Cellular Signalling 18 (2006) 2283–2291 www.elsevier.com/locate/cellsig

PLD2 forms a functional complex with mTOR/raptor to transduce mitogenic signals

Sang Hoon Ha a, Do-Hyung Kim b, Il-Shin Kim a, Jung Hwan Kim a, Mi Nam Lee a, Hyun Ju Lee a, Jong Heon Kim a, Sung Key Jang a, Pann-Ghill Suh a, Sung Ho Ryu a,⁎

a Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbook 790-784, Republic of Korea b Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA Received 26 April 2006; received in revised form 11 May 2006; accepted 11 May 2006 Available online 3 June 2006

Abstract

Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger (PA), generated by D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265– 269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor. © 2006 Elsevier Inc. All rights reserved.

Keywords: PLD2; mTOR; Raptor; Complex formation

1. Introduction based on sequence similarity of their catalytic domains [4,5]. The mTOR pathway is an emerging target for the treatment of cancer, Mammalian target-of-rapamycin (mTOR) is a serine/threonine diabetes and obesity [6–10], and recent studies have demonstrated protein kinase [1–3] and a member of a novel superfamily of its role as a mediator of lifespan control in C. elegans [11] and signaling proteins termed PI 3-kinase related kinases (PIKKs), Drosophila [12]. Despite the significance of this pathway in such diverse biological processes, the mechanism of its regulation by upstream signals remains to be addressed. Recently, significant Abbreviations: mTOR, mammalian target-of-rapamycin; PLD, ; raptor, regulatory associated for mTOR; PIKK, phosphatidylinositol 3-kinase- progress was made in our understanding of the regulatory related protein kinase; Rheb, Ras enriched in brain; PC, ; PA, mechanisms of mTOR signaling and this revealed the participa- phosphatidic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propa- tion of the mTOR complex (mTOR/raptor) containing raptor nesulfonic acid; EMEM, Dulbecco's modified Eagle's medium; EDTA, ethylene [13,14] and GβL [15] in response to upstream signals for the diamine tetraacetic acid; PMSF, phenylmethlysulfonyl fluoride; PAGE, polyacryl- appropriate control of cell growth and the other mTOR complex amide gel electrophoresis; BCS, bovine calf serum; FBS, fetal bovine serum; PBS, phosphate buffered saline; DTT, dithiothreitol. (mTOR/rictor) containing rictor [16] and GβL [15] for the control ⁎ Corresponding author. Tel.: +82 54 279 2292; fax: +82 54 279 0645. of actin cytoskeleton. These upstream signals derived from E-mail address: [email protected] (S.H. Ryu). insulin, nutrients, and/or mitogens are likely to be mediated by the

