PKC-dependent Activation of Sphingosine Kinase 1 and Translocation to the Plasma Membrane

Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C6-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling. Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C6-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling. Sphingolipids are ubiquitously found in mammalian cell membranes, where they were originally thought to serve only as structural components. Subsequent studies clearly demonstrated evidence of important roles for sphingolipids and their metabolites as signaling molecules involved in such cellular processes as cell growth, differentiation, senescence, and apoptosis (1Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1510) Google Scholar, 2Hannun Y.A. Obeid L.M. Biochem. Soc. Trans. 1997; 25: 1171-1175Crossref PubMed Scopus (59) Google Scholar, 3Spiegel S. Cuvillier O. Edsall L.C. Kohama T. Menzeleev R. Olah Z. Olivera A. Pirianov G. Thomas D.M., Tu, Z. Van Brocklyn J.R. Wang F. Ann. N. Y. Acad. Sci. 1998; 845: 11-18Crossref PubMed Scopus (191) Google Scholar, 4Dickson R.C. Lester L.R. Biochim. Biophys. Acta. 1999; 1438: 305-321Crossref PubMed Scopus (131) Google Scholar). One particularly interesting sphingolipid metabolite, sphingosine-1-phosphate (S1P), 1The abbreviations used are: S1P, sphingosine-1-phosphate; SK, sphingosine kinase; hSK, human SK; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; GFP, green fluorescent protein; EGFP, enhanced GFP; EDG, endothelial differentiation gene; TNF, tumor necrosis factor. has emerged as a highly bioactive lipid implicated in both extracellular and intracellular signaling processes (5Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (667) Google Scholar). S1P has been shown to be secreted into serum from platelets; extracellular S1P, as a first messenger, binds members of the endothelial differentiation gene (EDG) receptor family (now termed S1P1, 2, 3, 4, and5) on the surface of endothelial cells, triggering such cellular processes as differentiation, migration, and mitogenesis (6Hla T. Prostaglandins. 2001; 64: 135-142Crossref PubMed Scopus (119) Google Scholar). S1P is also suggested to act as a second messenger participating in signaling cascades leading to cytoskeletal changes, motility, release of intracellular calcium stores, and protection from apoptosis (7Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar, 8Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar, 9Hobson J.P. Rosenfeldt H.M. Barak L.S. Olivera A. Poulton S. Caron M.G. Milstein S. Spiegel S. Science. 2001; 291: 1800-1803Crossref PubMed Scopus (386) Google Scholar, 10Olivera A. Kohama T. Edsall L. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (468) Google Scholar). Whether acting intracellularly or extracellularly, S1P is generated from phosphorylation of sphingosine by the action of SK. Therefore, because of the involvement of S1P in such diverse and vital cellular processes, production of S1P through increased SK cellular activity has become the subject of much interest. Mammalian cells growing in culture possess intrinsic SK activity, generating low basal levels of S1P. Meanwhile, the agonist-induced increase in SK activity results in significant increase in S1P levels, responsible for cell signaling effects. Reported agonists of SK include platelet-derived growth factor (11Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (829) Google Scholar), tumor necrosis factor-α (12Xia P. Wang L. Gamble J.R. Vadas M.A. J. Biol. Chem. 1999; 274: 34499-34505Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar,13Xia P. Gamble J.R. Rye K.-A. Wang L. Hii C.S. Cockerill P. Khew-Goodall Y. Bert A.G. Barter P.J. Vadas M.A. Proc. Natl. Acad. Sci.U. S. A. 1998; 95: 14196-14201Crossref PubMed Scopus (361) Google Scholar), nerve growth factor (14Rius R.A. Edsall L.C. Spiegel S. FEBS Lett. 1997; 417: 173-176Crossref PubMed Scopus (98) Google Scholar, 15Edsall L.C. Pirianov G.G. Spiegel S. J. Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar), muscarinic acetylcholine agonists (16Meyer zu Heringdorf D. Lass H. Alemany R. Laser K.T. Neumann E. Zhang C. Schmidt M. Rauen U. Jakobs K.H. van Koppen C.J. EMBO J. 1998; 17: 2830-2837Crossref PubMed Scopus (202) Google Scholar), serum (11Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (829) Google Scholar), and phorbol esters (17Mazurek N. Megidish T. Hakomori S. Igarashi Y. Biochem. Cell Biol. 1994; 198: 1-9Google Scholar, 18Beuhrer B.M. Bardes E.S. Bell R.M. Biochim. Biophys. Acta. 1996; 1303: 233-242Crossref PubMed Scopus (79) Google Scholar). However, the mechanisms by which SK is activated and where in the cell S1P is generated remain unknown. In this study we have addressed PMA-induced activation of SK. PMA stimulated a significant increase in SK