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Involvement of Phospholipase D in Sphingosine 1-Phosphate-induced Activation of Phosphatidylinositol 3-Kinase and Akt in Chinese Hamster Ovary Cells Overexpressing EDG3*

Open AccessPublished:September 21, 2001DOI:https://doi.org/10.1074/jbc.M105673200
      Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI3K), and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)-induced activation of the G protein-coupled EDG3 receptor to stimulation of PI3K and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells but not vector-transfected cells induced activation of PLD, PI3K, and Akt in a time- and dose-dependent manner. Akt phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002 (2-(4-monrpholinyl)-8-phenyl-4H-1-benzopyran-4-one), indicating that Akt activation was dependent on PI3K. S1P-induced activation of PI3K and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas both PI3K and Akt activation were not inhibited by 2-butanol without such reaction. Co-expression of wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of phosphatidic acid, resulted in increases in PI3K activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI3K and Rac, was inhibited by 1-butanol, but not by 2-butanol. These results demonstrate that PLD participates in the activation of PI3K and Akt stimulation of EDG3 receptor.
      PA
      phosphatidic acid
      PBut
      phosphatidylbutanol
      PI3K
      phosphatidylinositol 3-kinase
      PLD
      phospholipase D
      PLDSc
      Streptomyces chromofuscas PLD
      S1P
      sphingosine 1-phosphate
      CHO
      Chinese hamster ovary
      PI3P
      PI3-phosphate
      Hydrolysis of phosphatidylcholine by phospholipase D (PLD)1 to generate phosphatidic acid (PA) and choline has been implicated in a variety of cellular responses, including rapid responses such as secretion and cytoskeletal reorganization as well as proliferation, differentiation, and apoptosis (
      • Exton J.H.
      ,
      • Jones D.
      • Morgan C.
      • Cockcroft S.
      ,
      • Liscovitch M.
      • Czarny M.
      • Fiucci G.
      • Tang X.
      ). Two mammalian PLDs, PLD1 and PLD2, have been identified that differ in terms of cellular localization and function (
      • Exton J.H.
      ,
      • Jones D.
      • Morgan C.
      • Cockcroft S.
      ,
      • Liscovitch M.
      • Czarny M.
      • Fiucci G.
      • Tang X.
      ,
      • Frohman M.A.
      • Sung T.C.
      • Morris A.J.
      ,
      • Meier K.E.
      • Gibbs T.C.
      • Knoepp S.M.
      • Ella K.M.
      ). A number of reports have pointed to the ability of PA to modify, in cell-free systems, the activities of components playing key roles in signal transduction, including serine/threonine protein kinases (
      • Ghosh S.
      • Strum J.C.
      • Sciorra V.A.
      • Daniel L.W.
      • Bell R.M.
      ,
      • Limatola C.
      • Barabino B.
      • Nista A.
      • Santoni A.
      ,
      • Waite K.A.
      • Wallin R.
      • Qualliotine-Mann D.
      • McPhail L.C.
      ), protein phosphatases (
      • Bokoch G.M.
      • Reilly A.M.
      • Daniels R.H.
      • King C.C.
      • Olivera A.
      • Spiegel S.
      • Knaus U.G.
      ,
      • Kishikawa K.
      • Chalfant C.E.
      • Perry D.K.
      • Bielawska A.
      • Hannun Y.A.
      ), GTPase-activating proteins (
      • Tsai M.-H., Yu, C.-L.
      • Wei F.-S.
      • Stacey D.W.
      ), lipid kinases (
      • Melendez A.
      • Andres F.
      • Gillooly D.J.
      • Harnett M.M.
      • Allen J.M.
      ,
      • Moritz A.
      • De Graan P.N.E.
      • Gispen W.H.
      • Wirtz K.W.A.
      ), phospholipases (
      • Jones G.A.
      • Carpenter G.
      ,
      • Litosch I.
      ), and NADPH oxidase (
      • Palicz A.
      • Foubert T.R.
      • Jesaitis A.J.
      • Marodi L.
      • McPhail L.C.
      ). However, it remains unclear whether PA exertsin vivo regulatory effects on these signaling molecules. A recent study has demonstrated that in insulin-stimulated cells, PA derived from PLD2 activation takes part in the Ras-mitogen-activated protein kinase signaling pathway by promoting recruitment to the membrane and activation of Raf-1 (
      • Rizzo M.A.
