associated with the plasma membrane [18] and generates phosphatidylinositol–4,5-bisphosphate (PIP2) [19] which plays a pivotal role in receptor endocytosis. The level of PIP2 reaches a maximum at the initiation of endocytosis [20] and it causes the recruitment of endocytic proteins onto the plasma membrane [21,22]. Recently it was also reported that activation of PIP5- kinase through interaction with AP2 and local generation of PIP2 is important for the recruitment of endocytic machineries and synaptic vesicle internalization [23]. Taken together, we propose that PA generation by PLD1 activation is a key molecular event that causes the local accumulation of PIP2 through direct activation of PIP5-kinase and enhancing AP2-PIP5-kinase path- way by placing these two molecules in close proximity to facilitate EGFR endocytosis. Materials and Methods RNA interference Pairs of 21-nucleotide sense and antisense RNA oligomers which correspond to human PLD1a coding nucleotides 1455–1475 were chemically synthesized and annealed by Dharmacon Research, Inc. The oligonucleotides for PLD1 were as follows: sense, 59-AAG GUG GGA CGA CAA UGA GCA-39, and antisense, 59-UGC UCA UUG UCG UCC CAC CUU-39. Luciferase GL2 duplex (Dharmacon Research, Inc.) was used as negative control. Cell culture and transfection HeLa cells obtained from the American Type Culture Collection were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 100 units/ml penicillin, 100 mg/ml strepto- mycin, and 10% fetal bovine serum (GIBCO-BRL). HeLa cells were introduced with synthetic siRNA (20 nM duplex) for PLD1 using METAFECTENETM reagent (Biontex) according to the manufacturer’s instructions and then cultured for 48 hours to achieve PLD1 silencing. To express the PLD1 constructs, cells were transiently expressed with rat-origin PLD1 cDNAs using Lipofectamine PlusTM reagent (Invitrogen) one day after siRNA transfection. Generation of plasmids and GST fusion proteins R304A and R304K point mutants of PLD1 were generated by mutating the arginine residue at 304 to Ala or Lys respectively. Briefly, the R304A mutant was obtained using forward primer: 59- TGA TAA TCT TTC AGC TAC ACT GAT TTT A-39 and reverse primer: 59-TAA AAT CAG TGT AGC TGA AAG ATT ATC A-39. A similar strategy was used to generate the R304K mutant using forward primer: 59-TGA TAA TCT TTC AAA AAC ACT GAT TTT A-39 and reverse primer: 59-TAA AAT CAG TGT TTT TGA AAG ATT ATC A-39. The resulting products were cloned into pCDNA3.1 (Invitrogen) or pEGFP-C1 (Clontech) vector. Glutathione S-transferase (GST) fusion proteins were prepared using glutathione-Sepharose 4B (Amersham Biosciences). GST fusion PH domains of PLCb1, IRS, and dynamin were kindly provided by Dr. Brian K. Kay (The University of Wisconsin, Madison, WI) and GST-m2 was generous gift from Dr. Juan S. Bonifacino (National Institutes of Health, Bethesda, MD). In vitro binding analysis In vitro binding was performed in 500 ml of buffer A (50 mM Hepes-NaOH (pH 7.5), 3 mM MgCl2, 2 mM CaCl2, 3 mM EGTA, and 80 mM KCl) containing 1% Triton X-100 at 4uC for 3 h. In experiments adding C6-PA (Avanti Polar Lipids), pellets PLD1 and EGFR Endocytosis were washed with buffer A containing the same concentrations of C6-PA used in the incubation. Immunoprecipitation and western blotting For immunoprecipitation, HeLa cells were serum starved for 12 h, treated with EGF (20 nM) (Daewoong Pharmaceutical Co.), and lysed by sonication with buffer A containing protease inhibitors, 1% Triton X-100, and 1% cholic acid. Cell lysates were centrifuged at 100,000 g for 30 min at 4uC and the same amounts of supernatant were incubated with anti-PLD [24] or anti-EGFR (Upstate Biotechnology) antibody immobilized on Protein A-Sepharose beads (Amersham Biosciences). For immu- noblotting, anti-m2 (BD Transduction Laboratories), anti-a- adaptin (BD Transduction Laboratories), anti-clathrin heavy chain (BD Pharmingen), and anti-GST (Santa Cruz Biotechnology, Inc.) antibodies were used. Horseradish peroxidase-conjugated goat anti- rabbit or goat anti-mouse antibodies were purchased from Kirkegaard and Perry Laboratories Inc. Proteins were visualized by chemiluminescence using Enhanced Chemiluminescence kit (Amersham Bioscience). In vitro adaptor recruitment assays HeLa cells transfected with wild type PLD1 were homogenized with buffer B (buffer A with protease inhibitors and 0.25 M sucrose) using a Dounce homogenizer and then sonicated. A postmitochondrial supernatant was prepared by centrifuging the homogenate at 1500 g for 10 min and the resulting supernatant was further centrifuged at 100,000 g for 15 min in a TL-100.2 rotor (Beckman Instruments). After washing with buffer B, membrane pellets were resuspended in 50 ml of rat brain cytosol (10 mg/ml) prepared in buffer A containing protease inhibitors. After 15 min at 37uC, reactions were stopped by adding 1 ml of cold buffer A and membranes were collected by centrifugation as described above. Pellets were resuspended in 50 ml of Laemmli sample buffer and 10 ml aliquots were subjected to SDS-PAGE and western blotting. Adaptor recruitment assays in cells Recruitment of endocytic proteins onto cell membranes was performed as described below. After depletion of endogenous PLD1, HeLa cells were transfected with vector, wild type, K898R, R158Q, or R304A PLD1. The cells were then treated with EGF (20 nM) during 2 min and washed twice with ice-cold buffer A. Preparation of cell membrane was performed as described in ‘‘In vitro adaptor recruitment assays.’’ After washing with buffer B, membrane pellets were resuspended in 50 ml of Laemmli sample buffer and 10 ml aliquots were subjected to SDS-PAGE and western blotting. Internalization of EGFR HeLa cells grown in 60 mm dishes were starved for 12 h and EGF (20 nM) was treated. Cells were washed twice with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBS2+) and incubated with 1 mg/ml Sulfo-NHS-SS-biotin (Pierce) for 30 min at 4uC. Nonreactive biotin was quenched with ice-cold PBS2+ and 0.1 M glycine for 20 min at 4uC. The cells were then lysed with radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, and 1% sodium deoxycholate) containing protease inhibitors and protein concentrations were determined. Biotiny- lated and nonbiotinylated proteins were separated from equal amounts of cellular protein by incubation with Immunopure immobilized streptavidin (Pierce) for 4 h at 4uC. The biotin- PLoS ONE | www.plosone.org 6 September 2009 | Volume 4 | Issue 9 | e7090