Cellular Signalling 21 (2009) 675–684

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Cellular Signalling

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The sphingosine 1-phosphate receptor 5 and sphingosine kinases 1 and 2 arelocalised in centrosomes: Possible role in regulating cell division

Laura Gillies a, Sue Chin Lee a, Jaclyn S. Long a, Nicholas Ktistakis a, Nigel J. Pyne b,⁎, Susan Pyne a

a Cell Biology Group, SIPBS, University of Strathclyde, 27 Taylor St, Glasgow, G4 0NR, UKb Department of Signaling, Babraham Institute, Cambridge, CB2 4AT, UK

⁎ Corresponding author. Tel.: +44 141 5482659; fax: +E-mail address: n.j.pyne@strath.ac.uk (N.J. Pyne).

0898-6568/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.cellsig.2009.01.023

a b s t r a c t

a r t i c l e i n f o

Article history:

We show here that the end Received 28 November 2008Received in revised form 3 January 2009Accepted 4 January 2009Available online 12 January 2009

Keywords:Sphingosine 1-phosphateCentrosomalMitosisSphingosine kinaseSphingosine 1-phosphate receptor 5

ogenous sphingosine 1-phosphate 5 receptor (S1P5, a G protein coupled receptor(GPCR) whose natural ligand is sphingosine 1-phosphate (S1P)) and sphingosine kinases 1 and 2 (SK1 andSK2), which catalyse formation of S1P, are co-localised in the centrosome of mammalian cells, where theymay participate in regulating mitosis. The centrosome is a site for active GTP–GDP cycling involving the G-protein, Gi and tubulin, which are required for spindle pole organization and force generation during celldivision. Therefore, the presence of S1P5 (which normally functions as a plasma membrane guaninenucleotide exchange factor, GEF) and sphingosine kinases in the centrosome might suggest that S1P5 mayfunction as a ligand activated GEF in regulating G-protein-dependent spindle formation and mitosis. Theaddition of S1P to cells inhibits trafficking of S1P5 to the centrosome, suggesting a dynamic shuttlingendocytic mechanism controlled by ligand occupancy of cell surface receptor. We therefore propose that thecentrosomal S1P5 receptor might function as an intracellular target of S1P linked to regulation of mitosis.

© 2009 Elsevier Inc. All rights reserved.

1. Introduction

The centrosome is considered to be a multi-platform scaffold for anumber of signaling pathways involved in regulating cell cycleprogression and checkpoint control [1,2]. The centrosome is also thesite of active GTP–GDP cycling essential for spindle force generation,spindle pole organization and the regulation of microtubule dynamicsduring mitosis [3,4]. Genetic studies in C. elegans and D. melanogasterhave identified the key components regulating GTP–GDP cyclingduring asymmetric cell division as being a non-GPCR guaninenucleotide exchange factor (GEF), termed RIC-8; a guanine nucleotidedissociation inhibitor (GDI), termed GPR; Giα and a GTPase accelerat-ing protein (GAP), RGS7 [3–5]. These components were found toregulate the pulling forces associated with the correct positioning andorientation of the mitotic spindle during asymmetric cell division.According to the current model, RIC-8 induces guanine nucleotideexchange of Giα-GDP (GOA-1/GPA-16 in C. elegans) in complex with aGDI (GPR1/2 in C. elegans, Pins in D. melanogaster) which thenassociates with an RGS (RGS7). RGS7 accelerates the intrinsic GαGTPase via its GAP activity. RIC-8 is unable to induce guaninenucleotide exchange in G protein heterotrimers and thus, theassociation of GPR may serve to exclude Gβγ from the complex [6].In vitro studies of mammalian RIC-8 indicate that this GEF mayregulate the activity of the nuclear mitotic apparatus protein, NuMA

44 141 5522562.

l rights reserved.

by altering protein–protein interactions. When presented with acomplex of NuMA-LGN-Giα-GDP, RIC-8 promotes formation of GTP-Giα causing the dissociation of the complex and the release of NuMA.Tall and Gilman [7] suggest that the dynamic release of NuMA fromthe LGN-Giα complex allows it to activate downstream effectors suchas dynein and dynactin to generate spindle forces during asymmetriccell division. Considering that RIC-8 is unable to induce guaninenucleotide exchange in heterotrimeric G-proteins [6], the questionremains as to how free Giα becomes available to NuMA or LGN in thefirst place. Indeed Oxford and Webb [8] have suggested that an initialround of GEF/RGS activity is required to split heterotrimeric Giα andthereby allow the Go-Loco motif of LGN to access isolated Giα-GDPand present it to RIC-8 for the second round of GEF activity.

In addition to modulating pulling forces on microtubules byinteracting with motor protein complexes, G-proteins have also beenshown to modulate microtubule dynamics directly in vitro. Forinstance, Gα stimulates tubulin GTPase and increases microtubuledynamics in the absence of GTP or can enhance microtubulepolymerisation when GTP levels are elevated [9]. On the other hand,Gβγ promotes tubulin polymerisation and stabilizes microtubules viaa prenylation-dependent process [10]. siRNA knock down of Gαsubunits in nematode zygotes results in anterior and posteriormicrotubules with equal stability whereas in the wild type, anteriormicrotubules reside longer at the cell cortex than posterior micro-tubules indicating that Gα subunits also regulate microtubuledynamics in vivo [11]. The regulation of microtubule dynamics byGDP-GTP cycling requires heterotrimeric G-protein activation regard-less of whether the heterotrimer is bound to tubulin [12]. Therefore it

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is possible that a GPCR might function as a GEF in the centrosome toactivate G-protein to modulate microtubule dynamics.