0898-6568/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2006.05.021 2284 S.H. Ha et al. / Cellular Signalling 18 (2006) 2283–2291 tuberous sclerosis complex 1/2 [17] and Rheb [18–22]. Though 5′-UGC UCA UUG UCG UCC CAC CUU-3′, which correspond to human the functional connection between mTOR complex and these PLD1a coding nucleotides 1455–1475. All siRNA sequences were subjected to BLAST in the NCBI database and complete matches were only found for PLD2 upstream regulators is known, their physical connection is not sequences. Luciferase GL2 duplex was purchased from Dharmacon Research, completely understood [6–9]. Recently, Rheb was shown to Inc. and was used as a negative control. For add-back experiment for PLD2 interact with mTOR/raptor and activate kinase activity of mTOR silencing, three residues of human PLD2 cDNA (nucleotides 703–723 of PLD2; [23,24]. AAGAGGTGGCTGGTGGTGAAG) are substituted to AAGAGATGGCTAG wt In addition, mTOR requires the lipid second messenger TAGTGAAG for add back mutants of PLD2 [31]. This mutation is silencing mutations. This is subcloned into mammalian expression vector pcDNA3.1 phosphatidic acid (PA) for its activation [25,26]. PA is an enzy- (Invitrogen) and digested with restriction KpnI and XbaI. These matic product of phospholipase D (PLD) [27,28]. Mammalian mutations are confirmed through nucleotide sequence analysis. PLD isozymes identified to date, PLD1 and PLD2, sense a variety of signals, such as neurotransmitters, hormones and 2.4. Cell culture and plasmid/siRNA transfection growth factors, to regulate multiple physiological events such as proliferation, secretion, respiratory burst and actin cytoskeletal COS7 cells were maintained in a 5% CO2 humidified atmosphere at 37 °C reorganization [27,28]. Here, our effort to elucidate which iso- and fed DMEM supplemented with 10% bovine calf serum. HEK293 cells and OVCAR-3 cells were fed DMEM supplemented with 10% fetal bovine serum. zyme is mainly involved in mitogen-induced mTOR activation Cells grown on tissue culture dishes was transiently transfected using identifies that PLD2 is a major isozyme to transduce mitogenic LipofectAMINE, as described previously [31,32]. Cells were allowed to express signaling to mTOR/raptor and this is achieved by complex the recombinant proteins for 24 h after transfection and then deprived of serum formation between PLD2 and mTOR/raptor. As a result, we for additional 24 h. The cells were then subjected to co-immunoprecipitation suggest the physical/functional connections between mitogen- analysis. For knockdown with siRNA, cells were grown for 36–48 h before serum deprivation. induced PA generation and its effector mTOR and would pro- vide further insight into mTOR-related metabolic diseases such 2.5. Co-immunoprecipitation as cancer, diabetes and obesity. After harvesting COS7 cells, total extracts were prepared by brief sonication 2. Materials and methods in ice-cold lysis buffer (40 mM HEPES pH7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na3VO4, 2.1. Materials 0.5% CHAPS, 1 mM PMSF, protease inhibitor cocktails) [13]. Clarified extracts were mixed with 2 μg of the respective antibodies. Then protein A-Sepharose The enhanced chemiluminescence kit and dipalmitoylphosphatidyl beads were added to isolate the antibody complex. After four washings with lysis [methyl-3H] were purchased from Amersham Biosciences. Horseradish buffer, the final immunoprecipitates were washed once with wash buffer (50 mM peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgA, IgM, and HEPES pH7.5, 150 mM NaCl), and then subjected to SDS-PAGE. IgG were from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD). Polyclonal antibody was raised against PLD as previously described [29]. 2.6. Western blot analysis Antibodies against mTOR, pS6K1 (pThr 389), S6K1, p4EBP1(pThr 37/46), and 4EBP1 and rapamycin were from Cell Signaling. Protein A-Sepharose was from Proteins were separated by SDS-PAGE on 8–16% gradient gels, and the RepliGen (Needham, MA). CHAPS was from Sigma. Dulbecco's modified separated proteins were transferred onto nitrocellulose membranes and blocked Eagle's medium (DMEM) and LipofectAMINE were from Invitrogen. C-6 with TTBS buffer (10 mM Tris–HCl, pH 7.6, 150 mM NaCl, and 0.05% Tween- phosphatidic acid was from Avanti, and recombinant 4EBP1 was purchased from 20) containing 5% skimmed milk powder. Membranes were then incubated with Stratagen. primary antibody at the concentration recommended by the manufacturer for 4 h at room temperature. Unbound antibody was washed away with TTBS buffer. 2.2. Plasmids Membranes were subsequently incubated with horseradish peroxidase-conju- gated secondary antibody for 1 h at room temperature, washed five times with Mammalian expression vectors for PLD1wt,PLD2wt,PLD2ΔN184, TTBS buffer, and developed using an ECL system. PLD2ΔN308, and PLD2K758R were used as described previously [30–32]. Ex- pression vectors for HA–mTORwt, myc–mTORwt, myc–raptorwt, HA–raptorwt 2.7. In vivo PLD assay and HA–raptor194YDC/AAAmt were gifts from Dr. David M. Sabatini (MIT) [13]. To introduce the TOS motif mutation in PLD2, pCDNA3.1(+)/PLD2 containing In vivo PLD activity was assayed by measuring the formation of wild type PLD2 was PCR amplified using the following oligomers; sense phosphatidyl–butanol as described previously [32]. In brief, cells were loaded (5′GGC CGA GAC CAA GTT TGT TAT CGC3′), antisense (F265A:5′CCA with [3H]myristic acid (2 μCi/ml) for 8 h and then washed twice with DMEM. TCG ATC CGC ACG CCG TGC CGT GCC TCC GTG CTC CTT TTC CCC Labeled cells were incubated with 0.4% butanol for 10 min to measure basal ACT TGC ACC TCA GCG CCA GG3′), antisense (E266R: 5′CCA TCG ATC PLD activity. Total lipids were extracted with 1.2 ml of methanol:1 M NaCl: CGC ACG CCG TGC CGT GCC TCC GTG CTC CTT TTC CCC ACT TGC chloroform (1:1:1 by volume) and then separated by thin-layer chromatography ACC CTA AAG CCA GG3′). DNA fragments generated by PCR and on silica gel plates. The amount of [3H]phosphatidyl–butanol formed was pCDNA3.1(+)/PLD2(WT) were treated with XhoI and Cla I. expressed as a percentage of total [3H]lipid to account for cell labeling efficiency differences.

