Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway

Abstract

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex, mTORC1, regulates cell growth in response to mitogenic signals and amino acid availability. Phospholipase D (PLD) and its product, phosphatidic acid, have been established as mediators of mitogenic activation of mTORC1. In this study, we identify a novel role for PLD1 in an amino acid-sensing pathway. We find that amino acids activate PLD1 and that PLD1 is indispensable for amino acid activation of mTORC1. Activation of PLD1 by amino acids requires the class III phosphatidylinositol 3-kinase hVps34, which stimulates PLD1 activity through a functional interaction between phosphatidylinositol 3-phosphate and the Phox homology (PX) domain of PLD1. Furthermore, amino acids stimulate PLD1 translocation to the lysosomal region where mTORC1 activation occurs in an hVps34-dependent manner, and this translocation is necessary for mTORC1 activation. The PX domain is required for PLD1 translocation, mTORC1 activation, and cell size regulation. Finally, we show that the hVps34-PLD1 pathway acts independently of, and in parallel to, the Rag pathway in regulating amino acid activation of mTORC1.

Figure 1.

PLD1 is activated by amino acids in the mTORC1 pathway. (A) HEK293 cells were serum starved overnight followed by the following treatments: deprivation of amino acids (AA) for 2 h, restimulation with amino acids for 30 min, or stimulation with 100 nM insulin for 30 min. In vivo PLD assays were performed to measure total cellular PLD activity. (B) Various types of cells as indicated were serum starved overnight followed by amino acid withdrawal for 2 h and then stimulated with amino acids for 30 min. Total cellular PLD activity was measured. (C) Recombinant PLD1 or PLD2 was transiently expressed, and the cells were treated as in B. Recombinant PLD activities were measured as described in Materials and methods. (A–C) All data are mean ± SD or representative blots from three to five independent experiments. A one-sample t test was performed to compare each sample with the control. *, P < 0.05; **, P < 0.01. (D) Cells were transduced with lentiviruses expressing two independent shRNAs against PLD1 and a scrambled (scram) sequence as a negative control, selected with puromycin, serum starved overnight, and amino acid deprived for 2 h followed by amino acid stimulation for 30 min. Cell lysates were analyzed by Western blotting. (E) Cells were treated as in D, except that Leu deprivation and stimulation were performed in lieu of amino acid deprivation/stimulation. (F) Four types of cells were treated as in D and analyzed by Western blotting. (D–F) All experiments were performed at least three times, and representative blots are shown. Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein.

Figure 2.

hVps34 is necessary for amino acid activation of PLD1 upstream of mTORC1. HEK293 cells were treated as described below, and in vivo PLD assays and Western analysis of cell lysates were performed in parallel. (A) Serum-starved cells were subjected to amino acid withdrawal for 2 h and were then stimulated with amino acids for 30 min. 10 mM 3-MA and 100 nM rapamycin were added 60 and 30 min before stimulation, respectively, where indicated. (B and C) Cells were transduced with lentiviruses expressing two independent shRNAs against hVps34 and a scrambled (scram) sequence as a negative control, selected with puromycin, serum starved overnight, and amino acid deprived for 2 h followed by 30 min of amino acid (AA) stimulation (B) or insulin (100 nM) stimulation (C). (D) Cells transduced with lentiviruses expressing PLD1-shRNA or scramble control and selected with puromycin were transiently transfected with an Myc-hVps34/V5-hVps15 bicistronic construct or empty vector. The cells were then serum starved overnight and amino acid deprived for 2 h followed by amino acid stimulation for 30 min. Cell lysates were subjected to Western analysis. (E) Cells were transduced with lentiviruses expressing hVps34-shRNA or scramble control, selected with puromycin, serum starved overnight, and then stimulated with 300 µM PA for 30 min. Cell lysates were subjected to Western analysis. (F) Cells were treated as in E but amino acid deprived for 2 h followed by amino acid stimulation, PA (300 µM) stimulation, or both for 30 min. Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein. (A–C) All data are mean ± SD or representative blots from three to five independent experiments. A one-sample or paired t test was performed to compare the indicated pairs of data. *, P < 0.05; **, P < 0.01.

