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Phosphatidylinositol 3-Kinase/AKT Pathway Regulates the Endoplasmic Reticulum to Golgi Traffic of Ceramide in Glioma Cells

2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês

10.1074/jbc.m808934200

ISSN

1083-351X

Autores

Paola Giussani, L. Brioschi, Rosaria Bassi, Laura Riboni, Paola Viani,

Tópico(s)

Calcium signaling and nucleotide metabolism

Resumo

Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER. Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER. Gliomas are the most frequent brain tumors derived from the transformation of glial cells or their precursors (1Singh S.K. Hawkins C. Clarke I.D. Squire J.A. Bayani J. Hide T. Henkelman R.M. Cusimano M.D. Dirks P.B. Nature. 2004; 432: 396-401Crossref PubMed Scopus (5995) Google Scholar, 2Vescovi A.L. Galli R. Reynolds B.A. Nat. Rev. Cancer. 2006; 6: 425-436Crossref PubMed Scopus (817) Google Scholar, 3Kondo T. Eur. J. Cancer. 2006; 42: 1237-1242Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The World Health Organization classifies gliomas according to their cellular features and their grade of malignancy (from I to IV) (4Kleihues P. Louis D.N. Scheithauer B.W. Rorke L.B. Reifenberger G. Burger P.C. Cavenee W.K. J. Neuropathol. Exp. Neurol. 2002; 61: 215-225Crossref PubMed Scopus (1546) Google Scholar). Glioblastoma multiforme (GBM), 3The abbreviations used are: GBM, glioblastoma multiforme; ER, endoplasmic reticulum; PI3K, phosphatidylinositol-3-OH kinase; PIP2, phosphatidylinositol(4,5)P2; PIP3, phosphatidylinositol(3,4,5)P3; PH, pleckstrin homology; PTEN, phosphatase and tensin homologue; Cer, ceramide; Wm, wortmannin; BFA, brefeldin A; BSA, bovine serum albumin; CERT, ceramide transfer protein; [3H]Sph, d-erythro-[3-3H]sphingosine; [14C]SM, [choline-methyl-14C]sphingomyelin; [3H]C6-Cer, N-[hexanoyl-6-3H]d-erythro-hexanoyl-sphingosine; HPTLC, high performance thin layer chromatography; NBD-C6-Cer, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) sphingosine; FAPP, four phosphate adaptor protein; EGF-FAPP1-PH, PH domain of FAPP1 protein fused with enhanced green fluorescent protein; DN-AKT, dominant negative mutant of Akt; PBS, phosphate-buffered saline; siRNA, small interfering RNA; SMS, sphingomyelin synthase; GCS, glucosylceramide synthase; SMase, sphingomyelinase; PI4K, phosphatidylinositol 4-OH kinase; PI4P, phosphatidylinositol 4-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin; SREBP, sterol regulatory element-binding protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; N-SMase, neutral SMase; A-SMase, acidic SMase.3The abbreviations used are: GBM, glioblastoma multiforme; ER, endoplasmic reticulum; PI3K, phosphatidylinositol-3-OH kinase; PIP2, phosphatidylinositol(4,5)P2; PIP3, phosphatidylinositol(3,4,5)P3; PH, pleckstrin homology; PTEN, phosphatase and tensin homologue; Cer, ceramide; Wm, wortmannin; BFA, brefeldin A; BSA, bovine serum albumin; CERT, ceramide transfer protein; [3H]Sph, d-erythro-[3-3H]sphingosine; [14C]SM, [choline-methyl-14C]sphingomyelin; [3H]C6-Cer, N-[hexanoyl-6-3H]d-erythro-hexanoyl-sphingosine; HPTLC, high performance thin layer chromatography; NBD-C6-Cer, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) sphingosine; FAPP, four phosphate adaptor protein; EGF-FAPP1-PH, PH domain of FAPP1 protein fused with enhanced green fluorescent protein; DN-AKT, dominant negative mutant of Akt; PBS, phosphate-buffered saline; siRNA, small interfering RNA; SMS, sphingomyelin synthase; GCS, glucosylceramide synthase; SMase, sphingomyelinase; PI4K, phosphatidylinositol 4-OH kinase; PI4P, phosphatidylinositol 4-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin; SREBP, sterol regulatory element-binding protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; N-SMase, neutral SMase; A-SMase, acidic SMase. also referred to as grade IV astrocytoma, is the most frequent class of malignant primary brain tumor and one of the most aggressive forms of cancer. There is a poor prognosis