2.3. RNA interference 2.8. In vitro kinase assay for mTOR activity. Pairs of 21-nucleotide sense and antisense RNA oligomers were synthesized and annealed by Dharmacon Research, Inc. (Lafayette, CO). The oligonucleo- Recombinant myc–mTOR was expressed with the indicated proteins and tides used for PLD2 were: sense, 5′-AAG AGG UGG CUG GUG GUG AAG-3′ then immunoprecipitated using anti-myc antibody, as previously described [13]. and antisense, 5′-CUU CAC CAC CAG CCA CCU CUU-3′, which correspond Recombinant 4EBP1 was used as a substrate for in vitro kinase assays. Activities to human PLD2 coding nucleotides 703–723. The oligonucleotides used for were measured using anti-phospho-4EBP1 antibody (phosphor-37/46). The PLD1 were: sense, 5′-AAG GUG GGA CGA CAA UGA GCA-3′, and antisense, kinase assay was performed by mixing buffer containing 25 mM Hepes pH7.4, S.H. Ha et al. / Cellular Signalling 18 (2006) 2283–2291 2285

50 mM KCl, 10 mM MgCl2, 4 mM MnCl2, 20% glycerol, 2 mM DTT, 0.1 mM completely rescue mTOR signaling despite PLD2 expression. ATP, 1 μg 4EBP1 with the indicated immunoprecipitates and then incubated at However, PA could not trigger S6K1 phosphorylation in PLD2- 30 °C for 15 min. knocked-down HEK293 and COS7 cells (Fig. 1B). Summariz- ing, the isozyme-specific knockdown of PLD by using siRNA 3. Results suggests that PLD2 has a profound role in mTOR signaling, and that PLD2 might have another mechanism to activate mTOR 3.1. PLD2 is mainly involved in mTOR activation signaling in accord with PA generation.