      • Shome K.
      • Vasudevan C.
      • Stolz D.B.
      • Sung T.C.
      • Frohman M.A.
      • Watkins S.C.
      • Romero G.
      ).
      Sphingosine 1-phosphate (S1P), a metabolite of sphingomyelin, acts as a second messenger and also as a high-affinity agonist for the EDG family of G protein-coupled cell surface receptors, which includes EDG1, EDG3, EDG5, EDG6, and EDG8 (
      • An S.
      ,
      • Pyne S.
      • Pyne N.J.
      ,
      • Spiegel S.
      • Milstien S.
      ). The cellular responses elicited by S1P include stimulation of mitogenesis, cell differentiation, smooth muscle contraction, regulation of cell migration, inhibition of tumor cell invasion (
      • An S.
      ,
      • Pyne S.
      • Pyne N.J.
      ,
      • Spiegel S.
      • Milstien S.
      ,
      • Chun J.
      • Contos J.J.
      • Munroe D.
      ,
      • Hla T.
      • Lee M.J.
      • Ancellin N.
      • Liu C.H.
      • Thangada S.
      • Thompson B.D.
      • Kluk M.
      ,
      • Igarashi Y.
      ,
      • Moolenaar W.H.
      ), activation of several enzymes such as phospholipase C, adenylate cyclase, mitogen-activated protein kinase family protein kinases, phosphatidylinositol 3-kinase (PI3K), and PLD, and Cas tyrosine phosphorylation (
      • Banno Y.
      • Fujita H.
      • Ono Y.
      • Nakashima S.
      • Ito Y.
      • Kuzumaki N.
      • Nozawa Y.
      ,
      • Koh J.
      • Satao K.
      • Watanabe T.
      • Tomura H.
      • Kuwabara A.
      • Kimura T.
      • Tamama K.
      • Ishizuka T.
      • Murata N.
      • Kanda T.
      • Kobayashi I.
      • Ohta H.
      • Ui M.
      • Okajima F.
      ,
      • Ohmori T.
      • Yatomi Y.
      • Okamoto H.
      • Miura Y.
      • Rile G.
      • Satoh K.
      • Ozaki Y.
      ,
      • Okamoto H.
      • Takuwa N.
      • Gonda K.
      • Okazaki H.
      • Chang K.
      • Yatomi Y.
      • Shigematsu H.
      • Takuwa Y.
      ,
      • Okamoto H.
      • Takuwa N.
      • Yatomi Y.
      • Gonda K.
      • Shigematsu H.
      • Takuwa Y.
      ,
      • Orlati S.
      • Porceli A.M.
      • Hrelia S.
      • Van Brocklyn J.R.
      • Spiegel S.
      • Rugolo M.
      ,
      • Rakhit S.
      • Conway A.M.
      • Tate R.
      • Bower T.
      • Pyne N.J.
      • Pyne S.
      ). A potential role for S1P in cell proliferation and survival has been suggested based on its ability to antagonize the apoptosis-inducing effects of ceramide (
      • Goetzl E.J.
      • Kong Y.
      • Mei B.
      ) and to activate mitogen-activated protein kinase followed by activation of c-Fos, activations that are mediated via EDG3 and EDG5 (
      • An S.
      • Zheng Y.
      • Bleu T.
      ). However, the mechanisms of S1P action underlying cell survival activity are not fully understood. Recent studies have demonstrated that PLD participates in cell proliferation and antiapoptosis (
      • Lee S.D.
      • Lee B.D.
      • Han J.M.
      • Kim J.H.
      • Kim Y.
      • Suh P.G.
      • Ryu S.H.
      ,
      • Nakashima S.
      • Nozawa Y.
      ,
      • Yamakawa H.
      • Banno Y.
      • Nakashima S.
      • Sawada M.
      • Yamada J.
      • Yoshimura S.
      • Nishimura Y.
      • Nozawa Y.
      • Sakai N.
      ,
      • Zhang Y.
      • Redina O.
      • Altshuller Y.M.
      • Yamazaki M.
      • Ramos J.
      • Chneiweiss H.
      • Kanaho Y.
      • Frohman M.A.
