PLD1 Regulation by AMPK Figure 4. The effects of PLD1 on ERK activation by various stimuli. A. After HEK-293 cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h, cells were treated with 1-BtOH or t-BtOH for 15 min and then lysed with lysis buffer. Cell lysates were then analyzed by western blotting with the indicated antibodies. pACC (Ser-79) was used as a positive control to show the activation of AMPK pathway induced by glucose deprivation. Actin was used as a loading control. These data represent the mean of three independent analyses. B. HEK-293 cells were transfected with the indicated siRNAs of control (Luciferase) or PLD1. At 48 h after transfection, cells were incubated in glucose free-DMEM with or without 25 mM glucose and the indicated concentrations of exogenous PA for 2 h. Cells were harvested and then lysed with lysis buffer. pACC (Ser- 79) was used as a positive control and the protein amounts were normalized against actin. Cell lysates were subjected to SDS-PAGE and analyzed by western blotting with the indicated antibodies. These data represent one of three experiments in independent analyses. C. HEK-293 cells were transfected with the indicated siRNAs of control (Luciferase) or PLD1. At 48 h after transfection, cells were treated with the indicated concentrations of 2-DOG for 10 min. Then, cells were lysed with lysis buffer and lysates were subjected to SDS-PAGE and then immunoblotted with the indicated antibodies. pACC (Ser-79) was used as a positive control to show the activation of the AMPK pathway induced by treatment with 2-DOG. Actin was used as a loading control for western blotting. The data shown represent one of three independent experiments. D. HEK-293 cells were transfected with the indicated siRNAs for control (Luciferase) or PLD1. At 48 h after transfection, cells were treated with the indicated concentrations of mannitol as a negative control to induce hyperosmolar stress for 10 min. Cells were prepared according to the methods described in panel A and cell lysates were then immunoblotted with the indicated antibodies. These data shown are from one of three independent experiments. E. HEK-293 cells were transfected with the indicated siRNAs of control (Luciferase) or PLD1. At 48 h after transfection, cells were treated with the indicated concentrations of H2O2 for 10 min as a positive control to induce oxidative stress. Cells were prepared according to the methods described in panel A and cells lysates were then immunoblotted with the indicated antibodies. pACC (Ser-79) was used as a positive control to show the activation of the AMPK pathway induced by H2O2 treatment. Actin was used as a loading control for western blotting. These data shown are from one of three independent experiments. F. HEK-293 cells were transfected with the indicated plasmids for PLD1-WT, PLD1-S505Q or PLD1-K860R. At 48 h after transfection, cells were treated with 25 mM of 2-DOG or 0.2 mM of H2O2 for 10 min. Cell lysates were prepared as described in A and then immunoblotted with the indicated antibodies. pERK activation was normalized against the expression of ERK. The relative intensity of pERK was quantified as described in the materials and methods. The data shown represent one of three independent experiments. Bar graphs reflect the average of three independent experiments. *, p,0.05 versus PLD1-WT-transfected cells. doi:10.1371/journal.pone.0009600.g004 glucose uptake under glucose deprivation conditions in muscle cells, C2C12 and L6 GLUT4-myc myotube cells. Understanding the molecular mechanism involved in glucose uptake may provide insight into the regulation of glucose homeostasis and shed light on diseases related to type 2 diabetes. Emoto and Huang previously reported that the expression of PLD1 facilitates insulin-stimulated glucose uptake in 3T3L1 adipocytes [26], [27]. However, their studies were limited in that they did not identify the upstream regulator of PLD1 and its roles were only studied in adipocytes. In general, muscle is known to respond to acute and transient glucose deprivation, while adipose tissue responds to chronic reactions. In the present study, we demonstrated that PLD1 phosphorylation is responsible for acute signaling mechanisms in muscle cells and that AMPK is a novel regulator of PLD1 that functions in a glucose dependent manner. Despite extensive research on upstream stimuli that activate AMPK, few studies have been conducted to investigate the downstream substrate(s) of AMPK-mediated glucose uptake. Acetyl-coA carboxylase (ACC) is a well-known AMPK substrate that is involved in lipid metabolism. Recent reports have revealed that AS 160, which is a known substrate for AKT, is also a downstream molecule of AMPK in contraction-stimulated glucose uptake [28-30]. However, the recent identification of multiple functions of AMPK in energy sensing pathways indicates that it plays a more comprehensive role in such pathways. In this study, energy-deficient conditions mimicked by glucose deprivation induced AMPK activation, which subsequently stimulated PLD1 lipase activity. This AMPK-mediated PLD1 activity was involved in glucose uptake under energy-depleted conditions (Figure 5). Taken together, these findings indicate that AMPK activity driven by contraction can regulate AS 160 and that AMPK stimulation induced by glucose deprivation may control PLD1. However, further investigations are needed to determine how two different effectors, AS 160 and PLD1, are distinguished by AMPK in response to specific stimuli. AMPK plays a pivotal role in the maintenance of glucose homeostasis, showing strong responses to glucose deprivation in muscle cells. AMPK is known to have a well-conserved consensus motif for phosphorylation. It has been reported that PLD activity is regulated through phosphorylation or protein-protein interac- tion [2], [9]. In this study, we demonstrated for the first time that the phosphorylation of PLD1 by AMPK is important for its activity (Figure 3). The PLD1-loop region corresponding to amino acids 499–604 is directly phosphorylated by AMPK (Figure 2C) and the phosphorylation sites for AMPK are only observed in the PLD1 sequence because the loop region is a unique characteristic of PLD1. We investigated the phosphorylation sites of the PLD1- loop region using the AMPK consensus motif, [IMLVP or hydrophobic]-X-[HRK]-X-X-[ST]-X-X-X-[IMLVP or hydro- phobic], through a site scan and a BLAST search [31–34]. Throughout the immunoprecipitation assays and in vitro and in vivo phosphorylation assays, we found that the PLD1-loop region had two phosphorylation sites and one of them (the Ser-505 residue) is actually phosphorylated by AMPK (Figures 2E and F). Although many investigators have suggested that the PLD isoforms have different regulatory mechanisms due to their different structures, the specific role of the PLD1-loop region has not yet been defined. Here, we identified the phosphorylation site within the PLD1-loop region that regulates PLD1 activity and is involved in AMPK- mediated glucose uptake. However, additional studies may be necessary to characterize the distinct roles of the PLD1-loop region in various cell types. Insulin is known to stimulate glucose uptake in the skeletal muscle cells through PI-3K-dependent AKT activation. However, glucose uptake following extracellular stresses such as glucose deprivation and AICAR has been reported to occur independently of AKT [3], [29], [35]. Despite these reports, the detailed signaling pathway through which glucose uptake occurs in response to extracellular stress has not yet been clarified. Farese et al. showed that the activation of AMPK by AICAR induces stimulation of ERK, which belongs to the MAPK family, through a specific pathway [21], [23]. In the present study, western blotting and gene silencing with PLD1-siRNA revealed that AMPK-mediated PLD1 activity results in the generation of PA and that PLD1 stimulates ERK activity in response to glucose deprivation (Figure 4). PA has been reported to stimulate the MAPK pathway through the recruitment of raf-1 to the membrane. In addition, AMPK stimulated by AICAR, oxidative stress (H2O2) or glucose deprivation has been found to be associated with activation of the MAPK pathway [36–38]. These data appear to explain the difference between the two distinct coupled signaling pathways, insulin-AKT activation and glucose deprivation-ERK stimulation. PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9600