PLD hydrolyzes phosphatidylcholine to generate PA [27,28] 3.2. PLD2 forms a complex with mTOR through its TOS and this process constitutes a link whereby mitogenic signals motif-like sequence impinge on the mTOR pathway [25]. However, the mechanism by which PA activates mTOR in cells remains unknown [26].To We also investigated why PA could no longer activate mTOR gain further insight into this process, we compared the effects of in the absence of PLD2 expression. One possibility concerns the two mammalian PLD isozymes, PLD1 and 2 [27,28], on mTOR proximity of PLD2 around the mTOR complex. PA contains a activation using isozyme-specific siRNAs in human embryonic long acyl chain that might restrict its membrane mobility; more- kidney (HEK) 293 cells, human ovarian cancer-derived over, PA is a transient species [27,28,33]. These properties of PA OVCAR-3 cells, and monkey epithelial COS7 cells. As shown encouraged us to speculate that mTOR might be localized near in Fig. 1A, the knockdown of PLD2 dramatically reduced the PLD to monitor the PA produced in response to upstream sig- phosphorylations of S6K1 and 4EBP1, well-known mTOR nals; moreover, such a possibility suggests the existence of a effectors [6–9], as compared with those of PLD1 knockdown, physical connection between mTOR complex and PLD. To test suggesting that PLD has an essential role in mTOR activation. this possibility, endogenous mTOR was immunoprecipitated Next, we questioned whether exogenous PA can rescue mTOR with anti-mTOR antibody, and it was found that these immuno- signal abrogation in PLD2-knocked-down cells. We reasoned precipitates from COS7 (Fig. 2A) and HEK293 (data not shown) that if the role of PLD2 in mTOR activation is solely that of contained endogenous PLD2. The interaction between mTOR PA generation, then the exogenous addition of PA should and PLD2 is sensitive to Triton X-100, but stable in CHAPS- containing lysis conditions, like the mTOR–raptor interaction [13,14]. Recombinant PLD2 also formed a complex with recombinant mTOR, and this interaction was specific for PLD2 as recombinant PLD1 did not interact with mTOR (Fig. 2B). To identify the region of PLD2 responsible for the inter- action with mTOR, N-terminal truncated PLD2 fragments were generated [30] and used for site-mapping analysis. This process resulted in the identification of a PH domain-containing region in PLD2 (i.e., a.a. 185–308) (Fig. 2C). Interestingly, in PLD2, but not in PLD1, this region was found to contain FEVQV (a.a. 265–269), which is a TOS motif pattern present in both S6K1 and 4EBP1 that allows binding with mTOR through raptor (Fig. 2D) [34–36]. Therefore, we produced point mutants, PLD2F265A and PLD2E266R, and examined their binding with mTOR. We found that both of these PLD2 point mutants abrogated its interaction with mTOR (Fig. 2D), thus supporting the notion that these residues are important for PLD2–mTOR binding, and that they constitute a TOS motif, as found in S6K1 and 4EBP1 [34–36].

3.3. Raptor provides docking site for PLD2 in the mTOR complex

mTOR exists as two protein complexes in mammalian cells [9], i.e., cell growth-related mTOR functions in cooperation with raptor [13,14], whereas cytoskeletal organization-related mTOR Fig. 1. PLD2 plays an essential role in mTOR activation. (A) siRNAs for PLD1 function cooperates with rictor/mAVO3 [16]. Our identification or PLD1 were transfected into HEK293, OVCAR-3, or COS7 cells using of a TOS motif-like sequence in PLD2 suggests that PLD2 may lipofectamine. After 36 h, cells were deprived of serum for 24 h, and then were form a complex with mTOR by interacting with raptor. In the lysed in CHPAS-containing lysis buffer, as described in materials and methods. present study, we found; 1) that recombinant PLD2 specifically siRNA for luciferase was used as a negative control. Equal protein loadings were interacts with recombinant raptor even under Triton X-100 lysis verified versus actin. (B) HEK293 and COS7 cells were transfected with the indicated siRNAs using lipofectamine. After 36 h, cells were deprived of serum conditions (Fig. 3A); 2) that the interaction between PLD2 and for 24 h. 100 μM of C-6 PA solubilized in DW was then treated for 30 min. raptor is insensitive to Triton X-100 (Fig. 3B) and that it does not Resulting lysates were subject to SDS-PAGE. require an interaction between raptor and mTOR (Fig. 3C); 3) 2286 S.H. Ha et al. / Cellular Signalling 18 (2006) 2283–2291