Figure 3.

hVps34 activates PLD1 in cells. (A) HEK293 cells were cotransfected with HA-PLD1 and increasing amounts of bicistronic Myc-hVps34/V5-hVps15 cDNA followed by in vivo PLD assays and Western analysis of cell lysates in parallel samples. (B) Cells were cotransfected with HA-PLD1 and Myc-hVps34/V5-hVps15, serum starved overnight, amino acid (AA) deprived for 2 h, and then stimulated with amino acids for 30 min. In vivo PLD assays were performed. (C) Cells were transduced with lentiviruses expressing hVps34-shRNA, selected with puromycin, and transfected with wt or kinase-dead Myc-hVps34 followed by serum starvation overnight, amino acid deprivation for 2 h, and then stimulation with amino acids for 30 min. In vivo PLD assays and Western analysis of cell lysates were performed in parallel. Scram, scrambled. (A–C) All data are mean ± SD or representative blots from three to five independent experiments. A one-sample or paired t test was performed to compare the indicated pairs of data. *, P < 0.05; **, P < 0.01. Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein.

Figure 4.

PI3P activates PLD1 in cells. (A) HEK293 cells were cotransfected with HA-S6K1 and GFP-2×FYVE (the latter at two different amounts), serum starved overnight, amino acid (AA) deprived for 2 h, and then stimulated with amino acids for 30 min. Western analysis was performed. (B) Cells were transfected with GFP-FYVE at two different amounts with or without Myc-hVps34/V5-hVps15 cDNA and then treated as in A. In vivo PLD assays were performed. (C) Cells were transfected with HA-PLD1 followed by serum starvation overnight or amino acid withdrawal for 2 h. The cells were then stimulated with 15 µM PI3P or PI or carrier alone for 30 min. In vivo PLD assays were performed. (D) Cells were transduced with lentiviruses expressing hVps34-shRNA or scramble (scram), selected with puromycin, and then transfected with HA-PLD1 followed by serum starvation overnight and amino acid withdrawal for 2 h. The cells were then stimulated with 15 µM PI3P or carrier alone in the presence of amino acids for 30 min. In vivo PLD assays were performed. (B–D) All data are mean ± SD or representative blots from three to five independent experiments. A one-sample or paired t test was performed to compare the indicated pairs of data. *, P < 0.05; **, P < 0.01. (A, C, and D) Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein.

Figure 5.

The PX domain is required for PLD1 activation by PI3P and hVps34. (A) A schematic representation of the domain structures of PLD1 and its mutants used in this study. (B) HEK293 cells were transfected with wt and mutant PLD1 followed by serum starvation overnight and stimulation with 15 µM PI3P for 30 min. In vivo PLD assays were performed. Western blots show recombinant PLD1 protein expression. (C) Cells were cotransfected with Myc-hVps34 and various PLD1 mutants followed by serum starvation overnight and in vivo PLD assays. Western blots show expression of recombinant hVps34 and various PLD1 proteins. (A–C) All data are mean ± SD from three to five independent experiments. A one-sample or paired t test was performed to compare the indicated pairs of data. *, P < 0.05; **, P < 0.01. (D) Cells were transfected with Myc-hVps34 (top) or Flag-PLD1 (bottom). Immunoprecipitation (IP) was performed with anti-Myc or -Flag antibody followed by immunoblotting for recombinant hVps34 and endogenous PLD1 or recombinant PLD1 and endogenous hVps34, respectively. Predicted molecular masses of proteins are indicated for Western blots.

Figure 6.

Amino acids regulate PLD1 translocation to the lysosomal region in an hVps34-dependent fashion. (A) HEK293 cells were transfected with HA-PLD1 followed by serum starvation overnight and amino acid (AA) deprivation for 2 h. Some cells were transduced with lentiviruses expressing hVps34-shRNA (hVps34 knockdown [KD]) and selected with puromycin before transfection of HA-PLD1, whereas others were treated with 10 mM 3-MA for 1 h before amino acid stimulation. Upon amino acid stimulation for 30 min, cells were fixed and immunostained with anti-HA and -LAMP2 antibodies. (B) Cells were treated as in A without transfection and immunostained with anti-mTOR and -LAMP2 antibodies. (C) Cells were transfected with HA-PLD1, treated as in A, and then immunostained with anti-HA and -mTOR antibodies. (A–C) The merged images were pseudocolored as follows: LAMP2 in red and PLD1 and mTOR in green (A and B) and PLD1 in red and mTOR in green (C). Enlarged images of the merges are shown in the rightmost columns. Bars, 5 µm; (enlarged images) 0.5 µm. (D) Percentage of LAMP2-colocalized cells among HA- (for PLD1) or mTOR-positive cells was quantified for experiments shown in A and B. Cells with the majority of HA-PLD1 or mTOR signals overlapping with LAMP2 signals were scored as colocalizing. Mean results of three independent experiments are shown with error bars representing SD.