      ), but little information is available regarding the involvement of PLD in signaling related to proliferation and survival. However, a wealth of evidence has demonstrated that PI3K does play an important role in this signaling. More specifically, the serine/threonine kinase Akt/protein kinase B has been found to be a critical downstream effector of PI3K in cell survival signaling (
      • Marte B., M.
      • Downward J.
      ). In addition, we have recently demonstrated that S1P induces PI3K activation via EDG1, EDG3, and EDG5. S1P induced membrane ruffling and cell migration in a PI3K- and Rac-dependent manner (
      • Okamoto H.
      • Takuwa N.
      • Yokomizo T.
      • Sugimoto N.
      • Sakurada S.
      • Shigematsu H.
      • Takuwa Y.
      ). Interestingly, PLD has also been shown to be involved in this membrane ruffling (
      • Honda A.
      • Nogami M.
      • Yokozeki T.
      • Yamazaki M.
      • Nakamura H.
      • Watanabe H.
      • Kawamoto K.
      • Nakayama K
      • Morris A.J.
      • Frohman M.A.
      • Kanaho Y.
      ). Thus, it is of interest to determine whether PLD is involved in S1P-induced activation of the PI3K/Akt pathway and, if so, how.
      In the present study, we demonstrate that inhibition of PA accumulation and expression of a catalytically inactive mutant of PLD2 strongly inhibit EDG3-mediated stimulation of PI3K and Akt, whereas exogenous PLD alone induces stimulation of PI3K and Akt. These observations indicate a novel role for PLD in the PI3K signaling pathway.

      DISCUSSION

      PLD is activated rapidly in response to diverse extracellular stimuli, including hormones, growth factors, neurotransmitters, cytokines, antigens, and certain physical stresses (
      • Exton J.H.
      ,
      • Jones D.
      • Morgan C.
      • Cockcroft S.
      ,
      • Liscovitch M.
      • Czarny M.
      • Fiucci G.
      • Tang X.
      ,
      • Frohman M.A.
      • Sung T.C.
      • Morris A.J.
      ,
      • Meier K.E.
      • Gibbs T.C.
      • Knoepp S.M.
      • Ella K.M.
      ). The initial product of PLD, PA, is thought to serve a signaling function. However, the intracellular targets for this lipid messenger have not been clearly identified. In this study, we demonstrated that PLD stimulation is necessary for S1P-induced activation of PI3K and its downstream effector Akt, and is sufficient for inducing activation of both PI3K and Akt under certain conditions. This is the first report to indicate the involvement of PLD and PA in in vivo activation of PI3K and its effector Akt. This conclusion is based on the following three major findings: (a) S1P-induced activation of PI3K and Akt was inhibited by 1-butanol, which served as a phosphatidyl group acceptor in the PLD-catalyzed transphosphatidylation reaction to reduce the levels of PA. In contrast, 2-butanol, which did not serve as a phosphatidyl group acceptor, was ineffective; (b) S1P-induced activation of Akt was suppressed by the overexpression of a dominant negative PLD2 mutant; and (c) treatment of cells with exogenous PLDSc induced activation of PI3K and Akt, and Akt activation was completely abolished by a PI3K inhibitor, LY294002. Moreover, S1P-induced membrane ruffling, which was dependent upon PI3K (
      • Okamoto H.
      • Takuwa N.
      • Yokomizo T.
      • Sugimoto N.
      • Sakurada S.
      • Shigematsu H.
      • Takuwa Y.
      ), was abolished by 1-butanol, but not by 2-butanol. These observations point to an essential role for PLD and PA in PI3K activation induced by the G protein-coupled receptor agonist S1P.
      Many studies have indicated that S1P induces cell proliferation, suppression of apoptosis, modulation of cell motility, and cell shape changes (
      • An S.
      ,
      • Pyne S.
      • Pyne N.J.
      ,
      • Spiegel S.
      • Milstien S.
      ,
      • Chun J.
      • Contos J.J.
      • Munroe D.
      ,
      • Hla T.
      • Lee M.J.
      • Ancellin N.
      • Liu C.H.
      • Thangada S.
      • Thompson B.D.
      • Kluk M.
      ,
      • Igarashi Y.
      ,
      • Moolenaar W.H.
      ,
      • Okamoto H.