Sphingosine 1-phosphate (S1P) binds to a family of five GPCRstermed S1Pn (where n=1–5) [13,14], that are differentially coupled toheterotrimeric G-proteins (Gi, Gq and G12/13) to regulate variouseffectors, such as MAP kinases, linked to diverse cellular processessuch as proliferation, cell survival and differentiation. Our immuno-fluorescence studies on the sub-cellular distribution of endogenousS1P receptors indicates that S1P5 is localised in the centrosome ofmammalian cells where it might function as a GEF to regulate Giα-mediated microtubule and spindle dynamics. This study describesthese findings and defines regulatory mechanisms controlling S1P5trafficking to the centrosome.

2. Materials and methods

2.1. Materials

All biochemicals were obtained from the Sigma Chemical Company(Poole, UK) unless otherwise stated. Cell culture supplies andLipofectamine 2000 were obtained from Invitrogen (Paisley, UK).S1P and D-erythro-sphingosine were purchased from Avanti PolarLipids (Alabaster, USA), G418 from Merck Biosciences Ltd (Notting-ham, UK) and Vectashield® with DAPI from Axxora (UK) Ltd(Nottingham, UK). Antibodies used were from suppliers as follows:anti-S1P5, anti-β tubulin and anti-γ-tubulin were from Santa CruzBiotechnology Inc. (Santa Cruz, USA); anti-ERK-2 from BD Biosciences(Oxford, UK); anti-SK1 and anti-SK2 from Abgent (San Diego, USA);anti-GFP from Axxora (UK) Ltd. (Nottingham, UK). [Methyl-3H]Thymidine (25 Ci/mmol) was purchased from GE Healthcare (LittleChalfont, UK). siRNA hSIP5 and hS1P2 were a gift from Dr ViswanathanNatarajan (University of Chicago). DharmaFECT™ 2 was purchasedfrom Thermo Fisher Scientific Ltd (Loughborough, UK).

2.2. Cell culture

Human embryonic kidney 293 (HEK 293) cells were maintained inminimumessentialmedium(MEM) supplementedwithessential aminoacids, penicillin (50 U/ml), streptomycin (50 µg/ml) and 10% (v/v) foetalcalf serum (FCS) whereas stable over-expressing GFP-tagged wild typeSK1 or GFP-tagged PA-binding deficient SK1mutant HEK 293 cells weremaintained in Dulbecco's modified Eagle medium (DMEM) supplemen-ted with 10% (v/v) FCS and 800 µg/ml G418. Mouse embryonicfibroblasts (MEF) and PC12 cells were maintained in DMEM supple-mentedwith penicillin (50U/ml), streptomycin (50 µg/ml) and 10% (v/v)FCS. In all cases, cells were quiesced in serum free medium for 24 hbefore experimentation.

2.3. Plasmids

Wild type murine SK1 was cloned into pcDNA4/myc-His (Invitro-gen) and sub-cloned into eGFP-C2 (Clontech) to produce plasmidsencoding an N-terminal GFP-tagged (GFP-SK1) form of SK1 [15].Similarly, standard PCR techniques were used to sub-clone S1P5 fromS1P5-pcDNA3 (a gift from Kevin Lynch, University of Virginia, USA) intoeGFP-C2 (to encode an N-terminal GFP-tagged form of S1P5, myc-S1P5). Site-directed mutagenesis was used to generate a phosphatidic

Fig.1. Centrosomal localisation of S1P5 inHEK 293 cells andMEF. (a) Immunofluorescencemicro(detected with anti-S1P5 antibody and FITC-conjugated secondary antibody, green) with endogconjugated secondary antibody, red) in HEK 293 cells. Nuclei were stained with DAPI (blue). CoInset:western blot analysis ofHEK293 cell lysatesusing anti-S1P5 antibody. ImmunofluorescencGFP-S1P5 and endogenous β-tubulin (detectedwith anti-β tubulin antibody and TRITC-conjugaGFP antibody and showing expression of GFP-S1P5; (b) Immunofluorescence microscopy imagsiRNA S1P5 (100 nM) and the lack of effect of siRNA S1P2 (200 nM) on centrosomal S1P5 expressinot with siRNA S1P2. Neither siRNA altered the expression level of ERK-2. Scale bar=50 µm. Pl

acid (PA)-binding deficient form of GFP-tagged SK1 (GFP-(PA-)SK1;Ktistakis et al. unpublished).

