Phospholipase D1 Is an Effector of Rheb in the Mtor Pathway

Phospholipase D1 is an effector of Rheb in the mTOR pathway

Y. Sun*†, Y. Fang*†, M.-S. Yoon*, C. Zhang*, M. Roccio‡, F. J. Zwartkruis‡, M. Armstrong§, H. A. Brown§, and J. Chen*¶

*Department of Cell and Developmental Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801; ‡Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center, Utrecht, 3508 GA, Utrecht, The Netherlands; and §Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232

Edited by William T. Greenough, University of Illinois at Urbana–Champaign, Urbana, IL, and approved May 1, 2008 (received for review December 28, 2007) The mammalian target of rapamycin (mTOR) assembles a signaling The tumor suppressors tuberous sclerosis complex 1 and 2 network essential for the regulation of cell growth, which has (TSC1/2) are negative regulators of mTOR signaling in the emerged as a major target of anticancer therapies. The tuberous control of cell size and cell cycle progression (11, 12). The sclerosis complex 1 and 2 (TSC1/2) proteins and their target, the small GTPase-activating protein (GAP) activity of TSC2 targets GTPase Rheb, constitute a key regulatory pathway upstream of the small G protein Rheb (Ras homologue enriched in brain), mTOR. Phospholipase D (PLD) and its product phosphatidic acid are which activates mTOR signaling and regulates cell size (11, 12). also upstream regulators of the mitogenic mTOR signaling. However, The TSC/Rheb pathway appears to be a signal integrator at how the TSC/Rheb and PLD pathways interact or integrate in the which multiple inputs including cellular energy levels, growth rapamycin-sensitive signaling network has not been examined be- factors, and hypoxia converge (3). Despite the initial speculation fore. Here, we find that PLD1, but not PLD2, is required for Rheb of the involvement of nutrient signals, TSC1/2 was not found to activation of the mTOR pathway, as demonstrated by the effects of be absolutely essential in mediating amino acid sufficiency RNAi. The overexpression of Rheb activates PLD1 in cells in the signals (13–15). The mTOR-associating protein Raptor may absence of mitogenic stimulation, and the knockdown of Rheb mediate nutrient-sensing signals (16), and hVps34, a class III impairs serum stimulation of PLD activation. Furthermore, the over- PI3K, has also emerged as a component of the amino acid- expression of TSC2 suppresses PLD1 activation, whereas the knock- regulated mTOR pathway parallel to TSC/Rheb (13, 17). The down or deletion of TSC2 leads to elevated basal activity of PLD. current model of Rheb activation of mTOR involves direct Consistent with a TSC-Rheb-PLD signaling cascade, AMPK and PI3K, interaction between these two proteins independent of Rheb both established regulators of TSC2, appear to lie upstream of PLD as GTP loading and activation of mTOR kinase by Rheb in a GTP-dependent manner (14, 18). In this study, we have identi- revealed by the effects of pharmacological inhibitors, and serum fied another mechanism of Rheb action in the amino acid- activation of PLD is also dependent on amino acid sufficiency. Finally, sensing mTOR signaling pathway. We find that PLD1 is required Rheb binds and activates PLD1 in vitro in a GTP-dependent manner, for Rheb activation of mTORC1 and that the TSC/Rheb pathway strongly suggesting that PLD1 is a bona fide effector for Rheb. Hence, and amino acid sufficiency are upstream regulators of PLD in our findings reveal an unexpected interaction between two cascades cells. Furthermore, we present evidence that Rheb binds and in the mTOR signaling pathways and open up additional possibilities activates PLD1 in vitro in a GTP-dependent manner. Our for targeting this important growth-regulating network for the observations strongly suggest that PLD1 is a bona fide effector development of anticancer drugs. for Rheb in the amino acid-sensing mTOR signaling pathway.

rapamycin ͉ tuberous sclerosis complex ͉ phosphatidic acid Results PLD1 Is Required for Rheb Activation of mTOR Signaling. To examine ammalian target of rapamycin (mTOR) is a Ser/Thr kinase the relationship between TSC/Rheb and PLD pathways, which Mserving a pivotal role in the regulation of cell growth and are both upstream regulators of mTORC1 signaling, we probed proliferation by mediating nutrient availability and mitogenic the requirement of PLD in Rheb-mediated S6K1 activation by signals (1–3). The best characterized downstream effectors of the using 1-butanol, an inhibitor of PA production through PLD. We rapamycin-sensitive mTOR signaling complex (mTORC1) are reported previously (4) that 1-butanol at low concentrations the ribosomal S6 kinase 1 (S6K1) and the eukaryotic translation (e.g., 0.5%) inhibits serum-stimulated S6K1 activation by Ϸ60%. initiation factor 4E-binding protein 1 (4E-BP1), both of which As shown in Fig. 1A, Rheb-stimulated S6K1 activation, as well as are critically involved in the regulation of protein synthesis in T389-S6K1 phosphorylation, in HEK293 cells was also inhibited response to mitogenic stimuli, nutrient sufficiency, and cellular by 1-butanol, the degree of which was comparable to that of energy levels (1–3). The lipid second messenger phosphatidic inhibition of serum stimulation. As a negative control, 2-butanol acid (PA) has been found to mediate mitogenic activation of had minimal effect. This observation suggested that PLD might mTOR signaling (4, 5). As one of the enzymes responsible for the be involved in the activation of S6K1 by Rheb. However, the production of cellular PA, phospholipase D (PLD) is an up- relatively nonspecific nature of 1-butanol as an inhibitor called stream regulator in the mTOR pathway in both mitogenesis (6, for more definitive evidence for the involvement of PLD. 7) and the mechanical stimulation of skeletal muscle growth (8).