activity associated with the membrane fraction of cell extracts. This activation was accompanied by translocation of SK to the plasma membrane and was associated with an increase in S1P secretion into the media. These results demonstrate for the first time PKC-mediated translocation of SK to the plasma membrane with a concomitant increase in secretion of S1P into the media, indicating that S1P may generate biological responses through an autocrine/paracrine signaling response. Human embryonic kidney (HEK) 293 and Hela cells were purchased from American Type Culture Collection. Eagle's minimum essential medium, high glucose Dulbecco's modified Eagle's medium, heat-inactivated fetal bovine serum, phosphate-buffered saline, LipofectAMINE 2000, and pcDNA4-myc/HIS were purchased from Invitrogen. A23187, bisindoylmaleimide, calphostin-c, 4α-phorbol-12,13-didecanoate, and phorbol-12-myristate-13-acetate (PMA) were purchased from Calbiochem. S1P, sphingosine, and C6-ceramide were purchased from Matreya, Inc. (Pleasant Gap, PA). γ-32PATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. 32Pi (10m Ci/ml) was purchased from Amersham Biosciences. 3-3Hd-erythro-sphingosine was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Restriction enzymes were purchased from Roche Molecular Biochemicals. HEK 293 cells were cultured in minimum Eagle's medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO2 incubator at 37 °C. Hela cells were cultured under similar conditions with high glucose Dulbecco's modified Eagle's medium. Cells were seeded 24 h before experimentation. Based on the complete coding sequence for human SK1 in GenBankTM (accession no.AF238083), gene-specific primers (forward, 5′-CGCCGCAGGGAATGACACC-3′, and reverse, 5′-GCCTGTCCCCCAAAGCATAC-3′) were used to amplify the entire open reading frame by PCR from human fetal kidney Marathon-ready cDNA (CLONTECH). An additional PCR reaction was required to clone the SK cDNA into theHindIII and EcoRI restriction sites of the mammalian expression vector pEGFP-C3 (CLONTECH) or pcDNA3 (Invitrogen), using the following primers: forward, 5′-TTAAAGCTTATGGATCCAGCGGGCGGC-3′, and reverse, 5′-TTTGAATTCTCATAAGGGCTCTTCGGCGGTGG-3′. Transient expression of SK was accomplished using LipofectAMINE 2000 (Invitrogen) in accordance with the manufacturer's instructions. Following 48 h of expression, transfection efficiencies were determined to be typically between 40–60%. SK activity was determined as previously described by Olivera et al. (19Olivera A. Kohama T., Tu, Z. Milstein S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Briefly, cell extracts were assayed using 1 mmd-erythro-sphingosine-bovine serum albumin (0.3%) and γ-32PATP (10 μCi, 20 mm) containing 200 mm MgCl2. Lipids were extracted and then resolved by TLC on silica gel G60 with 1-butanol/methanol/acetic acid/water (80:20:10:20, v/v). Forty-eight hours after transfection, cell medium was replaced with phosphate-free medium. Following a 2-h incubation, 1 mCi of32Pi was added to the medium. After 2 h, cells were stimulated with vehicle or 300 nm PMA for 1 h. Cells were then washed once with ice-cold phosphate-buffered saline and lysed in 500 μl of lysis buffer (50 mm Tris, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 10 mm NaF, 10 mm okadaic acid, 10 mm β-glycerol phosphate) containing protease inhibitors. Lysate was centrifuged at 14,000 × g for 15 min, and supernatant was immunoprecipitated with a polyclonal anti-GFP antibody (CLONTECH) conjugated to protein A for 4 h at 4 °C. The immunoprecipitate was then resuspended in Laemmli buffer and separated on a 4–15% SDS-polyacrylamide gel, transferred to nitrocellulose, and analyzed by autoradiography. The antibody was prepared by the Medical University of South Carolina antibody facility. Briefly, a synthetic oligopeptide corresponding to the last 20 amino acids of the C-terminal (CVEPPPSWKPQQMPPPEEPL) of the hSK1 (GenBankTM accession no. AAF73423) was conjugated to keyhole limpet hemocyanin (KLH) and injected into New Zealand White rabbits. Antiserum was affinity-purified over a cyanogen bromide-activated agarose column bound with the same oligopeptide and eluted with 100 mmglycine (pH 2.5). Blots were probed with 0.3 μg/ml preimmunized rabbit serum, 0.3 μg/ml immunized rabbit serum, immunized rabbit serum blocked with 2 μg/ml synthetic oligopeptide, or 0.2 μg/ml mouse anti-c-Myc (SC-40, Santa Cruz). Immunoreactive bands were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz) and an ECL Plus detection system (Amersham Biosciences). Forty-eight hours after transfection, cells were treated with 300 nm PMA for the indicated times. Cells were immediately washed twice with ice-cold phosphate-buffered saline and then scraped in 500 μl of lysis buffer (20 mmTris, pH7.5, 10 mm EDTA, 2 mm EGTA, mm 1 mm phenylmethylsulfonyl fluoride, 10 nm okadaic acid, 1 mm mm NaF, 1 mm Na3VO4, and 10 μg/ml and Cell were by at × g for 10 were separated into membrane and by at × g for at 4 °C. The membrane were resuspended in μl of lysis buffer containing and on for membrane were by at for 10 at 4 and the resuspended in μl of lysis A dominant negative mutation was into SK in the expression vector via (forward, and reverse, generating a of to as previously by et al. J.R. P. Gamble J.R. Vadas M.