We then investigated the mechanism whereby PA induces the interaction between PLD1 and m2. Stahelin et al. [14] suggested that the PX domain of PLD1 has a binding pocket for anionic phospholipids such as PA. Thus, we hypothesized that the occupation of the PX domain by PA generated by the activation of PLD1 might contribute to the binding of the PH domain and m2. To explore this possibility, we checked the effect of PA on the PLD1-m2 interaction using exogenous PA, and found that PA increased this interaction in a dose-dependent manner (Figure 3C). Furthermore, the addition of 20 nM of PA induced binding between m2 and the lipase inactive mutant PLD1 (K898R), as shown in Figure 3D. Interestingly, PLD1 (R158Q) mutant, which has full lipase activity (data not shown) but reduced affinity for PA [14], could not interact with m2 in the presence of 20 nM of PA. These results indicate that PLD1 is a direct target of PA, its own product, and that the PA binding enhances its interaction with m2. The PLD1-m2 interaction is important for adaptor recruitment onto membranes AP2 localizes to the cytosol when cells are in an un-stimulated state, thus AP2 should translocate onto the plasma membrane before it acts as an adaptor linking EGFR to the endocytic machinery [15]. Also, it is generally accepted that recognition of EGFR by m2 is an essential process in the clathrin-mediated endocytosis of EGFR [4]. To understand the role of PLD1-m2 interaction in EGFR endocytosis, we first performed in vitro membrane recruitment analysis using membrane preparation of wild type PLD1 and rat brain cytosol. The addition of purified PLD1 fragment (296–312) corresponding to the region involved in m2 binding abolished the positive effect of wild type PLD1 on the recruitment of m2 onto the membrane in a dose-dependent manner (Figure 4A); however, the same fragment carrying the R304A mutation had no effect. To confirm this, we performed adaptor recruitment analysis in cells. HeLa cells depleted of endogenous PLD1 were transfected with wild type, K898R, R158Q, or R304A PLD1 and the localization of endocytic proteins were monitored after 2 min of EGF treatment. Wild type PLD1, but none of the mutants with little affinity for m2 (i.e., K898R, R158Q, and R304A), enhanced the recruitment of m2, a- subunit of AP2 (a-adaptin), and clathrin heavy chain (CHC) onto membrane (Figure 4B). These results suggest that the interaction between PLD1 and its reaction product PA promotes the recruitment of adaptors to membranes by enhancing the interaction between PLD1 and m2. PLD1-m2 interaction is important for PLD1-mediated EGFR endocytosis Upon EGF stimulation, m2 translocates onto the plasma membrane and recognizes EGFR. So we checked the role of PLD1 in the m2-EGFR interaction. EGF-dependent EGFR-m2 association was observed in wild type PLD1-transfected cells, but not in cells transfected with PLD1 (R304A), PLD1 (R158Q), or PLD1 (K898R) although these PLD1 constructs exist in EGFR complex (Figure 5A). Next, we measured the internalization rate of EGFR during 3 min of EGF treatment. Cells expressing PLD1 (R304A), PLD1 (R158Q), or PLD1 (K898R) neither of which could interact with m2 showed about 20% of EGFR internalization rate compared to wild type PLD1-expressing cells (Figure 5B). These PLD1 constructs were expressed in comparable levels in cells (Figure 5B, inset) and had comparable lipase activity. In addition, the transient expression of a PLD1 fragment 296–312 resulted in the potent inhibition of EGFR internalization rate (Figure 5D) and the EGFR-m2 association (Figure 5C) although PLD1 and EGFR Endocytosis Figure 4. The PLD1-m2 interaction is important for adaptor recruitment onto membranes. (A) Purified GST-fusion PLD1 296–312 or PLD1 296–312 (R304A) was added to recruitment assay mixture containing membrane preparation from wild type PLD1-expressing HeLa cells and rat brain cytosol (10 mg/ml). Membrane-recruited m2 was detected by western blotting. Data are representative of two indepen- dent experiments. (B) Translocation of endocytic proteins onto plasma membrane was analyzed. HeLa cells transfected with vector, wild type, K898R, R158Q, or R304A PLD1 were treated with EGF (20 nM) during 2 min and the membrane preparations were subjected to SDS-PAGE and immunoblotted using the indicated antibodies. doi:10.1371/journal.pone.0007090.g004 this construct had little effect on the lipase activity of PLD. Taken together, these results indicate that the interaction between PLD1 and m2 is a key determinant for the EGF-induced internalization rate of EGFR in cells. Discussion PLD1 is a key factor in the regulation of EGFR endocytosis. PLD1 is activated upon EGF stimulation and it hydrolyzes PC to PA at the plasma membrane [16]. Nonetheless, the identity of PA effector protein and the mechanism by which PA regulates the EGFR endocytosis are still unknown. The present study shows that PLD1 itself is an effector of PA and auto-regulatory interaction between the PX domain of PLD1 and PA promotes the binding of another domain of PLD1, the PH domain, with m2 and thereby facilitates EGFR endocytosis. The findings that the PLD1-m2 association is dependent on its lipase activity and that the effector of PA is PLD1 itself establish the key role of PLD1 in EGFR endocytosis and also provide new mechanistic insight. To our knowledge, this is the first example of an auto-regulatory mechanism for a signaling enzyme. On the basis of our results, we propose that PLD1 is a complex molecular device with functional triad composed of catalytic, PX, and PH domain, each of which senses and mediates discrete steps of EGFR endocytosis (Figure 5E). In our model, the catalytic domain of PLD1 first generates PA from PC in response to EGFR PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e7090