PLD generates PA and choline by hydrolyzing phosphatidyl- Author contributions: Y.S., Y.F., F.J.Z., H.A.B., and J.C. designed research; Y.S., Y.F., M.-S.Y., choline (PC) in response to a variety of stimuli (9, 10). Two major C.Z., M.R., and M.A. performed research; M.R., F.J.Z., M.A., and H.A.B. contributed new isoforms of PC-specific PLD have been identified in mammals, reagents/analytic tools; and Y.S., Y.F., and J.C. wrote the paper. namely PLD1 and PLD2. PLD1 can be directly activated by three The authors declare no conﬂict of interest. families of proteins, the conventional protein kinase C (cPKC), This article is a PNAS Direct Submission. Rho GTPases (RhoA, Cdc42, and Rac1), and ARF GTPases (9, †Y.S. and Y.F. contributed equally to this work. 10), whereas PLD2 exhibits a high basal activity. Previously, we ¶To whom correspondence should be addressed. E-mail: [email protected]. have shown that PLD1 is required for mTOR signaling and cell This article contains supporting information online at www.pnas.org/cgi/content/full/ size regulation and that Cdc42 activates S6K1 by regulating 0712268105/DCSupplemental. PLD1 (6). © 2008 by The National Academy of Sciences of the USA

8286–8291 ͉ PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712268105 Downloaded by guest on September 27, 2021 120 vector Flag-Rheb 120 A ) B A C 100 3.0 100 * 2.5 80 *

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S6 0 0 PLD1 A L79 FBS: - +++ --1-BtOH 2-BtOH Scram Scram Rheb #1Rheb #2 pT389 S6K1 shRNA: Flag-Rheb S6K1 GTPases Rheb pT389-S6K1 mTOR PLD1 pT389 S6K1 Myc-S6K1 Tubulin Tubulin