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). HEK 293 cells were culture 24 h to After 48 h, cells were under an with a and were using PerkinElmer that was for nm and nm for fluorescent Hela cells on culture were washed with phosphate-buffered saline and and for 20 in ice-cold at °C. Cells were blocked with fetal bovine serum for 20 to with μg/ml rabbit for 1 h and 10 μg/ml anti-rabbit conjugated with for 1 h, both in fetal bovine HEK 293 were seeded at 1 × mm and with 2 of as described After 48 h, the of was to 2 and cells were treated with 300 nm PMA for the indicated times. before the of the time cells were with 300 Lipids were extracted by the of and the was under Lipids were separated using in mm calcium corresponding to S1P were scraped and the using a the of the phorbol PMA to SK in HEK 293 PMA in a increase in cell SK activity of or cells into and membrane was a increase in SK activity in the fraction and a significant increase in SK activity in the membrane fraction 1 This PMA-induced increase in membrane-associated SK activity the increase in plasma membrane-associated PKC activity following PMA not the increase in membrane-associated SK activity was accompanied by SK translocation to the membrane PMA we hSK1 from human fetal kidney Marathon-ready cDNA (CLONTECH) and the with Transient of increased the cell SK activity by more over vector cells not indicating that the hSK1 was a we treated HEK 293 cells with PMA and the protein using PMA translocated to the plasma this was as early as 15 and was complete by 1 h whether translocation of by was by we a polyclonal antibody the C-terminal of hSK1 of from HEK 293 cells demonstrated a increase of in the membrane fraction in response to PMA with concomitant in the fraction and a significant in the membrane fraction 3, SK was previously shown to be as well as to be associated with the H.M. J.P. M. Olivera A. Nava Milstein S. Spiegel S. J. Scopus Google Scholar). that the cytoskeletal of SK to the plasma membrane in response to of in the membrane HEK 293 cells with pcDNA3 2 and or 3, and were to with a rabbit polyclonal detection of hSK1 which was blocked by with the oligopeptide 4, and HEK 293 cells were with or and treated in the or of 300 nm PMA for 1 h. were then into and membrane extracts as described under of fraction were to using the were analyzed by are with with the of this highly we the of endogenous SK the cell in response to PMA. the Hela cell that of endogenous SK for significant levels of SK were found to to the plasma membrane in response to PMA, similar to the effects with the protein These results demonstrate for the first time that SK to the plasma membrane in response to PMA PMA is a known of PKC, leading to phosphorylation of involved in whether SK was a of PKC, cells hSK1 were with in the of PMA or more a increase in with PMA as with the cells PKC activity using as a were not the role of PKC in mediating SK translocation, we the effects of the PKC inhibitors (bisindoylmaleimide and and the indirect inhibitor C6-ceramide on PMA-induced translocation of to the plasma membrane. of HEK 293 cells with of PKC PMA or induced translocation of to the plasma membrane. of preincubation with of the inhibitors to 1 h of with PMA translocation to the plasma membrane Therefore, results demonstrate that PMA-induced SK translocation to the plasma membrane is on activation of of PKC on SK translocation in HEK 293 nm phorbol 300 nm 300 nm 300 nm the and of In at 300 cells were results were by in a the and of In at 300 cells were results were by whether the activity of the SK was necessary for translocation, we a that a mutation in accordance with by al. J.R. P. Gamble J.R. Vadas M.