PLD1 Regulation by AMPK Figure 2. AMPK phosphorylates PLD1 but not PLD2. A. After treatment with 25 mM 2-DOG for 10 min, HEK-293 cells were lysed with buffer containing 1% Triton X-100 and 1% cholic acid. After determination of the total protein concentrations, equal amounts of extracts were immunoprecipitated with anti-PLD antibody. After brief centrifugation and washing, the precipitates were subjected to SDS-PAGE and immunoblotted with anti-PLD and anti-AMPK antibodies. The total lysates show approximately 1% of the proteins used for immunoprecipitation. B. AMPK was incubated with purified PLD1 (100 ng) in kinase buffer A (50 mM Tris/HCl, pH 7.4, 1 mM dithiothreitol, 0.02% Brij-35 and 10 mCi of [c-32P]ATP (3,000 Ci/mmol)) in the presence or absence of AMP (200 nM). This reaction was terminated by the addition of SDS sample buffer. The precipitates were then subjected to SDS-PAGE and exposed to photographic film for autoradiography. Ponceau S staining shows the protein amounts in each sample. C. GST-truncated PLD1 fusion mutants were expressed in bacteria and then purified using glutathione sepharose beads. The truncated PLD1 mutants were followed by F1 (amino acid [a.a.] 1 to 331), F2 (a.a. 332 to 498), F3 (a.a. 499 to 604), F4 (a.a. 605 to 712), F5 (a.a. 713 to 825), F6 (a.a. 826 to 927) and F7 (a.a. 928 to 1036). AMPK was incubated with an equal amount of each GST-truncated PLD1 fusion mutants (1 mg) in kinase buffer A in the presence of AMP (200 nM). This reaction was terminated by the addition of SDS sample buffer and exposed to photographic film for autoradiography. D. Schematic diagram of the PLD1 sequences. Human PLD1 sequences corresponding to the loop region were aligned with those of rat and mouse. Candidate phosphorylation sites of PLD1 for AMPK are shown in the boxes. E. HEK-293 cells were transfected with the indicated plasmids for vector, PLD1-WT, S558A, S505Q or S505A. At 48 h after transfection, cells were lysed with buffer and the lysates were immunoprecipitated with anti-PLD antibody. After brief centrifugation and washing, AMPK was incubated with the PLD1-immunocomplexes in kinase buffer A. This reaction was terminated by the addition of SDS sample buffer and the precipitates were then subjected to SDS-PAGE and exposed to photographic film for autoradiography. The relative intensity of the autoradiogram 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 vector-transfected cells. F. HEK-293 cells were transfected with the indicated plasmids of PLD1-WT or PLD1-S505Q. At 24 h post- transfection, cells were infected with control (empty vector), AMPK-WT or AMPK-DN adenovirus. At 24 h after infection, cells were loaded with [32P] orthophosphate (3 mCi/ml) for 5 h and then treated with 25 mM of 2-DOG for 10 min. Next, the extracts were prepared with lysis buffer and immunoprecipitated with anti-PLD antibody. After brief centrifugation and washing, the precipitates were then subjected to SDS-PAGE, transferred to nitrocellulose membranes and exposed to photographic film for autoradiography. The expression levels of AMPK and PLD1 were analyzed by western blotting and ponceau S staining. Data represent one of three independent experiments. doi:10.1371/journal.pone.0009600.g002 AMPK-Induced PLD1 Activation Is Required for ERK Stimulation Based on the finding that AMPK stimulates PLD1 through direct phosphorylation (Figures 1 and 2), we investigated the downstream target molecules of AMPK-induced PLD1 activation to determine the reasons for PA formation. Previous studies have found that the activation of PLD may implicate the mitogen activated protein kinase (MAPK) pathway through raf-1 activation via recruitment of PLD-generated PA binding [24], [25]. We attempted to determine if stimulation of ERK is regulated by PLD activity under glucose deprivation conditions. To accomplish this, we utilized 1-butanol (1-BtOH) as an inhibitor of PLD and t- butanol (t-BtOH) as a negative control in the presence or absence of glucose. 