vector Flag-Rheb PLD1 activity S6K1 activity (%) C B 3.0 PLD2 mRNA 100 2.5 1 80 shRNA 2.0 60 Scram PLD2 #1 PLD2 #2 Scram PLD2 #1 PLD2 #2 0.8 1.5 40 Flag-Rheb 1.0 0.6 0.5 20 PLD1 0.0 0 0.4 wt C181S D60K wt C181S D60K pT389-S6K1 Fold change 0.2 Rheb Rheb S6K1 PLD1 pT389-Myc-S6K1 0 S6K1 mTOR shRNA: Scram PLD2 #1 PLD2 #2 Tubulin Fig. 2. Rheb activates PLD1 in cells. (A) HEK293 cells were cotransfected with HA-PLD1 and various epitope-tagged GTPases as indicated, followed by incu- Fig. 1. PLD1 is required for Rheb activation of S6K1. (A) HEK293 cells were bation in serum-free medium containing 3H-palmitic acid for 1 day, and in vivo transfected with Myc-S6K1 with or without FLAG-Rheb for 24 h and serum- PLD assays were performed as described in Materials and Methods. Expression starved overnight. Where indicated, cells were pretreated with 0.5% 1-buta- of the recombinant proteins was conﬁrmed by Western blotting using anti- nol (1-BtOH) or 2-butanol (2-BtOH) for 30 min before lysis. Myc-S6K1 was epitope tag antibodies. (B) FLAG-Rheb (WT C181S or D60K mutant) was immunoprecipitated from cell lysates and subjected to S6 kinase assays in cotransfected with HA-PLD1 or Myc-S6K1 into HEK293 cells, followed by in vitro.(B and C) HEK293 cells were infected with lentiviruses expressing two vivo PLD assays and in vitro S6 kinase assays, respectively. Expression of the independent shRNAs for PLD1 (B), PLD2 (C), or a scrambled sequence (Scram) recombinant proteins is shown by Western blots. (C) HeLa cells were infected as a negative control. The infected cells were selected with puromycin, trans- with lentiviruses expressing two independent shRNAs for Rheb or a scrambled fected with Flag-Rheb or pCDNA3 (empty vector), serum-starved, and PA- sequence (Scram). The infected and puromycin-selected cells were subjected stimulated. The cell lysates were analyzed by Western blotting. Quantitative to in vivo PLD assays as described in A with or without serum stimulation for RT-PCR was performed to quantify knockdown efﬁciency of PLD2. 30 min. In parallel experiments, the cell lysates were subjected to Western blot analyses. *, Student’s t tests were performed to compare PLD activity to that of ScramϩFBS (P Ͻ 0.01). Of the two known mammalian PLD isoforms, we previously showed that PLD1 is required for serum-stimulated S6K1 activation in HEK293 cells (6), although PLD2’s role in mTOR PLD1: Either Rheb was an upstream activator of PLD1 or PLD1 signaling is also possible (7, 19). To test whether either PLD acted in parallel to Rheb in the mTOR signaling network. To mediates Rheb activation of S6K1, we knocked down PLD1 and probe the possibility that Rheb might be an upstream regulator PLD2 separately in HEK293 cells via lentivirus-mediated of PLD1, we asked whether the overexpression of Rheb could shRNA expression. As shown in Fig. 1B, knockdown of PLD1 led activate PLD1. A recombinant HA-tagged PLD1 was transiently to a reduction of S6K1 activation (as measured by T389 phos- expressed in HEK293 cells together with various small G pro- phorylation) in response to Rheb overexpression. The different teins, including WT Rheb, WT Cdc42, and constitutively active levels of PLD1 knockdown resulting from the two independent Cdc42 (L61), Rap1 (V12), and Rab5A (L79). As we reported shRNAs correlated closely with the degrees of reduction of S6K1 previously (6), the constitutively active Cdc42-L61, but not WT activation upon Rheb overexpression, confirming the knock- Cdc42, activated PLD1 by Ϸ2-fold in these cells in the absence down specificity. Exogenous PA, the product of PLD, rescued of any stimulus (Fig. 2A). Significantly, the overexpression of T389 phosphorylation in the presence of PLD1 knockdown (Fig. WT Rheb activated PLD1 by Ϸ2.5-fold. Rap1-V12 and Rab5A- 1B, last two lanes), suggesting that the function of PLD1 in Rheb L79, previously shown to be unable to activate S6K1 when activation of S6K1 is most likely dependent on the phospholipase overexpressed in cells (21), did not have any effect on PLD1. activity. In contrast, knockdown of PLD2 by two independent Thus, the ability of the small G proteins to activate PLD1 shRNAs had no effect on Rheb activation of S6K1 (Fig. 1C). Although we could not directly assess the degree of PLD2 correlated well with their capacity to activate S6K1. protein knockdown due to the lack of an effective antibody Two Rheb mutants, C181S and D60K, were examined for their and/or low abundance of PLD2 in these cells, the mRNA levels ability to activate PLD1 when overexpressed. Mutation of the of PLD2 were reduced by 90% and 60%, making it highly likely CAAX motif (C181S) presumably eliminates farnesylation of that the protein would be dramatically reduced based on our Rheb, and this mutant has been reported to have diminished

experience with knockdown of many other genes. Taken to- capacity to activate S6K1 (22, 23). Another mutant, D60K, has CELL BIOLOGY gether, these results strongly suggest that PLD1, but not PLD2, been reported to be inactive toward S6K1 (24, 25). As shown in is required for Rheb-mediated S6K1 activation through the Fig. 2B, C181S significantly diminished, whereas D60K abol- production of PA. Of note, we have found that PLD1, and not ished Rheb’s ability to activate PLD1 in cells, and the activities PLD2, also is required for serum-stimulated mTORC1 activa- of these two mutants toward PLD1 correlated well with their tion in HEK293 cells (data not shown), as well as the activation abilities to activate S6K1. These observations are consistent with of a myogenic mTOR pathway in C2C12 myoblasts (20). the notion that Rheb activates S6K1 via PLD1. To further validate Rheb’s role in the activation of PLD in the Rheb Activates PLD1 in Cells. At least two alternative possibilities cell, we knocked down Rheb in HeLa cells and assessed its effect existed to explain the dependence of Rheb-S6K1 activation on on serum activation of endogenous PLD. As shown in Fig. 2C,

Sun et al. PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 ͉ 8287 Downloaded by guest on September 27, 2021 PLD1 activity (%) S6K1 activity (%) * A A B * * 120 100 200 * 100 80 80 * 120 60 * 150 60 40 40 100 80 20 20 (%) tivity 0 0 50 40 PMA: -+++ -+++