Figure 7.

PX domain is necessary for amino acid–induced PLD1 lysosomal translocation and mTORC1 signaling. (A) HEK293 cells were transfected with various mutants of HA-PLD1 followed by serum starvation overnight and amino acid (AA) deprivation for 2 h. Upon amino acid stimulation for 30 min, cells were fixed and immunostained with anti-HA and -LAMP2 antibodies. Images were processed as described in Fig. 5. Bars, 5 µm; (enlarged images) 0.5 µm. (B) Percentage of LAMP2-colocalized cells among HA-PLD–positive cells was quantified for experiments shown in A, as described in Fig. 6 D. (C) Cells were transfected with wt-, ΔPX-, or Δloop-PLD1, serum and amino acid starved, and then stimulated with amino acids for 30 min followed by in vivo PLD assays. (D) Cells were cotransfected with Myc-S6K1 and wt-, ΔPX-, or Δloop-PLD1, treated as in C, and subjected to Western analysis. Quantification of Western band intensities was performed by densitometry of x-ray film images using ImageJ software (National Institutes of Health). The levels of pS6K1 relative to total S6K1 are shown. Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein. (B–D) Three independent experiments are shown with error bars representing SD. *, P < 0.05; **, P < 0.01.

Figure 8.

hVps34 and PLD1 regulate cell size. (A) HEK293 cells were transduced with lentiviruses expressing shRNAs for raptor, hVps34, or PLD1, puromycin selected, and then subjected to cell size measurement of median forward scatter-height. The result of overnight treatment with 100 nM rapamycin is included as a control. Representative histograms are also shown, with cell counts in arbitrary units. (B) Cells were transfected with wt- or ΔPX-PLD1 together in pCDNA3 (vector), selected with G418 for 3 d, and then subjected to cell size measurement as described in A. A one-sample t test was performed to compare each data with the control. Three independent experiments were performed, and the results of mean ± SD are shown in the graphs. *, P < 0.05; **, P < 0.01.

Figure 9.

PLD1 and Rag pathways act in parallel to mediate amino acid activation of mTORC1. (A) HEK293 cells were transduced with lentiviruses expressing shRNAs, selected with puromycin, serum starved, and amino acid (AA) deprived followed by amino acid stimulation for 30 min. Cell lysates were analyzed by Western blotting. scram, scrambled. (B) Cells were treated as in A, and in vivo PLD assays were performed. *, P < 0.05; **, P < 0.01. (C, left) Cells were transduced by lentiviruses expressing shRNAs, selected with puromycin, and then transfected with HA-PLD1 followed by serum starvation and amino acid deprivation. Upon amino acid stimulation for 30 min, cells were fixed and immunostained with anti-HA or -mTOR together with anti-LAMP2 antibodies. The merged images were pseudocolored as follows: LAMP2 in red and PLD1 and mTOR in green. Bars, 5 µm; (enlarged images) 0.5 µm. (right) Percentage of LAMP2-colocalized cells among HA- or mTOR-positive cells was quantified for three independent experiments as described in Fig. 6 D, and the results of mean ± SD are shown in the graph. (D) Cells were transduced with lentiviruses expressing shRNAs, selected with puromycin, and then transfected with Rag B/C. Upon serum starvation and amino acid deprivation followed by amino acid stimulation for 30 min, cells were lysed for Western analysis. Predicted molecular masses of the proteins are indicated for Western blots. S6K1 migrated on SDS-PAGE as a 70-kD protein.

Figure 10.

A proposed model for amino acid–sensing mTORC1 signaling. Upon amino acid stimulation, two parallel pathways converge on the lysosome to activate mTORC1.