for patients with GBM, the median survival being 9–12 months, and this does not significantly improve even after aggressive treatment with multiple approaches, including surgery, chemotherapy, and radiotherapy (5Davis F.G. McCarthy B.J. Expert Rev. Anticancer Ther. 2001; 1: 395-401Crossref PubMed Scopus (106) Google Scholar). This poor survival rate seems to be mainly due to the marked invasiveness and proliferation of GBM, as well as to their apoptotic resistance and aberrant angiogenesis. These malignancy features could be related to the varying mutations frequently found in GBM tumors that impact different key pathways involved in the control of cell proliferation, survival, differentiation, migration, and DNA repair (6Rao R.D. James C.D. Semin. Oncol. 2004; 3: 595-604Crossref Scopus (71) Google Scholar, 7Guha A. Mukherjee J. Curr. Opin. Neurol. 2004; 17: 655-662Crossref PubMed Scopus (41) Google Scholar, 8Brantley E.C. Benveniste E.N. Mol. Cancer Res. 2008; 6: 675-684Crossref PubMed Scopus (184) Google Scholar, 9Hegi M.E. Liu L. Herman J.G. Stupp R. Wick W. Weller M. Mehta M.P. Gilbert M.R. J. Clin. Oncol. 2008; 26: 4189-4199Crossref PubMed Scopus (626) Google Scholar).Among the disregulated signaling pathways in GBMs, PI3K-Akt-PTEN has been identified as being an important oncogenic pathway (10Vogt P.K. Trends Mol. Med. 2001; 7: 482-484Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 11Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. Cancer Res. 2000; 60: 3504-3513PubMed Google Scholar, 12Knobbe C.B. Merlo A. Reifemberger G. Neuro-Oncol. 2002; 4: 196-211Crossref PubMed Google Scholar). PI3Ks are members of a unique and conserved family of lipid kinases that phosphorylate the 3′-hydroxyl group of phosphatidylinositol and phosphoinositides such as phosphatidylinositol(4,5)P2 (PIP2), which is converted to phosphatidylinositol(3,4,5)P3 (PIP3). The activation of PI3Ks can occur through growth factor receptors, G-protein-coupled receptors, cell adhesion molecules, and oncogenes such as Ras. The generated polyphosphoinositides bind to the pleckstrin homology (PH) domain of different proteins, including PDK1 (3′-phosphoinositide-dependent protein kinase 1) and the serine/threonine kinase Akt, thus facilitating the interaction between the two proteins and the activation of Akt by PDK1. Cell levels of phosphoinositides are controlled not only by PI3K but also by the tumor suppressor PTEN. which is a PIP3 phosphatase. PTEN negatively regulates the PI3K/Akt pathway by converting PIP3 back to PIP2. The activation of Akt promotes cell growth and survival and inhibition of apoptosis and also regulates glucose and lipid metabolism at both the transcriptional and post-transcriptional levels; furthermore, Akt activation can exert some control over cell movement and vesicle trafficking within cells (13Engelman J.A. Luo J. Cantley L.C. 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Morente M. Egia A. Blazquez C. Garcia S. Giroux V. Malicet C. Villuendas R. Gironella M. González-Feria L. Piris M.A. Iovanna J.L. Guzman M. Velasco G. Cancer Cell. 2006; 9: 301-312Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). This suggests that the modulation of Cer levels in the ER/Golgi compartment can be crucial to glioma cell decision toward survival or death. This study investigates the role of the PI3K-Akt-PTEN pathway in the regulation of the Cer metabolism in C6 glioma cells; this is a cell line in which the PI3K/Akt pathway is constitutively activated due to the lack of PTEN expression (42Kubiatowski T. Jang T. Lachyankar M.B. Salmonsen R. Nabi R.R. Quesenberry P.J. Litofsky N.S. Ross A.H. Recht L.D. J. Neurosurg. 