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D ac D TSC2 N1651S PL TSC2 N1651S 0 0 TSC2 TSC2 Wort2-DG Rap -AA -AA -AA+AA -serum -serum +20% dialysed FBS PLD1 S6K1 +20% FBS pT389-S6K1 pT389-S6K1

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TSC2 ac PLD 0 PLD1 0 TSC2: +/+ -/- Scram TSC2_CTSC2_ shRNA: Scramble TSC2_C shRNA: N Fig. 4. PLD activity is regulated by upstream signals in the mTOR pathway. (A) HEK293 cells were serum-starved overnight, followed by in vivo PLD assays Fig. 3. TSC2 lies upstream of PLD1. (A) FLAG-TSC2 or the N1651S mutant was as described in Materials and Methods. The cells were stimulated by 20% FBS cotransfected with HA-PLD1 or Myc-S6K1 into HEK293 cells, followed by in before lipid extraction. Some cells were pretreated with 100 nM wortmannin vivo PLD assays and in vitro S6 kinase assays, respectively. Expression of the (Wort), 100 mM 2-deoxyglucose (2-DG), or 100 nM rapamycin (Rap) for 30 min recombinant proteins is shown by Western blots. *, Student’s t tests were before serum stimulation. (B) Serum-starved HEK293 cells were subjected to performed to compare these data to that of PMA-stimulated cells without amino acids withdrawal for 60 min (ϪAA) where indicated, stimulated by 20% recombinant TSC2 expression (P Ͻ 0.01). (B) HEK293 cells were infected with dialyzed FBS for 30 min in the presence (ϩAA) or absence of amino acids, and lentiviruses expressing two shRNAs against TSC2, drug-selected, serum- then subjected to in vivo PLD assays. (C) HEK293 cells expressing an shRNA for starved, and then subjected to in vivo PLD assay. TSC2 knockdown efﬁciency TSC2 was treated as above and subjected to in vivo PLD assays. All data shown was assessed by Western blotting. *, Student’s t tests were performed to are the average results of three to ﬁve independent experiments, with error ϩ ϩ compare the PLD activity to that in scramble cells (P Ͻ 0.01). (C) TSC2 / and bars representing SD. *, Student’s t tests were performed to compare the data TSC2Ϫ/Ϫ EEF cells were transiently transfected with HA-PLD1, serum-starved, sets as indicated (P Յ 0.01). and subjected to in vivo PLD assays. The recombinant PLD1 activity was obtained by subtracting the activity of empty vector-transfected cells from that of HA-PLD1-transfected cells and then normalized against HA-PLD1 band serum (like insulin) is a well established mitogen for the acti- intensity on Western blots. vation of the PI3K-TSC-mTOR pathway. To better assess the role of TSC in the potential activation of PLD by Rheb, we knocked down TSC2 in HEK293 cells and knockdown of Rheb by two independent shRNAs led to dimin- examined the cellular PLD activity. As shown in Fig. 3B, TSC2 ished PLD activation, accompanied by reduced T389 phosphor- knockdown led to a significant elevation in PLD activity in ylation of S6K1. The effect of Rheb knockdown on S6K1 serum-starved cells, and the degree of increase was correlated phosphorylation in HeLa cells is consistent with observations by with the knockdown efficiency by two independent shRNAs. others (26). These data suggest that the mitogenic activation of Because of technical hurdles involved in quantifying recombi- PLD in cells depends on Rheb. nant PLD1 activity in TSC2 knockdown and PLD1 overex- Ϫ/Ϫ ϩ/ϩ TSC2 negatively regulates mTOR signaling by acting as a GAP pressed cells, we took advantage of the TSC2 or TSC2 for Rheb. The ability of Rheb to activate PLD1 prompted us to Eker rat embryonic fibroblasts (EEFs) (27), in which we tran- ask whether the overexpression of TSC2 would have a negative siently expressed HA-PLD1. Under serum-starvation conditions, the recombinant PLD1 activity was undetectable in effect on PLD1 activation. As shown in Fig. 3A, the overexpres- ϩ/ϩ Ϫ/Ϫ sion of TSC2 diminished phorbol 12-myristate 13-acetate TSC2 cells and was significantly activated in TSC2 cells C (PMA)-stimulated recombinant PLD1 activity and S6K1 acti- (Fig. 3 ). In contrast, the recombinant PLD2 activity was comparable in TSC2ϩ/ϩ and TSC2Ϫ/Ϫ cells [supporting infor- vation. A TSC patient-derived, GAP-inactive mutant of TSC2, mation (SI) Fig. S1]. Collectively, these observations cogently N1651S (23), had no inhibitory effect on PLD1, suggesting that state the model that Rheb activates PLD1 in cells. the GAP activity of TSC2 is involved in its regulation of PLD1. The effect of TSC2 overexpression was modest probably because Regulation of PLD by Signals Upstream of mTOR Signaling. Several TSC1 is required for the stabilization and maximal function of upstream signals regulate mTORC1 by impinging on the TSC/ TSC2 in cells. However, we were unable to reliably assess the Rheb axis, including cellular energy levels through AMPK and effect of TSC1/TSC2 coexpression on PLD1 activation due to growth factor signaling via the PI3K/Akt pathway (3). To technical difficulties in coexpressing those three recombinant examine the potential roles of PI3K and the cellular energy levels proteins. Alternatively, PMA as a stimulus may activate a on PLD activation, we performed in vivo PLD assays in HEK293 significant pool of PLD1 (e.g., through cPKC) in the cells cells to test the effects of the specific PI3K inhibitor wortmannin independent of the Rheb-mTOR pathway. It should be noted and the AMPK activator 2-deoxyglucose on serum stimulation that we used PMA in these experiments because of its robust of PLD. As shown in Fig. 4A, serum stimulation activated cellular activation of PLD1, which allowed for the detection of the PLD by Ϸ1.5-fold, comparable to reported serum activation of modest effect of TSC2 overexpression. Serum stimulation would PLD in other types of cells (28). This activation was dampened be performed for all other experiments described later because by wortmannin and drastically inhibited by 2-deoxyglucose;