Fig. 2. PLD2 localizes at the mTOR complex. (A) COS7 cell lysates (10 mg) were prepared under different lysis conditions using Triton X-100 or CHPAS and then subjected to co-IP analysis against anti-mTOR antibody. Resulting immunoprecipitates were subjected to SDS-PAGE. Arrows indicate the positions of blotted proteins. (B) Lysates from COS7 cells overexpressing myc–mTOR and PLD1 or PLD2 were co-immunoprecipitated with anti-myc antibody and immunoblotted with anti-PLD antibody. (C) Upper panel; Schematic view of PLD2 N-terminal deleted fragments versus whole PLD2. Lower panel; myc–mTORwt was transfected with the indicated PLD2 fragments and co-IP analysis was performed. (D) Upper panel; TOS motifs of human 4EBP1 and human S6K1 were compared with the putative TOS motif in human/rat/mouse PLD2. Corresponding regions in human/rat/mouse PLD1 are shown and asterisks are used to highlight differences. Lower panel; myc–mTOR was expressed with the indicated PLD2 mutants and the resulting lysates were subjected to co-IP analysis. Endogenous raptor was immunoblotted with anti-raptor polyclonal antibody. that the interaction between PLD2 and raptor under Triton X-100 complex with mTOR–raptor, and that in this context raptor lysis conditions is also dependent on TOS motif-like sequence of provides docking site for PLD2 by interacting with a TOS motif- PLD2 (Fig. 3D). These findings raise the possibility the PLD2– like sequence in PLD2. raptor interaction is independent of mTOR. However, endoge- Raptor contains a conserved N-terminal (RNC) domain, nous mTOR complex was found to contain raptor and PLD2 which is followed by three HEATrepeats in its central region and (Fig. 2A), and the interaction between mTOR and PLD2 was 7 WD40 repeats in the C-terminal portion [13]. Moreover, these also found to be dependent on its TOS motif-like sequence HEAT and WD40 repeats are protein–protein interaction motifs (Fig. 2D). These findings strongly suggest that PLD2 forms a and are present in many eukaryotic proteins [37]. To identify the S.H. Ha et al. / Cellular Signalling 18 (2006) 2283–2291 2287

Fig. 3. PLD2 uses raptor to form a complex with mTOR/raptor. (A) myc–raptor was expressed with PLD1 or PLD2 into COS7 cells. Resulting lysates were prepared using Triton X-100-containing lysis conditions and then subjected to co-IP analysis. (B) HA–raptor and PLD2 were expressed and lysed in lysis buffer containing different detergents, as indicated, and then subjected to co-IP analysis. Endogenous mTOR was blotted by anti-mTOR antibody. (C) PLD2 was expressed with eitherHA–raptorwt or HA–raptor194YDC/AAAmt and the resulting lysates were immunoprecipitated with anti-HA antibody. Bound mTOR and PLD2 were detected by immunoblotting. (D) HA– raptor was expressed with the indicated PLD2 constructs into COS7 cells. Cells were lysed in lysis buffer containing Triton X-100 and then subjected to co-IP analysis using anti-HA antibody. (E) HA–raptor1–646 and HA–raptor1020–1335 were expressed with PLD2, myc–S6K1, or myc–4EBP1. After co-IP analysis with anti-HA antibody, resulting immunoprecipitates were subjected to SDS-PAGE analysis. Anti-myc antibody was used to determine myc–S6K1 and myc–4EBP1 levels. Asterisks indicate expressed raptor fragments. (F) The indicated recombinant proteins were expressed into COS7 cells. Resulting lysates were obtained under CHAPS-containing conditions, divided in two, and subjected to co-IP analysis with anti-HA or anti-myc antibodies. (G) The indicated proteins were expressed in COS7 cells and the resulting lysates were subjected to co-IP analysis with anti-myc antibody. 2288 S.H. Ha et al. / Cellular Signalling 18 (2006) 2283–2291 S.H. Ha et al. / Cellular Signalling 18 (2006) 2283–2291 2289