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Transient expression of this in HEK 293 cells not in in basal activity of SK, that Importantly, this abrogated PMA-induced of SK activity, that as a dominant negative PMA of cells clearly induced translocation of the to the plasma membrane SK activity is not required for PMA-induced translocation to the plasma membrane. has been shown that of results in release of S1P, is not known S1P is whether PMA regulation of SK was accompanied by S1P release into the media. such a was to the of S1P in response to PMA. HEK 293 cells were treated with PMA by with for 10 PMA induced only a increase in the of into intracellular S1P as with cells A and In was a 4-fold increase in S1P release into the from cells as with cells and PKC inhibitors (bisindoylmaleimide and the intracellular of S1P and S1P release into the These that PMA-induced activation and translocation of SK to the plasma membrane are accompanied by S1P release into the in a where then cell surface receptors. In this study, we have demonstrated that PMA a significant increase in membrane-associated SK activity, and this increase is accompanied by a translocation of SK to the plasma membrane. In we have shown that PMA phosphorylation of SK. PKC inhibitors SK translocation, indicating that this is mediated by SK activity is not required for its translocation, as shown by the of a dominant negative to to the plasma membrane. SK activation and translocation by PMA in a 4-fold increase in S1P secretion into the media. a novel mechanism of SK activation and for the first time that this translocation is accompanied by release of S1P into the media. SK has been a protein that is generating basal levels of S1P the In SK is also thought to be a regulated protein in which of cells with of a of known SK results in a increase in S1P over the basal levels, a cell signaling leading to diverse biological processes calcium cell growth, differentiation, motility, and (6Hla T. Prostaglandins. 2001; 64: 135-142Crossref PubMed Scopus (119) Google Scholar, M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar, 8Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar, 9Hobson J.P. Rosenfeldt H.M. Barak L.S. Olivera A. Poulton S. Caron M.G. Milstein S. Spiegel S. Science. 2001; 291: 1800-1803Crossref PubMed Scopus (386) Google Scholar, 10Olivera A. Kohama T. Edsall L. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (468) Google Scholar). is in the and from this study that agonists are to SK activity the cell, is not this activation where S1P is or S1P cell signaling cascades that such diverse biological we have demonstrated for the first time that PMA-induced activation of SK1 leads to PKC-mediated translocation of SK to the plasma membrane and SK1 phosphorylation and is accompanied by secretion of S1P into extracellular media. a role for agonist-induced translocation of SK to the membrane. In a study using platelet-derived growth Rosenfeldt et al. H.M. J.P. M. Olivera A. Nava Milstein S. Spiegel S. J. Scopus Google demonstrated an of SK at membrane that S1P production in through to receptors. agonists SK also be mediated by translocation of SK. In this was in and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that of in cells was accompanied by SK translocation to the plasma membrane. The results that PKC activation may a mechanism for translocation of SK to the plasma membrane in response to many that that SK is in response to PMA the that SK is a of this we on this because in studies on SK phosphorylation by PKC are Whether phosphorylation of SK is required for its translocation also is to be These studies are under in mechanism by which SK be found in the of the cell membrane was suggested in a by Ancellin et al. N. N. S. G. Hla T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in which SK was shown to be extracellularly. However, in that study was of of that In by et mechanism of SK regulation was suggested in studies to its This a signaling by which SK with a of the receptor signaling and SK activity, leading to activation of and protection from apoptosis P. Wang L. N. F. Gamble J.R. Vadas M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies that SK activation in the of the plasma membrane may be a for to its Whether the for SK, sphingosine, is at the plasma membrane to be studies that and are activated in response to the same that SK (11Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (829) Google Scholar, E. M. S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, N. M. S. S. A. R. T. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, F. Obeid L.M. Y.A. FEBS Lett. 2001; PubMed Scopus Google Scholar, Y. D. S. Biol. Cell. 2001; PubMed Scopus Google Scholar), the that sphingosine be generated in or to the plasma membrane concomitant with SK These studies are with S1P generated in a for secretion of the into a mechanism of S1P release from a study we previously L.C. C. C. J. Y.A. Obeid L.M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), that the receptor was to S1P, its to biological activity through receptors. One a of of S1P through or through known or not by which the cell may S1P in response to SK agonists to generate biological activity through receptors. In we a novel mechanism by which PMA SK and translocation to the plasma membrane in a This translocation is accompanied by secretion of S1P into the media, autocrine/paracrine signaling.