8288 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712268105 Sun et al. Downloaded by guest on September 27, 2021 B Rheb Cdc42 GST A 3 * γ GTP S 2.5 GDPβS * 2 A Lysate Pulldown HA-PLD1 GST mTOR 1.5 GST-Rheb

mTOR GST proteins 1 PLD1

Relative PLD1 activity 0.5 GST-Rheb * 20 0 GST No PLD1 GTPγS: - + - + - + - + - + - + 15 pT389-S6K1 * None GST Rheb Cdc42 H-Ras RalA 10

5 TSC1/2 B mitogens 0 Relative amount of HA-PLD1 Amino Akt Rheb Acids Fig. 5. Rheb interacts with PLD1. (A) HEK293 cells were transfected with GST PI3K Cdc42, cPKC or GST-Rheb for 24 h. The cell lysates were incubated with glutathione beads ? Vps34 PLD1 and analyzed by Western blotting for GST proteins and endogenous PLD1. (B) Lysates of CHO cells expressing HA-PLD1 were incubated with purified GST, PDK1, aPKC GST-Cdc42, or GST-Rheb in the presence of GTP␥SorGDP␤S, followed by GST mTOR pulldown using glutathione beads and Western blot analyses of HA-PLD1, S6K1 endogenous mTOR, and Coomassie blue staining of GST proteins. The band intensity for PLD1 was quantified by densitometry. The average results of four Fig. 6. Rheb activates PLD1 in vitro.(A) HA-PLD1 was immunoprecipitated independent experiments are shown, with error bars representing SD. *, P Ͻ from serum-starved CHO cells induced to express HA-PLD1. Purified GST fusion 0.01 by Student’s t test. of GTPases, either unloaded or loaded with GTP␥S, were added to the immunocomplex, and in vitro PLD assays were carried out as described in Materials and Methods. The relative activities are shown with that of GST-added similar inhibition of S6K1 T389 phosphorylation was also ob- reaction as 1; background activities (‘‘No PLD1’’) are shown and have not been served (Fig. 4A). As expected, rapamycin had no effect on PLD subtracted from the data. The average results of three to six experiments are activity, although it abolished S6K1 activation. These observa- shown, with error bars representing SD. *, These data are significantly higher than that of GST when analyzed by Student’s t tests (P Ͻ 0.01). (B) A proposed tions are consistent with PLD lying downstream of TSC/Rheb model for PLD1 involvement in Rheb-mTOR signaling. and upstream of mTOR. Because amino acid sufficiency is essential for mTORC1 signaling, although the exact sensing mechanism is unclear, we examined independent of GTP loading, which is consistent with previous the requirement of amino acids for PLD activation. A short-term reports (14, 18). amino acid withdrawal using amino acid-free DMEM significantly suppressed serum stimulation of PLD activation in HEK293 cells, Rheb Activates PLD1 in Vitro. The physical interaction between and replenishing amino acids in the deprived cells restored PLD Rheb and PLD1 prompted us to ask whether Rheb could directly activation (Fig. 4B). Furthermore, the enhancement of PLD activity activate PLD1 in vitro. Stably expressed HA-PLD1 was immu- by TSC2 knockdown also was inhibited by amino acid deprivation noprecipitated from serum-starved cells and incubated with (Fig. 4C). These results reveal regulation of PLD by amino acid bacterially purified GST-Rheb in the presence or absence of availability and suggest that PLD may partly mediate amino acid GTP␥S. As shown in Fig. 6A, PLD1 was markedly activated by sensing in the mTOR pathway. Because exogenous PA is not able GST-Rheb, and this activation was absolutely dependent on the ␥ to override the amino acid requirement for S6K1 activation (4), presence of GTP S. The degree of PLD1 activation by Rheb was amino acid signals likely impinge on other targets in the mTORC1 somewhat higher than that by GST-Cdc42. Neither H-Ras nor pathway in addition to PLD. RalA was able to activate PLD1. The lack of activation by RalA, despite its reported interaction with PLD1, is consistent with Rheb Binds PLD1. Taken together, our observations placed PLD1 observations by others (29). Taken together, Rheb is likely a downstream of Rheb on a linear pathway. To dissect the direct activator of PLD1 and is at least as potent as Cdc42. mechanisms by which Rheb may regulate PLD1, we asked Discussion whether Rheb and PLD1 physically interact. GST pulldown As an essential growth-regulating signaling network and an assays were performed with lysates of HEK293 cells transiently attractive target of anticancer drug development in recent years, expressing GST-Rheb or GST. As shown in Fig. 5A, GST-Rheb, the molecular wiring of the rapamycin-sensitive pathways is of but not GST, associated with endogenous PLD1. GST-Rheb also tremendous interest and remains an intriguing puzzle. Our study interacted with mTOR, as reported by others previously (14, 18). described here has revealed a surprising connection between two To address whether GTP loading of Rheb is required for the previously established signaling pathways in this intricate net- CELL BIOLOGY Rheb–PLD1 interaction, in vitro GST pulldown assays were work. We have demonstrated that PLD1 is required for Rheb performed with bacterially expressed and purified GST fusion activation of mTOR signaling to S6K1 and that the TSC/Rheb proteins and lysates of CHO cells expressing HA-PLD1. The pathway controls PLD1 activation in cells. We have further GST proteins were loaded with GTP␥SorGDP␤S before provided biochemical evidence that Rheb binds and activates incubation with HA-PLD1-containing lysates and subsequent PLD1 in a GTP-dependent manner. In conclusion, our findings glutathione pulldown. GST-Cdc42, a well characterized PLD1- strongly suggest that PLD1 is a bona fide effector for Rheb (Fig. binding protein, was used as a positive control. As shown in Fig. 6B), filling a sizable gap in our knowledge of the signal trans- 5B, GST-Rheb pulled down PLD1 in a GTP-dependent manner. duction by the nutrient-sensing and growth-promoting mTOR Binding of mTOR to GST-Rheb also was observed, and it was network.

