Phospholipase D (PLD) is a ubiquitous enzyme that catalyzes the hydrolysis of phosphatidylcholine (PC) to produce metaboli- cally active phosphatidic acid (PA). PLDs are membrane-bound proteins and the relative distributions of PLD isoforms (PLD1 and PLD2) are distinct in various cell types [15]–[16]. Consistent with its diverse locations, PLD exerts multiple cellular functions in different cell types [17]–[19]. Previous studies have suggested that PLD activity is regulated by various stress signals including serum withdrawal, glucose availability, oxidative stress and pressure overload [20]–[22]. As a result, it has been proposed that PLD plays a role under stressful conditions. However, the regulatory mechanisms involved in PLD activity for glucose uptake under glucose deprivation conditions in skeletal muscle cells have not been studied to date. In this study, we attempted to gain insight into the relationships between AMPK, PLD1 and glucose uptake. Based on the results of the present study, we suggest that AMPK-induced PLD1 activity is required for the regulation of glucose uptake via ERK activation in muscle cells and that PLD1 has a novel site that is phosphorylated by AMPK. Taken together, these findings imply that the AMPK- PLD1 pathway may contribute to the control of glucose homeostasis. Results AMPK Stimulates PLD1 Activity Previous studies have shown that PLD activity was regulated when energy imbalance was induced by serum withdrawal or glucose starvation [22], [23]. To determine the specific role of PLD isoforms under glucose deprivation, we examined PLD activity after the ectopic expression of PLD1 or PLD2 isoforms in HEK-293 cells. Endogenous PLD activity led to a significant increase in vector-transfected cells under glucose deprivation, as indicated by increased byproducts (PBtOH) of PLD-mediated transphosphatidylation (Figure 1A). PLD1 activity increased approximately 2-fold in PLD1-transfected cells under glucose deprivation conditions, whereas PLD2 activity changed very slightly under the same conditions (Figure 1A). Since, it is well known that AMPK is regulated by changes in the physiological glucose concentration [4], [9], we investigated the relationship between PLD isoforms and AMPK under different glucose concentrations. We first silenced PLD1 or PLD2 in HEK-293 cells and then examined PLD activity. As shown in Figure 1B, endogenous PLD1 activity was enhanced in PLD2-silenced cells in response to decreasing glucose concentrations, whereas endogenous PLD2 activity was unaffected in PLD1-silenced cells. The expression levels of PLD, AMPK, pACC (Ser-79) and pAMPK (Thr-172) were analyzed by western blotting. pACC (Ser-79) and pAMPK (Thr-172) were used as positive controls to show the activation of AMPK pathway induced by glucose deprivation. Figure 1A shows that PLD2 had a higher basal activity (1.6-fold) than PLD1 in the presence of glucose (25 mM) when PLD isoforms were overexpressed in HEK-293 cells. As shown in Figure 1B, endogenous PLD2 activity in PLD1 siRNA- transfected cells was also higher than endogenous PLD1 activity in PLD2 siRNA-transfected cells in the presence of both 25 mM and 12.5 mM glucose. Substantially, the basal activities of both PLD1 and PLD2 are quite different since there are distinct regulatory mechanisms of PLD isoforms. Additionally, it has previously been shown that the conditions used in Figure 1A and B do not lead to saturation of PLD2 isoform. Therefore, these results indicate that the activity of PLD1, but not that of PLD2, is inversely dependent upon the glucose concentration. Further- more, to determine if the stimulation of PLD is regulated by PLD1 Regulation by AMPK AMPK activity, we infected HEK-293 cells with AMPK adenoviruses using AMPK-wild type (WT) and AMPK-dominant negative (DN). In control adenovirus-infected cells, PLD activity significantly increased when glucose was deprived. In AMPK- WT adenovirus-infected cells, PLD activity increased approxi- mately 1.8-fold in the presence of glucose when compared to control adenovirus-infected cells. Under glucose deprivation, PLD activity increased about 1.7-fold in AMPK-WT, whereas no change in the PLD activity in AMPK-DN was observed (Figure 1C). These results suggest that PLD activity is regulated by AMPK activation. AMPK Directly Phosphorylates PLD1 To delineate the regulatory mechanism of PLD1 by AMPK, we performed co-immunoprecipitation experiments using HEK- 293 cells, which express PLD1 as a major isoform. When 2- deoxyglucose (2-DOG) was used as a glucose mimic to induce ATP depletion, endogenous PLD was physically associated with AMPK in a 2-DOG-dependent manner (Figure 2A). To determine if AMPK directly phosphorylates PLD1, we incubated PLD1 and AMPK with or without AMP. As shown in Figure 2B, AMPK phosphorylated the purified PLD1 in vitro. Specifically, AMPK enhanced the phosphorylation of PLD1 in the presence of AMP. However, AMPK was unable to phosphorylate the purified PLD2 directly in the presence of AMP (data not shown). To verify the potential phosphorylation sites, we performed in vitro phosphorylation assays using GST-truncated PLD1 fusion mutants. Figure 2C demonstrates that the F3 fragment corre- sponding to amino acid region 499–604 was specifically phosphorylated by AMPK in the presence of AMP. Based on this finding, we surveyed the candidate phosphorylation sites of the PLD1-loop corresponding to the 499–604 amino acid region by comparing it with an AMPK consensus motif. Interestingly, the PLD1-loop contained two candidate phosphorylation sites including the Ser-505 and the Ser-558 residues (Figure 2D). However, no possible phosphorylation sites were observed in the corresponding region of the PLD2 sequence because the central loop is a unique region of PLD1. To define the phosphorylation sites of PLD1, we generated several constructs including PLD1- S505A, PLD1-S505Q and PLD1-S558A. As shown in quantified results of Figure 2E, both PLD1-WT and PLD1-S558A were phosphorylated by AMPK in the presence of AMP whereas both PLD1-S505A and PLD1-505Q were not phosphorylated under the same conditions. We also utilized AMPK adenovirus to determine if AMPK phosphorylates PLD1 on the Ser-505 residue in vivo. After treatment with 2-DOG, PLD1 was phosphorylated by AMPK-WT, whereas PLD1-S505Q remained unphosphory- lated (Lane 3 and 4 in Figure 2F). These findings demonstrate that AMPK phosphorylates PLD1 on the Ser-505 residue in vivo. Based on these results, we suggest that PLD1 may be a novel substrate of AMPK. AMPK Regulates PLD1 Activity through Phosphorylation To determine if PLD1 activity is substantially regulated by phosphorylation, we measured the PLD1 activity using several PLD1-mutants including PLD1-WT, S505Q, S505E and S558A in the presence or absence of 25 mM glucose. As shown in Figure 3A, PLD1-WT activity was stimulated by the removal of glucose, whereas PLD1-S505Q activity was not. PLD1-S505Q still showed a basal level of activity similar to that of PLD1-WT in the presence of glucose. However, PLD1-S505A had no basal activity, regardless of the glucose concentrations (data not shown). These results indicate that the substitution of Ser to Ala on the Ser-505 residue is related to the basal activation of PLD1. In addition, PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9600