2001; 95: 480-488Crossref PubMed Scopus (103) Google Scholar). We show that the PI3K/Akt pathway is capable of regulating the Cer metabolism and its levels in ER by promoting the biosynthesis of complex sphingolipids acting on the vesicular ER to Golgi transport of Cer.EXPERIMENTAL PROCEDURESMaterials—All reagents were of analytical grade as follows: Dulbecco's modified Eagle's medium (DMEM), wortmannin (Wm), brefeldin A (BFA), bovine serum albumin (BSA), fatty acid-free BSA, and G418 were purchased from Sigma. Fetal calf serum (FCS) was from Cambrex (Walkersville, MD). LY294002 and LY303511 were from Cayman Chemical (Ann Arbor, MI). The antibodies recognizing phospho-Akt (Ser-473) were from Cell Signaling Technology, Inc. (Danvers, MA); polyclonal antibodies against Cer transfer protein (CERT) were from Bethyl Laboratories (Montgomery, TX). Lipofectamine 2000 and the Stealth RNAi were from Invitrogen. Complete Mini Protease inhibitor mixture tablets were from Roche Diagnostics. d-Erythro-[3-3H]sphingosine ([3H]Sph) (21.2 Ci/mmol) and [choline-methyl-14C]sphingomyelin ([14C]SM) (52 mCi/mmol) were from PerkinElmer Life Sciences; N-[hexanoyl-6-3H] d-erythro-hexanoylsphingosine ([3H]C6-Cer) (60 Ci/mmol) was from ARC (St. Louis, MO). High performance thin layer chromatography (HPTLC) silica gel plates were from Merck. 6-((N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) sphingosine (NBD-C6-Cer) and BODIPY-C5-Cer were from Invitrogen. The plasmid encoding the dominant negative form of Akt (Thr-308 and Ser-473 mutated to Ala) was kindly provided by Prof. Stefanie Dimmeler, Department of Internal Medicine III, University of Frankfurt, Frankfurt, Germany. The plasmid encoding for the PH domain of four phosphate adaptor protein 1 (FAPP1) fused with enhanced green fluorescent protein (EGFP-FAPP1-PH) was kindly provided by Dr. Maria Antonietta De Matteis, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy.Cell Culture—The C6 glioma cell line was obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia (Brescia, Italy). Cells were routinely maintained in DMEM supplemented with 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an atmosphere of 5% CO2 and 95% humidified air.Plasmids and Transfection—C6 glioma cells were plated in 60-mm cell culture dishes and grown in DMEM supplemented with 10% FCS until they were 50–70% confluent. Then cells were transfected with expression plasmid encoding the dominant negative mutant of Akt (DN-AKT) or pcDNA3.1 empty vector using the Lipofectamine 2000 reagent according to the manufacturer's directions. Stable transfectants for DN-AKT and vector were selected in a 2–3-week period using G418 antibiotic (0.5 g/liter).PH Domain Binding Assay—C6 glioma cells plated on coverslips at 1.5 × 104 cell/cm2 were grown for 24 h in DMEM plus 10% FCS. Cells were transfected with 4 μg of plasmid encoding for the EGFP-FAPP1-PH domain using Lipofectamine 2000 reagent in accordance with the manufacturer's protocol. After 24 h the cells were treated for 1.5 h with or without 0.5 μm Wm or 5 μm Wm or 20 μm LY294002 at 37 °C. The cells were then washed (three times with PBS) and fixed with 0.5% glutaraldehyde solution in PBS for 10 min at 4 °C. The specimens were observed and analyzed with a fluorescence microscope (Olympus BX-50) equipped with a fast high resolution charge-coupled device camera (Colorview 12) and an image analytical software (analysis from Soft Imaging System GmbH).RNA Interference—Small interfering RNA (siRNA) duplexes for rat CERT (GenBank™ accession number XM 345143.1) S87, S522, and control nontargeting siRNAs (scrambled sequences of S87 and S522 oligonucleotides) described in Ref. 43Giussani P. Colleoni T. Brioschi L. Bassi R. Hanada K. Tettamanti G. Riboni L. Viani P. Biochim. Biophys. Acta. 2008; 1781: 40-51PubMed Google Scholar were used. C6 glioma cells plated at 3 × 104 cell/cm2 were maintained 24 h in DMEM plus 10% FCS and then transfected in the same medium with a 1:1 (by mol) mix of S87 + S522 (si-CERT) or the nontargeting corresponding sequences (si-control) using Lipofectamine 2000 according to the manufacturer's protocol. All the experiments were performed 72 h after transfection.Immunoblotting—Phospho-Akt immunoblotting were performed using C6 glioma cells lysed with Akt buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 10 mm