Sun et al. PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 ͉ 8289 Downloaded by guest on September 27, 2021 Previously found to mediate mitogenic signals in the mTOR ogy Center. Rapamycin and wortmannin were purchased from Calbiochem. pathway (4, 6), PLD1 may also receive nutrient signals as Insulin, PMA, 2-deoxyglucose, 1-butanol, 2-butanol, polybrene, and puromy- suggested by the sensitivity of PLD1 activity to amino acid cin were from Sigma. GTP␥S and GDP␤S trilithium salts were from Roche. 3 3 deprivation (Fig. 4 B and C). Amino acid signals appear to [Choline-methyl- H] dipalmitoyl-phosphatidylcholine ( H-DPPC) was from Amersham. [9,10-3H]-palmitic acid was from PerkinElmer. All other lipids were impinge on the mTORC1 pathway at least at two distinct levels. from Avanti. On the one hand, depletion of amino acids abolishes insulin- induced GTP-loading of Rheb (15). On the other hand, in the Plasmids. D60K mutation was introduced into FLAG-Rheb (see below) by using presence of activated Rheb, as found in TSC2 knockout cells, QuikChangeII Kit (Stratagene). pGEX-Rheb was generated by inserting the amino acid deprivation can still block mTOR signaling likely via PCR product of Rheb into pGEX-4T. pRK7-GST-Rheb was made by inserting the inhibition of the hVps34 pathway (13, 17). This dual level of PCR product of GST via XbaI and BamHI sites into pRK7-FLAG-Rheb. Myc-S6K1 regulation is fully consistent with our current and previous and HA-PLD1 were described previously (6). The following plasmids were observations: PLD activation by serum, as well as by TSC2 generous gifts from various laboratories: plasmids for mammalian expression knockdown, requires amino acid sufficiency, yet exogenous PA of FLAG-tagged Rheb, Rheb-C181S, TSC1, TSC2, TSC2-N1651S, and bacterial cannot override the requirement for amino acids in the activation expression vector for GST-Cdc42 were from J. Blenis (Harvard Medical School, of S6K. These situations should be discriminated from experi- Boston) (33). FLAG-Rab5A-L79 and FLAG-Rap1-V12 were from K. L. Guan (University of Michigan School of Medicine, Ann Arbor, MI) (21). Bacterial ments in which Rheb is overexpressed. Under such conditions, expression plasmids for GST-H-Ras and GST-RalA were from L. A. Quilliam Rheb is fully GTP loaded and activates mTOR even in the (Indiana University School of Medicine, Indianapolis) (34). absence of amino acids or mitogens (21, 23, 26, 30, 31). There- fore, it is not surprising that the interaction between overex- Cell Culture and Transfection. HEK293 cells were grown in DMEM containing pressed GST-Rheb and endogenous PLD1 (Fig. 5A) in the GST 10% FBS at 37°C with 5% CO2. All transient transfections were carried out with pulldown assays performed with HEK293 cell lysates was not PolyFect (Qiagen) when the cells were 60–70% confluent. The amount of DNA affected by amino acid deprivation or serum starvation of the transfected was between 0.5 and 1.5 ␮g for each plasmid, adjusted to ensure cells (Fig. S2). How exactly PLD1 receives amino acid signals is equal expression of the recombinant protein to be compared within each of obvious interest. Although regulation of PLD1 binding to experiment. Various treatments were applied to the cells 24 h after transfec- and/or activation by the GTP loading of Rheb in cells remains tion. Serum starvation of cells was carried out in serum-free DMEM overnight. Amino acid deprivation was achieved by incubating cells in amino acid-free a strong possibility to be further examined, a potential relation- DMEM (HyClone) for 1 h. For PA stimulation of the cells, 42 ␮l of 1-palmitoyl ship between PLD1 and hVps34 also merits future investigation 2-oleoyl phosphatidic acid in chloroform (25 mg/ml) was dried under nitrogen, (Fig. 6B). resuspended in 250 ␮l of DPBS, and sonicated in a water bath sonicator (80 While this article was in its final stage of preparation, a report watts) for 5 min. Freshly prepared PA vesicles were added to the cell medium appeared, revealing another direct target of Rheb-FKBP38, an to a final concentration of 100 ␮M. CHO cells stably expressing HA-PLD1 under endogenous inhibitor of mTOR (32). Rheb is shown to bind the control of the tetracycline promoter (a generous gift from M. Frohman, FKBP38 in a GTP-dependent and amino acid-sensitive manner, State University of New York, Stony Brook, NY) (35) were grown in F-12 and it activates mTOR by displacing