PLD2 binding sites in raptor, truncated raptor mutants were used production during kinase assays, because our in vitro kinase for site-mapping analysis (Supplementary Fig. S1) [13].We assay did not contain phosphatidylcholine, an essential substrate believe that the WD40 repeat of raptor is responsible for PLD2 for PLD. Thus this finding excludes mTOR activation by PA binding because we found that a.a. 1020–1335 of raptor, which during these conditions. We speculate that PLD2 activates encompasses the WD40 repeat, can interact with PLD2 mTOR before cell lysis via an unidentified mechanism, such as, (Supplementary Fig. S1). Next, we directly compared the a conformational change or a posttranslational modification. The interaction preferences of PLD2, S6K1 and 4EBP1 for raptor regulation of the mTOR–raptor interaction by PLD2 can be (Fig. 3E), because S6K1 and 4EBP1 primarily use their TOS excluded because PLD2 did not have any effect on the mTOR– motifs to interact with raptor [34–36]. As previously reported raptor interaction (Fig. 2D). Also, neither reducing PA [35], S6K1 and 4EBP1 were found to favor the N-terminal generation with 1-butanol [25–28] nor adding PA modulates region (a.a. 1–646) of raptor, though they showed different the mTOR–raptor interaction (data not shown). These results preference on raptor binding with PLD2. These results raised the suggest that both the physical interaction between PLD2 and possibility that PLD2 forms a complex with either S6K1 or raptor and the enzymatic activity of PLD2 are concurrently 4EBP1 through raptor, and this was found to be the case, as required for mTOR pathway stimulation. PLD2 was found in raptor immunoprecipitates with S6K1 or It has been reported that the mTOR–raptor interaction is not 4EBP1 (Fig. 3F). Likewise, immunoprecipitation with recom- changed by mitogen stimulation [13], which suggests the binant S6K1 or 4EBP1 also showed PLD2 bound raptor with existence of unidentified mechanism to sense mitogenic signal. S6K1 or 4EBP1 (Fig. 3F). As expected, PLD2–raptor–S6K1 Various mitogens are known to activate PLD, and lead to PA complex formation was found to require the integrity of the production [27,28]. Our finding that the participation of PLD2 PLD2 TOS motif-like sequence (Fig. 3G). In case of S6K1 and in mTOR activation encouraged us to determine whether PLD2 4EBP1, it was reported that they compete for interaction with mediates the mitogenic activation of mTOR signaling. First, we raptor [35]. However, PLD2 uses WD40 region of raptor to tested the dynamicity of the PLD2–mTOR interaction. As interact. Therefore, it is reasonable that PLD2 can form a shown in Fig. 4D, 20% bovine calf serum (BCS) potently complex with either S6K1 or 4EBP1 through raptor. Altogether, increased the interaction between PLD2 and mTOR within our detailed molecular analysis reveals that PLD2 binds the 1min of treatment and maintained this for 30min. The mTOR/raptor complex through raptor. phosphorylations of S6K1 and 4EBP1 followed at 5 min. As expected, knockdown of PLD2 profoundly reduced the 3.4. Both PLD2–raptor interaction and enzymatic activity of mitogen-dependent phosphorylation of S6K1 (Fig. 4E), where- PLD2 are required for mitogen-dependent activation of mTOR as PLD1 knockdown had only a modest effect on S6K1 phosphorylation, thus supporting the notion that PLD2 is a To determine whether the PLD2–raptor binding is an aspect mainly involved in mitogen-induced mTOR signaling. To of mTOR pathway activation, we examined the ability of PLD2 further check the importance of PLD2 in mitogen-dependent point mutants to stimulate S6K1 phosphorylation. PLD2F265A mTOR signaling, we rescued PLD2 expression using siRNA- and PLD2E266R have similar level of enzymatic activity resistant expression constructs [31], and found that mitogen- compared to PLD2wt (Fig. 4A), suggesting that the localization induced S6K1 phosphorylation was rescued only by PLD2wt of PLD2 at the mTOR complex is not important for its enzymatic (Fig. 4F). Adding-back of PLD2F265A,PLD2E266R,and property. However, PLD2F265A and PLD2E266R did not trigger PLD2K758R did not rescue S6K1 phosphorylation attenuation S6K1 phosphorylation versus PLD2wt (Fig. 4B). We expressed in PLD2-knocked-down cells. Interestingly, the exogenous PA- myc-tagged recombinant mTOR with the indicated PLD2 dependent phosphorylation of S6K1 was rescued by PLD2wt constructs, and the myc-immunoprecipitate obtained was used and PLD2K758R, but not by PLD2F265A and PLD2E266R (Fig. for in vitro kinase assays (Fig. 4C). The results obtained 4F), which again highlighted the requirement for PLD2 correlated with those of S6K1 phosphorylation in as much as interaction with raptor, and PA production at the mTOR PLD2F265A and PLD2E266R could not trigger mTOR kinase complex for PLD2-dependent mTOR activation. activity (Fig. 4C). It is possible that PLD2 itself has some effect on mTOR kinase activity as was raptor or GβL [13,15]. 4. Discussion However, lipase-inactive PLD2K758R, which is capable of binding mTOR–raptor, did not activate mTOR kinase activity Despite their functional connection, no physical connection (Fig. 4C), thus excluding any direct effect of PLD2 on mTOR has been made between PA production and mTOR activation kinase activity. In addition, this activation is not due to PA [25,26]. Our results suggest that PLD2 might function as a

