.
Expression and affinity purification of PI 4-kinases
293T or COS-7 cells were transfected with plasmids encoding FLAG-tagged PI 4-kinase constructs using FuGENE HD (Roche Diagnostics, Indianapolis, IN) according the manufacturer’s instructions. 24 hours post- transfection, cells were washed with PBS and then lysed in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% NP-40, 10% glycerol, 1 mM EDTA, 0.5 mM DTT, and Halt protease inhibitor cocktail (Pierce, Rockford, IL) for 15 minutes on ice. The lysate was centrifuged for 15 minutes at maximum speed in a microcentrifuge at 4ºC to remove nuclei and insoluble material. The supernatant was then mixed with protein G-conjugated Dynabeads (Invitrogen, Carlsbad, CA) prebound with anti-FLAG monoclonal antibody (clone M2, Sigma-Aldrich) for 1 hour at 4ºC.
The Dynabeads were then washed 3 times with lysis buffer (including protease inhibitors) and then once with kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 1 mM EGTA, 0.2% Triton X-100) supplemented with 0.5 mM DTT and 0.1% bovine serum albumin (BSA). Bound kinase was eluted with kinase buffer supplemented with 2 mM DTT, 0.1% BSA, and 500 μg/mL 3XFLAG peptide (Sigma-Aldrich) for 30 minutes at 4ºC. The elution was repeated twice more; the eluates were pooled, aliquoted, snap-frozen in liquid nitrogen, and stored at −80ºC for subsequent use.
ADP-Glo PI 4-kinase assay
White low-volume 384 well polystyrene plates (ProxiPlate-384 Plus, PerkinElmer, Waltham, MA) were used for the ADP-Glo assay. PI dissolved in chloroform was dried in Eppendorf brand microcentrifuge tubes under a nitrogen stream in a fume hood, resuspended to 2.5 mM in kinase buffer containing 0.2% Triton X-100, and then sonicated by four 15-second pulses with a Branson Sonifier microtip (Danbury, CT) until the suspension became translucent. 2.5 μL of kinase were mixed with 2.5 μL of kinase buffer/2 mM DTT/0.1% BSA with PI micelles (800 μM final concentration) and ultrapure ATP (Promega, Madison, WI). Enzyme concentrations were chosen so that the reactions of both PI4KA and PI4KB were linear during the 15-minute incubation, and all reactions were carried out in triplicate. Blank wells lacked enzyme but did include kinase buffer, substrate, and ATP. The plates were covered and the reactions were carried out at room temperature for up to 60 minutes. Reactions were stopped with the addition of 5 μL ADP-Glo reagent (Promega). After a 40 minute incubation at RT, 10 μL of Kinase Detection Reagent (Promega) were added and the plates were incubated for another 40 minutes at RT (60 minutes for ATP concentrations greater than 100 μM). Plates were read on a BioTek Synergy 2 plate reader (Winooski, VT) with a sensitivity of 150 and an integration time of 1 second per well. Data were analyzed using Prism 5.0 software (GraphPad, La Jolla, CA).
For kinase reactions involving inhibitors, 2 μL of kinase were preincubated with 1 μL of inhibitor at varying concentrations in kinase buffer for 10 minutes at RT prior to addition of 2 μL of kinase buffer with PI (800 μM final concentration), ATP (100 μM final), and inhibitor. Wortmannin stocks were prepared in DMSO and were diluted immediately before use due to its instability in aqueous solutions. The percent inhibition was calculated relative to an enzyme control without inhibitor. IC50s were calculated by four-parameter nonlinear regression using Prism 5 software (GraphPad Software, La Jolla, CA).
High-throughput PI4KA assay
For Z-factor determination, 4 μL of purified PI4KA in kinase buffer containing 1 mM PI micelles were dispensed into Corning 3674 low-volume 384- well plates using a Multidrop Combi dispenser (Thermo Scientific, Barrington, IL). One column was left without enzyme to measure background luminescence. 100 nL of DMSO were dispensed into 11 columns using a Biomek FX pin tool dispenser (Beckman Coulter, Brea, CA), while 100 nL of 500 μM wortmannin in DMSO (10 μM final concentration) were dispensed into 12 columns using a Mosquito dispenser (TTP LabTech, Cambridge, MA). After a 10 minute preincubation, kinase reactions were started by adding 1 μL of 500 μM ATP using the Multidrop Combi dispenser. Kinase reactions were carried out for 15 minutes at RT. Reactions were stopped with the addition of 5 μL ADP-Glo reagent. After a 40 minute incubation at RT, 10 μL Kinase Detection Reagent was added and the plate was incubated for another 40 minutes at RT. Plates were read on a Pherastar plate reader (BMG LABTECH, Cary, NC).
Radiometric PI 4-kinase assay
Radiometric assays for PI 4-kinase activity were performed as described in [8
]. In brief, 5 μL of purified PI4KA or PI4KB was added to 40 μL of kinase buffer (50 mM Tris-Cl pH 7.5, 20 mM MgCl2
, 1 mM EGTA, 1 mM PI, 0.4% Triton X-100, 0.5 mg/mL BSA) and preincubated with inhibitors for 10 minutes. Reactions were started with 5 μL of 1 mM [γ32
P]ATP and incubated at RT. Reactions were stopped after 30 minutes by the addition of 3 mL CHCl3
OH/concentrated HCl (200:100:0.75, v/v) followed by mixing and then adding 0.6 mL of 0.6 M HCl. After centrifugation, the aqueous phase containing unreacted [γ32
P]ATP was removed, and the organic phase containing PtdIns4P was reextracted with 1.5 mL of CHCl3
OH/0.6 M HCl (3:48:47, v/v). The lower organic phase was transferred to scintillation vials, dried, and the product [γ32
P]PtdIns4P was quantified by liquid scintillation.