      • Takuwa N.
      • Yokomizo T.
      • Sugimoto N.
      • Sakurada S.
      • Shigematsu H.
      • Takuwa Y.
      ,
      • Takuwa Y.
      • Okamoto H.
      • Takuwa N.
      • Gonda K.
      • Sugimoto N.
      • Sakurada S.
      ). The EDG receptors for S1P, including EDG3, have been shown to mediate S1P-evoked signaling events relevant to cell proliferation and survival, including the activation of extracellular signal-regulated kinase (
      • An S.
      • Zheng Y.
      • Bleu T.
      ). We have previously demonstrated that S1P-induced PLD activation is independent of extracellular signal-regulated kinase activation in NIH3T3 cells (
      • Banno Y.
      • Fujita H.
      • Ono Y.
      • Nakashima S.
      • Ito Y.
      • Kuzumaki N.
      • Nozawa Y.
      ). In the present study, we have demonstrated that S1P-induced activation of PLD is required for activation of PI3K and Akt in EDG3-expressing CHO-K1 cells. It is widely accepted that the activation of Akt plays a pivotal role in cell survival and protection against apoptosis by phosphorylating BAD and caspase-9 and by regulating signaling via transcription factors such as the forkhead family and nuclear factor κB (
      • Data S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.R.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ,
      • Kane L.P.
      • Shapiro V.S.
      • Stokoe D.
      • Weiss A.
      ). On the other hand, it has previously been shown that PLDs are also involved in cell survival signaling events; for example, overexpression of PLD2 suppresses H2O2- and hypoxia-induced apoptosis in PC12 cells (
      • Lee S.D.
      • Lee B.D.
      • Han J.M.
      • Kim J.H.
      • Kim Y.
      • Suh P.G.
      • Ryu S.H.
      ,
      • Yamakawa H.
      • Banno Y.
      • Nakashima S.
      • Sawada M.
      • Yamada J.
      • Yoshimura S.
      • Nishimura Y.
      • Nozawa Y.
      • Sakai N.
      ). Our results reveal a novel link between PLD and the PI3K/Akt pathway in S1P-mediated survival signaling.
      There is some existing evidence indicating a functional link between PLD and PI3K. For example, PLD and PI3K are regulated by receptor tyrosine kinases. Similar to PI3K, PLD is regulated by RalA, a downstream target of Ras, in platelet-derived growth factor- and epidermal growth factor-stimulated cells (
      • Lucas L.
      • del Peso L.
      • Rodriguez P.
      • Penalva V.
      • Lacal J.C.
      ,
      • Slaaby R.
      • Jensen T.
      • Harald H.S.
      • Frohman M.A.
      • Seedorf K.
      ), and PLD2 is tyrosine-phosphorylated by forming a physical complex with the epidermal growth factor receptor (
      • Lu Z.
      • Horna A.
      • Joseph T.
      • Sukezane T.
      • Frankel P.
      • Zhong M.
      • Bychenok S.
      • Xu L.
      • Feig L.A.
      • Foster D.A.
      ). Recent studies have demonstrated that PLD1 and PI3K play a role in GLUT4 translocation between the plasma membranes and intracellular vesicles in insulin-stimulated cells (
      • Emoto M.
      • Klarlind J.K.
      • Waters S.B.
      • Hu V.
      • Buxton J.M.
      • Chawla A.
      • Czech M.P.
      ). Furthermore, several studies have demonstrated through the use of PI3K inhibitors that PI3K is involved in agonist-stimulated PLD activation (
      • Cissel D.S.
      • Fraundorfer P.F.
      • Beaven M.A.
      ,
      • Gillooly D.J.
      • Melendez A.J.
      • Hockaday A.R.
      • Harnett M.M.
      • Allen J.M.
      ,
      • Kozawa O.
      • Blume-Jensen P.
      • Heldin C.H.
      • Ronnstrand L.
      ,
      • Nakamura M.
      • Nakashima S.
      • Katagiri Y.
      • Nozawa Y.
      ). These studies suggest, as a possible underlying mechanism, that the PI3K products phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate might regulate the activity of the Ras-related low molecular mass GTPases Rho and Arf, which have been shown to be involved in the activation of PLD (
      • Exton J.H.
      ,
      • Jones D.