FKBP38. Interestingly, both medium containing 10% FBS, 10 ␮g/ml blasticidin, and 300 ␮g/ml zeocin at ␮ PA, the product of PLD, and FKBP38 bind a region of mTOR 37°C with 5% CO2. At the cell density of 60–70%, 1 g/ml doxycycline was added in the medium to induce HA-PLD1 expression. TSC2ϩ/ϩ and TSC2Ϫ/Ϫ EEF that contains the rapamycin-FKBP12-binding (FRB) domain (4, cells (a generous gift from R. S. Yeung, University of Washington, Seattle) (27) 32). It is tempting to speculate that PA also may function to were cultured in DME/F-12 medium with 10% FBS under 5% CO2. Nucleofec- displace FKBP38 from mTOR and that Rheb activation of PLD1 tion was used to introduce DNA into the EEF cells (Amaxa nucleofector, and binding of FKBP38 synergize to achieve complete removal program D23, Kit L). of FKBP38 and maximal activation of mTOR. Alternatively, PA may be necessary for mTOR activation after FKBP38 is re- Lentivirus-Mediated RNAi. All shRNA was purchased from Sigma–Aldrich (Mis- moved; Rheb may play a dual role in eliminating the inhibitor sion shRNA). Lentivirus packaging and infection were performed as described and supplying the activator, and the two events may take place previously (36). See SI Materials and Methods for target sequences. independently, but concurrently, to activate mTOR. Three classes of proteins directly interact with and activate Quantitative RT-PCR. Quantitative RT-PCR was performed as described previously (36). The primers used were: hPLD2 forward, 5Ј-TCCATCCAGGCCATTCTGCAC; PLD1: cPKC, ARF, and Rho. We have now identified Rheb as Ј ␤ Ј a new regulator of PLD1. PLD1 appears indispensable for Rheb hPLD2 reverse, 5 -CGTTGCTCTCAGCCATGTCTTG; -actin forward, 5 -GCACTCT- TCCAGCCTT CCT; and ␤-actin reverse, 5Ј-AGGTCTTTGCGGATGTCCAC. activation of the mTOR pathway (Fig. 1B) and presumably cell size regulation. We found that Rheb activated PLD1 expressed S6 Kinase Assay. Myc-S6K1 was immunoprecipitated from transfected cells and immunoprecipitated from mammalian cells, but not PLD1 and subjected to in vitro S6 kinase assay by using a peptide substrate as purified from insect cells (data not shown), implying that a described previously (6). yet-to-be-identified cofactor or certain mammalian cell-specific posttranslational modification of PLD1 is required for the In Vivo PLD Assay. In vivo PLD activity was measured in a transphosphatidy- activation of PLD1 by Rheb. Thus, several intriguing questions lation assay adopted from methods published previously (37, 38) and modified remain to be addressed: Does Rheb compete for or synergize as described previously (20). To calculate recombinant PLD1 activity, the PLD with the other known regulators in binding and activating PLD1? activity in cells transfected with empty vector (pCDNA3) was subtracted from How does Rheb cross-talk with the other regulators in a cell to the activity in transfected cells under the same conditions. For EEF cell nucleo- ϫ 6 ␮ regulate PLD1 and mTOR signaling? Is Rheb involved in PLD1 fection, 1 10 cells were electroporated with 5 g pCDNA3 or HA-PLD1, seeded into a six-well plate, and cultured for 2 days, followed by serum functions other than cell size control, such as vesicle trafficking starvation in DME/F-12 with 0.1% FBS overnight. PLD1 activity was measured and cytoskeleton reorganization? These questions will guide as above and normalized against HA-PLD1 band intensity on Western blots future research efforts to reveal a clearer picture of the TSC- (quantified by using Image J). Rheb-PLD regulatory network. GST Pulldown Assays. GST fusions of small GTPases were purified from bacteria Materials and Methods by using glutathione Sepharose affinity chromatography. The purified GST- Antibodies and Other Reagents. The antibodies were obtained from the GTPase proteins (5 ␮g each) were first stripped of nucleotides by incubation in following sources: anti-FLAG M2 from Sigma; anti-HA (16B12) and anti-Myc 5 mM EDTA (in PBS) on ice for 15 min, followed by incubating with 20 mM (9E10.2) from Covance; anti-phospho-T389-S6K1, anti-PLD1, and anti-Rheb MgCl2and 100 ␮M guanine nucleotide (GTP␥SorGDP␤S) in 30 ␮l for 30 min at from Cell Signaling; anti-tubulin from Abcam; and anti-mTOR (FRB domain) 4°C and then for 10 min at 30°C. The nucleotide-loaded protein was then was generated at the University of Illinois at Urbana–Champaign Biotechnol- mixed with 20 ␮l of CHO cell lysates [lysis buffer: 40 mM Tris⅐Cl (pH 7.2), 1 mM