Fig. 4. PLD2 activates mitogen-induced mTOR signaling (A) In vivo PLD assays were performed in COS7 cells expressing the indicated PLD2 constructs. PBt formation; phosphatidyl–butanol formation (B) Lysates from panel (A) were subjected to Western blot analysis. (C) myc–mTOR was expressed with the indicated PLD2 fragments. After co-IP analysis, myc-immunoprecipitates were subjected to in vitro kinase assays for mTOR, as indicated in materials and methods. IVK; in vitro kinase assay. (D) myc–mTOR and HA–PLD2 were expressed in COS7 cells. After 24 h, cells were deprived of serum for 24 h and then were treated with 20% of BCS for the indicated times to stimulate mTOR signaling. After co-IP analysis with anti-myc antibody, bound HA–PLD2 was proven using anti-HA antibody. (E) The indicated siRNAs were transfected into COS7 cells, and 36 h later, cells were deprived of serum for 24 h. 20% of BCS was then treated and the resulting lysates were subjected to SDS-PAGE. (F) PLD2 siRNAs were transfected with the indicated PLD2 add-back constructs as described in materials and methods. 20% of BCS or 100 μM of C-6 PAwas used to stimulate mTOR signaling. (G) Proposed model explaining the role of PLD2 in the mitogen-dependent activation of mTOR signaling. 2290 S.H. Ha et al. / Cellular Signalling 18 (2006) 2283–2291 mediator of mitogen-induced mTOR activation. And, based on presents a novel regulatory point that can be targeted for the our findings, we propose a model in which PLD2 binds raptor treatment of metabolic diseases. through its TOS motif-like sequence (Fig. 4G), and that the mitogen-induced PLD2–raptor interaction allows PA accumu- Acknowledgements lation near mTOR, enabling PA-dependent mTOR activation and the resulting phosphorylation of S6K1. We thank Dr. David M. Sabatini (MIT, USA) for providing In terms of sensing mitogenic signal as a form of PA through expression vectors (mTOR and raptor). This work was supported complex formation, this appears an efficient means of respond- by the 21C Frontier Functional Proteomics program of the ing quickly and specifically. Moreover, the coordinated Ministry of Science and Technology in the Republic of Korea to organization of multiple proteins by scaffold proteins is S.H. Ryu as well as grant from the Tuberous Sclerosis Alliance important for signaling efficiency and specificity, as exemplified to D.-H. Kim. in KSR-mediated MAPK regulation [38]. KSR, a kinase suppressor of Ras, functions as a scaffold and thus helps Appendix A. Supplementary data assemble MAPK pathway components into a localized signaling complex [38]. It is likely that the interaction of the upstream Supplementary data associated with this article can be found, regulator (i.e., PLD2) with scaffold (i.e., raptor) is also important in the online version, at doi:10.1016/j.cellsig.2006.05.021. for localized mTOR signaling complex, since downstream effectors (i.e., S6K1 and 4EBP1) use same scaffold (i.e., raptor) References to localize at the mTOR complex. [1] E.J. Brown, M.W.Albers, T.B. Shin, K. Ichikawa, C.T. Keith, W.S. Lane, S.L. S6K1 and 4EBP1 use their TOS motifs to interact with raptor Schreiber, Nature 369 (1994) 756. in similar ways, and therefore compete with each other for raptor [2] M.I. Chiu, H. Katz, V.Berlin, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 12574. binding [34–36]. However, PLD2 was found to share raptor with [3] D.M. Sabatini, H. Erdjument-Bromage, M. Lui, P. Tempst, S.H. Snyder, S6K1 or 4EBP1 despite their possessing a TOS motif-like Cell 78 (1994) 35. sequence. 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