Figure 1. Wild type but not lipase inactive PLD1 facilitates EGFR endocytosis. (A) HeLa cells were transfected with control (Luc) or PLD1 (PLD1) siRNA to deplete endogenously expressed PLD1. After serum starvation for 12 h, EGF was treated at 20 nM for 0, 2, 5, or 10 min and biotinylation of cell surface proteins was performed. Biotinylated (Cell surface) proteins were separated using streptavidin beads and analyzed by western blotting. A representative immunoblot of three independent experiments is shown. (B) Quantitation of EGFR internalization in HeLa cells. The kinetics of the EGF-induced internalization of EGFR in HeLa cells transfected with either luciferase (closed circle) or PLD1 (open circle) siRNA. (C) HeLa cells depleted of endogenous PLD1 were incubated with 125I-EGF at 37uC. ke valueswere measured during linear 3-min time course and expressed as per cent of the mean values obtained for control-transfected cells. The data represent mean values from three independent experiments and the error bars represent standard deviations. (D) After depleting endoge- nous PLD1 using PLD1 siRNA, endogenous level of wild type or K898R PLD1 was expressed in HeLa cells. The internalization rate of EGFR was calculated as described in (C) and is shown as a bar graph reflecting the average of three independent experiments and standard deviations. Immunoblots in the inset indicate the expression levels of the constructs used. doi:10.1371/journal.pone.0007090.g001 PLD1 and EGFR Endocytosis of PLD1-depleted cells was 19.5% compared to control. To confirm the role of PLD1 and to check the involvement of the lipase activity of PLD1 in EGFR endocytosis, we transfected wild type or a lipase inactive mutant PLD1 (K898R) after depleting the endogenous PLD1 from HeLa cells by siPLD1, and measured the internalization of EGFR after 2 min of EGF treatment. The expression of wild type PLD1 in PLD1-depleted HeLa cells restored the internalization rate of EGFR. However, the expression of PLD1 (K898R) failed to recover EGFR internalization rate (Figure 1D). These results suggest that endogenous PLD1 facilitates the EGF-induced internalization of EGFR in HeLa cells in a lipase activity-dependent manner. PLD1 associates with the m2 subunit of AP2 in a PA-dependent manner The endocytosis of activated cell surface receptors is mediated by an adaptor protein AP2 that recognizes them via its m2 subunit and recruits dynamin, clathrin, and accessory proteins involved in vesicle formation [4]. PLD1 localizes near EGFR in the plasma membrane and is transiently activated upon EGF stimulation [11]. To understand the mechanism by which lipase activity of PLD1 facilitates EGFR endocytosis, we checked the possibility of EGF stimulation-dependent complex formation between PLD1 and the components of AP2. Interestingly, the m2 subunit of AP2 was found to transiently interact with endogenous PLD1 after 1–2 min of EGF stimulation in HeLa cells (Figure 2A). In contrast, the lipase inactive mutant PLD1 (K898R), which could not enhance EGFR endocytosis (see Figure 1C) failed to interact with m2 (data not shown). To investigate the involvement of the lipase activity of PLD1 in its binding to m2, we measured the kinetics of PLD1 activation under the same conditions after EGF treatment. Figure 2B shows that the activity of PLD1 dramatically stimulated after 1 min of EGF treatment which correlates well with the kinetics of PLD1-m2 interaction. To confirm the lipase activity- mediated interaction between PLD1 and m2, 1-butanol was used to inhibit the PA production. PLD preferentially utilizes primary alcohol (e.g., 1-butanol), but not tertiary alcohol (e.g., t-butanol), over water to generate a phosphatidylalcohol (e.g., phosphatidyl- butanol) instead of PA [12]. After 2 min of EGF stimulation, the association between PLD1 and m2 was detected in control and t- butanol treated cells, but not in 1-butanol treated cells (Figure 2C). Taken together, these results suggest that the production of PA by PLD1 is important for its interaction with m2. Association with PA enhances the direct interaction between PLD1 and m2 To test whether PLD1 directly interacts with m2 or not, in vitro pull-down analysis was performed using purified PLD1 and GST-m2. As shown in Figure 3A, GST-m2 was coprecipitated with PLD1, indicating that m2 binds directly to PLD1. PLD1 is com- posed of a phox homology (PX) domain, a pleckstrin homology (PH) domain, a central loop, and the conserved region (CR) I-IV [13]. To identify the region responsible for direct binding to m2, we used GST fusion PLD1 fragments as shown schematically in Figure S1A. The pull-down assay showed that the PH domain bound to m2 (Figure S1B and S1C) via a region spanning amino acids 296–312 (Figure S1D). Moreover, this m2-PLD1 PH domain interaction was highly specific, as the PH domains of other proteins did not show any binding to m2 (Figure S1E). When immunoprecipitation was performed in HeLa cells that were transfected with PLD1 constructs after depleting endogenous PLD1, m2 bound to wild type PLD1, but not to R304A or R304K mutant (Figure 3B). These results indicate that Arg 304 within the PH domain of PLD1 is directly involved in interaction with m2. PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e7090