8290 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712268105 Sun et al. Downloaded by guest on September 27, 2021 Na3VO4, 25 mM NaF, 25 mM b-glycerophosphate, 2 mM EDTA, 2 mM EGTA, 1 concentration 30 ␮M) were added and incubated at 37°C for 10 min. The PLD mM DTT, 1 mM PMSF, and 0.3% Triton X-100] containing HA-PLD1 (see above) reaction was initiated by adding the substrate in the form of phospholipid and incubated at 30°C for 30 min. Glutathione Sepharose beads were added vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanomine, 3 to the mixture and gently rocked at 4°C for 1 h, followed by washing with lysis PIP2, and dipalmitoyl PC (DPPC) in a molar ratio of 16:1.4:1. [ H]DPPC was buffer and eluting in SDS sample buffer containing 8 M urea. For in vivo included at 20,000 cpm per assay. After incubation at 37°C for 30 min, the pulldown, GST- or GST-Rheb-transfected HEK293 cells were lysed, and the reaction was stopped by the addition of 200 ␮l of 10% trichloroacetic acid and lysates were incubated with glutathione Sepharose beads, followed by wash- 100 ␮l of 10 mg/ml BSA, followed by centrifugation at 3,000 ϫ g for 10 min at ing with PBS. 4°C. A 0.3-ml aliquot of the supernatant was removed and analyzed by liquid scintillation counting. In Vitro PLD Assay. In vitro PLD assays were performed by measuring the 3 release of [ H]choline modiﬁed from previously reported methods (39, 40). ACKNOWLEDGMENTS. We thank Dr. Nissim Hay for helpful discussions, Dr. HA-PLD1 was immunoprecipitated from doxycycline-treated and serum- Lee Henage for performing in vitro PLD assays, Dr. Troy Hornberger for helpful starved CHO cells described earlier and washed in lysis buffer with 1% Nonidet suggestions on PA stimulation, and Drs. John Blenis, Kun-Liang Guan, Law- P-40. The immunocomplex was resuspended in 50 ␮l of reaction buffer [50 mM rence Quilliam, and Michael Frohman for generously providing various re- Hepes (pH 7.5), 3 mM EGTA, 80 mM KCl, 1 mM DTT, 2 mM CaCl2,and3mM agents. This work was supported by grants from the National Institutes of MgCl2], to which 5 ␮g of GST fusion protein with or without GTP␥S (ﬁnal Health and American Cancer Society.

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