sodium fluoride, 1 mm EDTA, 10 mm Na4P2O7, 1 mm Na3VO4, and the protease inhibitor mixture). Solubilized proteins were centrifuged at 14,000 × g at 4 °C for 10 min. Supernatants were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in Tris-buffered saline (10 mm Tris-HCl, pH 7.4, 140 mm NaCl) containing 0.1% Tween 20 (TBS-T) and 5% skim milk, and then incubated with primary antibodies against phospho-Akt overnight at 4 °C. Membranes were washed in TBS-T, and bound antibodies were visualized with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) and chemiluminescent substrate. The relative intensities of bands were quantified by densitometry. CERT immunoblotting was performed using si-control and si-CERT transfected cells lysed with CERT buffer (10 mm Tris-HCl, pH 7.4, 0.25 mm sucrose, 0.5 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin), processed, and analyzed as described previously (43Giussani P. Colleoni T. Brioschi L. Bassi R. Hanada K. Tettamanti G. Riboni L. Viani P. Biochim. Biophys. Acta. 2008; 1781: 40-51PubMed Google Scholar).[3H]Sphingosine and N-[3H]Hexanoyl-sphingosine Metabolism—C6 glioma cells plated at 1.5 × 104 cell/cm2 were grown 48 h in DMEM plus 10% FCS. Cells, preincubated for 30 min with 0.5 μm Wm, 20 μm LY294002, or 20 μm LY303511, in the presence or absence of 1 μg/ml BFA, were pulsed with [3H]Sph (0.3 μCi/ml) or [3H]C6-Cer (0.6 μCi/ml) for 30 min or 1 h maintaining the pretreatment conditions. si-control and si-CERT cells, preincubated for 30 min with 0.5 μm Wm or 20 μm LY294002, were pulsed with [3H]Sph (0.3 μCi/ml) for 1 h maintaining the pretreatment conditions. Stable transfectants for DN-AKT and vector were pulsed with [3H]Sph (0.3 μCi/ml) for 1 h. All experiments were performed at 37 °C. Stock solutions of [3H]Sph or [3H]C6-Cer in absolute ethanol were prepared and added to fresh medium. In all cases the final concentration of ethanol never exceeded 0.1% (v/v). At the end of pulse, cells were washed twice with PBS at 4 °C, harvested, and submitted to lipid extraction and partitioning as described previously (44Riboni L. Viani P. Tettamanti G. Methods Enzymol. 2000; 311: 656-682Crossref PubMed Scopus (37) Google Scholar). The methanolized organic phase and the aqueous phase were analyzed by HPTLC using chloroform/methanol/water (55:20:3 by volume) and butanol/acetic acid/water (3:1:1 by volume) as solvent system, respectively. Digital autoradiography of HPTLC plates was performed with Beta-Imager 2000 (Biospace, France), and the radioactivity associated with individual lipids was measured using the software provided with the instrument. The 3H-labeled sphingolipids were recognized and identified as described previously (44Riboni L. Viani P. Tettamanti G. Methods Enzymol. 2000; 311: 656-682Crossref PubMed Scopus (37) Google Scholar).In Vitro Enzyme Assays—Sphingomyelin synthase (SMS), glucosylceramide synthase (GCS), and sphingomyelinase (SMase) activities were assayed using as enzyme source a homogenate of the control (untreated cells), 0.5 μm Wm-, or 20 μm LY294002-treated cells. SMS activity was assayed as described previously (41Viani P. Giussani P. Brioschi L. Bassi R. Anelli V. Tettamanti G. Riboni L. J. Biol. Chem. 2003; 278: 9592-9601Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), with minor modifications. The incubation mixture contained 50 mm Tris-Cl, pH 7.4, 25 mm KCl, 0.5 mm EDTA, 2 nmol (0.05 μCi) of [3H]C6-Cer (as 1:1 complex with fatty acid-free BSA), and 20 μg of cell protein in a final volume of 50 μl. After 15 min of incubation at 37 °C, the reaction was stopped by adding 150 μl of chloroform/methanol (1:2, by volume) at 4 °C. GCS activity was assayed as described previously (41Viani P. Giussani P. Brioschi L. Bassi R. Anelli V. Tettamanti G. Riboni L. J. Biol. Chem. 2003; 278: 9592-9601Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) with minor modifications. The incubation mixture contained 50 mm Tris-Cl, pH 7.4, 25 mm KCl, 10 mm MnCl2, 5 mm UDP-Glc, 2 nmol (0.05 μCi) of [3H]C6-Cer (as 1:1 complex with fatty acid-free BSA), and 15 μg of cell protein in a final volume of 50 μl. After 15 min of incubation at 37 °C, the reaction was stopped by adding 150 μl of chloroform/methanol (1:2, by volume) at 4 °C. In all cases, after lipid extraction and phase separation, the [3H]lipids were resolved by HPTLC as described previously (41Viani P. Giussani P. Brioschi L. Bassi R. Anelli V. Tettamanti G. Riboni L. J. Biol. Chem. 2003; 278: 9592-9601Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Mg2+-dependent neutral SMase (N-SMase) and acidic SMase (A-SMase) were assayed according to Ref. 41Viani P. Giussani P. Brioschi L. Bassi R. Anelli V. Tettamanti G. Riboni L. J. Biol. Chem. 2003; 278: 9592-9601Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar with minor modifications. The N-SMase incubation mixture contained 20 mm Tris-Cl, pH 7.4, 10 mm MgCl2, 0.1% Triton X-100, 250 μm [14C]SM (0.1 μCi), and 5–20 μg of cell protein in a final volume of 25 μl. The A-SMase incubation mixture contained 200 mm acetate buffer (pH 5.0), 10 mm EDTA, 0.1% Triton X-100, 250 μm [14C]SM (0.1 μCi), and 2–10 μg of cell protein in a final volume of 25 μl. After 30 min of incubation at 37 °C, the reactions were stopped by adding 100 μl of chloroform/methanol (2:1, by volume) at 4 °C; the radioactivity associated with the aqueous phase represented by [14C]phosphocholine was measured.Analysis of the Intracellular Distribution of Fluorescent Ceramides—C6 glioma cells or stable transfectants for DN-AKT and vector were plated at 1.5 × 104 cells/cm2 and grown on a glass coverslip for 48 h in DMEM plus 10% FCS. The cells were then loaded with 5 μm BODIPY-C5-Cer or NBD-C6-Cer (1:1 complex with fatty acid free BSA) as described previously (43Giussani P. Colleoni T. Brioschi L. Bassi R. Hanada K. Tettamanti G. Riboni L. Viani P. Biochim. Biophys. Acta. 2008; 1781: 40-51PubMed Google Scholar). After loading, the cells were incubated 30 min at 37 °C in DMEM plus 10% FCS with or without 0.5 μm Wm or 20 μm LY294002 or 20 μm LY303511. The cells were then washed (three times with PBS) and fixed with 0.5% glutaraldehyde solution in PBS for 10 min at 4 °C. The specimens were immediately observed and analyzed with a fluorescence microscope (Olympus BX-50), as described above.Other Methods—Total protein amount was assayed with the Coomassie Blue-based reagent (Pierce), using BSA fraction V as standard. Radioactivity was measured by liquid scintillation counting. Statistical significance of differences was determined by the Student's t test.RESULTSEffect of PI3K/Akt on [3H]Sph Metabolism—To investigate the effect of PI3K/Akt on Cer metabolism in C6 glioma cells, we first utilized the chemical inhibition of PI3K. As the commonly used PI3K inhibitors Wm and LY294002 can also inhibit type III PI4Ks, although at higher doses (45Balla A. Balla T. Trends Cell Biol. 2006; 16: 351-361Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), and it has been shown that PI4KIIIβ plays a role in CERT-mediated Cer metabolism (46Toth B. Balla A. Ma H. Knight Z.A. Shokat K.M. Balla T. J. Biol. Chem. 2006; 281: 36369-36377Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), we initially set up working concentrations to specifically inhibit PI3K. To do this we evaluated the effect of different concentrations of Wm and LY294002 on PI4K activity by staining cells with the EGFP-FAPP1-PH domain, which specifically interacts with phosphatidylinositol 4-phosphate (PI4P) (47Godi A. Di Campli A. Konstantakopoulos A. Di Tullio G. Alessi D.R. Kular G.S. Daniele T. Marra P. Lucocq J.M. De Matteis M.A. Nat. Cell Biol. 2004; 6: 393-404Crossref PubMed Scopus (436) Google Scholar). As shown in Fig. 1, the EGFP-FAPP1-PH domain in the control cells is almost exclusively present in the perinuclear region, representative of the Golgi complex, supporting the preferential localization of PI4P in this subcellular compartment (48Cockcroft S. De Mat

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