      • Morgan C.
      • Cockcroft S.
      ,
      • Liscovitch M.
      • Czarny M.
      • Fiucci G.
      • Tang X.
      ,
      • Frohman M.A.
      • Sung T.C.
      • Morris A.J.
      ,
      • Meier K.E.
      • Gibbs T.C.
      • Knoepp S.M.
      • Ella K.M.
      ). We have observed that Clostridium difficile toxin B, which inactivates all Rho G proteins including Rho, Rac, and Cdc42 (
      • Just L.
      • Selzer J.
      • Wilm M.
      • Von Eichel-Streiber C.
      • Mann M.
      • Aktries K.
      ), blocks all S1P-induced activation of PLD, PI3K, and Akt in EDG3- CHO-K1 cells (data not shown). Our previous study, however, demonstrated that S1P-induced Rac activation occurs downstream from PI3K activation in EDG3-CHO-K1 cells (
      • Okamoto H.
      • Takuwa N.
      • Yokomizo T.
      • Sugimoto N.
      • Sakurada S.
      • Shigematsu H.
      • Takuwa Y.
      ). In the present study, PI3K inhibitors had no effect on S1P-induced PLD activation in EDG3-CHO-K1 cells (Fig. 4 D), whereas 1-butanol inhibited S1P-induced PI3K activation (Fig. 3 B). These observations indicate that PI3K exists downstream rather than upstream of PLD in EDG3-CHO-K1 cells.
      Among the PI3Ks, two isoforms of PI3K catalytic subunits, p110α and p110β, form heterodimers with the p85/p55 adaptor subunits, whereas another catalytic subunit, p110γ, is associated with the p101 adaptor. p85/p55-associated p110α and p110β are stimulated by tyrosine kinase-coupled transmembrane receptors upon their recruitment to the plasma membrane by the assembly of phosphotyrosine-containing multimolecular complexes. Ras is also thought to participate in the activation of p110α and p110β through its direct binding to these catalytic subunits. On the other hand, p110γ and p110β have been shown to be stimulated by the heterotrimeric G proteins (
      • Rameh L.E.
      • Cantley L.C.
      ,
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.
      • Hawkins P.T.
      ). Previous studies have shown that PA and lyso-PA inhibit PI3K activity in in vitro systems, whereas these lipids activate phosphatidylinositol 4-kinase, phospholipase C, protein kinase C, and Lck tyrosine kinase under the same conditions (
      • Lauener R.
      • Shen Y.
      • Duronio V.
      • Salari H.
      ,
      • Lavie Y.
      • Agranoff B.W.
      ). On the other hand, a recent study has demonstrated that anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate, PA, and phosphatidylserine can bind to p110 (
      • Kirsch C.
      • Wetzker R.
      • Klinger R.
      ). Therefore, it is an interesting possibility that PA produced by PLD activation participates in the recruitment of PI3K to the plasma membrane in S1P-stimulated cells. In the present study, we observed stimulation of PI3K activity in anti-phosphotyrosine antibody immunoprecipitates from S1P-stimulated EDG3-CHO-K1 cells (Fig. 3), indicating that S1P-stimulated PI3K activity is associated with a phosphotyrosine-containing protein or that PI3K is directly tyrosine-phosphorylated. A number of studies have demonstrated that exogenously added and endogenously generated PA induces enhancement of tyrosine phosphorylation in neutrophils and other cell types (
      • Ohguchi K.
      • Kasai T.
      • Nozawa Y.
      ,
      • Sergeant S.
      • Waite K.A.
      • Heravi J.
      • McPhail L.C.
      ,
      • Siddiqui R.A.
      • English D.
      ). A more recent study has demonstrated that exogenous PA induces tyrosine phosphorylation of the p85 regulatory subunit of PI3K in neutrophilic leukocytes (
      • Siddiqui R.A.
      • English D.
      ). Therefore, it is tempting to speculate that PA-dependent tyrosine phosphorylation may be involved in recruitment to the plasma membrane and stimulation of the PI3K in the EDG3-mediated response to S1P. However, additional experiments are required to prove this hypothesis.

      Acknowledgments

      We thank Dr. Michael A. Frohmann for providing the plasmid encoding wild-type and catalytically inactive mutants of PLD1 and PLD2.

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