PLD1 Regulation by AMPK Figure 1. The effects of glucose deprivation on PLD activity. A. HEK-293 cells were transfected with the indicated plasmids for vector, PLD1 or PLD2. At 40 h after transfection, cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h. Cells were then labeled with [3H]myristric acid for 4 h and the formation of [3H]PBtOH was quantified as described in the materials and methods. Quantitative data represent the means of three independent experiments. *, p,0.05 versus vector-transfected cells with glucose; #, p,0.05 versus vector-transfected cells without glucose. B. HEK-293 cells were transfected with siRNAs for PLD1, PLD2 or control (Luciferase). At 40 h after transfection, cells were incubated in glucose free-DMEM with the indicated concentrations of glucose (25 mM [4.5 g/L], 12.5 mM [2.25 g/L], 6.25 mM [1.125 g/L]) for 2 h. Cells were then labeled with [3H]myristric acid for 4 h and the formation of [3H]PBtOH was quantified. The expression levels of PLD and AMPK were analyzed by western blotting using antibodies directed against PLDs or AMPK. pACC (Ser-79) and pAMPK (Thr-172) were used as positive controls to show the activation of AMPK pathway induced by glucose deprivation. Actin was used as a loading control. Quantitative data represent the means of three independent experiments. *, p,0.05 versus control siRNA-transfected cells in the presence of glucose; #, p,0.05 versus control siRNA-transfected cells in the absence of glucose. C. HEK-293 cells were infected with control (empty vector), AMPK-WT or AMPK-DN adenovirus. At 40 h after infection, cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h. Cells were labeled with [3H]myristric acid for 4 h and the formation of [3H]PBtOH was then quantified. Data represent the means of three independent experiments. *, p,0.05 versus control adenovirus- infected cells with glucose; #, p,0.05 versus control adenovirus-infected cells without glucose. doi:10.1371/journal.pone.0009600.g001 PLD1-S505E displayed much higher activity than PLD1-S505Q, regardless of the glucose concentrations. PLD1-S558A activity increased by approximately 2.2-fold under glucose deprivation conditions. PLD1-WT activity enhanced about 2.1-fold under the same conditions. Based on these findings, the Ser-558 residue of PLD1 does not appear to be a regulatory site that is phosphorylated by AMPK. To confirm that PLD1 activity is regulated by ATP depletion, we measured the PLD1 activity after treatment with 2-DOG. As shown in Figure 3B, the PLD1-WT activity was stimulated by ATP depletion induced by 2-DOG, but PLD1-S505Q was unaffected under the same conditions. Taken together, these results suggest that AMPK stimulates PLD1 activity through phosphorylation of the Ser-505 residue under glucose deprivation. PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9600