1-butanol was used to prevent generation of the PLD products, PA. ERK activation, which was determined based on an increase in the phosphorylated ERK levels, was dramatically increased by glucose deprivation in both NT (not treated)-cells and t-BtOH-treated cells (Figure 4A). However, ERK activation was very slightly increased in 1-BtOH-treated cells under the same conditions. In the absence of glucose, ERK activation was attenuated in 1-BtOH-treated cells when compared to both NT- cells and t-BtOH-treated cells. pACC (Ser-79) was used as a positive control to show the activation of the AMPK pathway induced by glucose deprivation. These results indicate that PLD activation is required for ERK activation. To elucidate the effects of PLD1-generated PA on ERK activation, we silenced PLD1 with siRNA and then treated the cells with various concentrations of exogenous PA. As shown in Figure 4B, ERK activation was attenuated in PLD1 siRNA-transfected cells when compared to control siRNA-transfected cells. In addition, exogenous PA increased ERK activation in a dose dependent manner. These results imply that PA supplied by PLD activation is responsible for ERK activation. Next, we examined the specificity of ERK activation by the presence of various extracellular stressors including 2-DOG, mannitol and H2O2. ERK activation was reduced in PLD1 siRNA-transfected cells in response to 2-DOG when compared to control siRNA-transfected cells (Figure 4C). To exclude the influence of nonspecific 2-DOG by hyperosmolar stress, we utilized mannitol as a negative control to induce hyperosmolar stress. As shown in Figure 4D, ERK activation was slightly increased in PLD1 siRNA-transfected cells in response to treatment with mannitol when compared to control siRNA- transfected cells. These results imply that PLD1 activation induced by energy depletion but not hyperosmolar stress is required for ERK activation. H2O2 used as a positive control to induce oxidative stress also resulted in a reduction of ERK activation in PLD1 siRNA-transfected cells when compared to control siRNA- transfected cells (Figures 4E). To confirm the effects of PLD lipase activity on ERK activation, we included another construct, PLD1- K860R, which is a lipase-inactive mutant. As shown in Figure 4F, PLD1-WT showed approximately 3.3-fold greater levels of ERK activation in response to 2-DOG and about 4.2-fold greater ERK activation following H2O2 treatment. The ERK activation observed in PLD1-WT was similar to the activation observed in PLD1-S505Q under basal conditions (Lanes 1 and 4). PLD1- S505Q also showed approximately 1.1-fold greater levels of ERK activation in response to treatment with 2-DOG and about 2-fold greater ERK activation following H2O2 treatment. PLD1-K860R showed decreased ERK activation under basal conditions when compared to PLD1-WT (Lanes 1 and 7). PLD1-K860R showed very slightly higher ERK activation, despite treatment with 2- DOG or H2O2. Based on these observations, we suggest that ERK activation is dependent on PLD1 activity in response to 2-DOG or H2O2. Therefore, these results imply that AMPK-mediated PLD1 activation contributes to the stimulation of ERK under energy depletion conditions caused by glucose deprivation and oxidative stress. AMPK-Induced PLD1 Activation Is Important for 14C-Glucose Uptake in Differentiated C2C12 and L6 GLUT4-myc Myotube Cells Previous studies have reported that AMPK activity is involved in glucose homeostasis in a variety of cells [1], [10], and that PLD activity is required for glucose uptake in muscles and adipocytes [21], [23], [26]. Based on these previous findings, we investigated the mechanisms by which AMPK and PLD regulate the glucose uptake using 2-deoxy[14C-glucose] as an indicator for the glucose uptake assay. As shown in Figure 5A, PLD1-WT showed an approximately 2.8-fold increase in 14C-glucose uptake in the presence of glucose when compared to vector-transfected cells (Columns 1 and 2). PLD1-S505Q showed about 2.7-fold greater in 14C-glucose uptake under the same conditions (Columns 1 and 3). PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9600