2.4. Transfection

Briefly, cells were grown to 70% confluence and transfected for 24 hwith the relevant plasmid construct (or vector, in controls) usingLipofectamine 2000 in 1% (v/v) FCS containing medium, followed by24 h in serum free medium. For the siRNA experiments, cell weregrown to 50% confluence, transfected for 48 h with siRNA (100 nM)using DharmaFECT™ 2 in 10% (v/v) FCS containing medium, asrecommended by the manufacturer, followed by 24 h in serum freemedium. Mock transfected cells were treated with DharmaFECT™ 2alone.

2.5. Immunoblotting

Analysis of proteins by SDS-PAGE and western blotting wasperformed as previously described by us [16].

2.6. Immunofluorescence microscopy

Cells were plated onto autoclaved 13 mm glass coverslips andgrown to 60% confluence before stimulation as required. Cells werefixed/permeabilised with methanol at −20 °C for 5 min beforeincubation in blocking solution (5% FCS, 1% bovine serum albumin(BSA), in phosphate-buffered saline (PBS)) for 30 min at roomtemperature. Coverslips were then incubated with primary antibody(1:100 dilution in blocking solution), as required, overnight at 4 °C.Coverslips were washed with PBS and incubated with FITC- orTRITC-conjugated anti-mouse IgG or anti-rabbit IgG secondaryantibody (1:100 dilution in blocking solution), as appropriate, atroom temperature for 1 h. Coverslips were washed in PBS, excessmoisture removed with tissue and mounted on glass slides usingVectashield® hard set mounting medium with DAPI. Immunofluor-escence was visualised using a Nikon (Surrey, UK) E600 epifluores-cence microscope.

2.7. Immunoprecipitation

Cells were grown to confluency on 140 mm diameter petri dishes,washed with ice cold PBS and harvested into 1 ml of immunopreci-pitation buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2,1% (v/v) NP40, 10% (v/v) glycerol, 20 mM Tris pH 8, 1 mg/ml BSA,0.5mMNa3VO4, 0.2mMPMSF,10 µg/ml leupeptin,10 µg/ml aprotinin)supplementedwith 20 µM Taxol to promotemicrotubule stabilisation/de-polymerisation and shaken on ice for 30 min. Lysates wereharvested, homogenized using a 23 gauge needle and syringe andcentrifuged at 21,000 g for 10min at 4 °C to remove cellular debris. Thesupernatants were pre-cleared by mixing with protein A or Gsepharose beads, as appropriate, for 20 min at 4 °C and the beadsdiscarded. Primary antibody (0.4–0.5 µg) was added to the super-natants and mixed at 4 °C for 1 h followed by the addition of protein Aor G sepharose beads, as appropriate, and mixing at 4 °C for a furtherhour. The immunoprecipitated protein complex was harvested bycentrifugation at 21,000 g for 15 s and washed with buffer A (10 mMHEPES pH 7, 100 mM NaCl 0.2 mM PMSF, 0.5% (v/v) NP40) followed by

scopy images demonstrating the centrosomal co-localisation (yellow) of endogenous S1P5enous β-tubulin or γ-tubulin (detectedwith respective anti-tubulin antibodies and TRITC-ntrol images show cells treated with TRITC or FITC conjugated secondary antibodies alone.emicroscopy images are also includeddemonstrating the co-localisation of over-expressedted secondary antibody, red) inMEF. Inset: western blot analysis of MEF lysates using anti-es demonstrating the absence of centrosomal S1P5 in HEK 293 cells after treatment withon/localisation. Thewestern blots confirm reduced expression of S1P5with siRNA S1P5, butease refer to the online version for colour images.

Fig. 2. Centrosomal localisation of SK1 and SK2 in HEK 293 cells. Immunofluorescence microscopy images demonstrating the centrosomal co-localisation (yellow) of endogenous(a) SK1 and (b) SK2 (detected with anti-SK1 or anti-SK2 antibody and FITC-conjugated secondary antibody, green) with endogenous β-tubulin or γ-tubulin (detected with respectiveanti-tubulin antibodies and TRITC-conjugated secondary antibody, red) in HEK 293 cells. Nuclei were stainedwith DAPI (blue). Inset shows co-immunoprecipitation of γ-tubulinwithSK1 using respective antibodies. ABO and GBO signify no protein A or protein G sepharose beads; (c) Western blot and SK1 activity measurements of HEK 293 cells stably GFP-SK1 orGFP-(PA-)-SK1. Also shown are immunofluorescence microscopy images demonstrating the centrosomal localisation of GFP-SK1 and GFP-(PA-)-SK1. Scale bar=50 µm;(d) Immunofluorescence image showing the co-localisation of S1P5 with GFP-SK1 or GFP-(PA-)-SK1. Scale bar=10 µm; (e) Western blot analysis of fractions from a discontinuoussucrose density gradient using HEK 293 cells over-expressing GFP-SK1 showing the presence of GFP-SK1, S1P5 and γ-tubulin in centrosomal fractions (CL, cell lysate); (f) Anti-GFPantibody immunoprecipitation analysis of fractions from a discontinuous sucrose density gradient using HEK 293 cells over-expressing GFP-(PA-)-SK1, western blotted with anti-GFP,γ-tubulin and S1P5 antibodies. Inset: immunoprecipitation using centrosomes with and without anti-GFP antibody; (g) Immunoprecipitation of HEK 293 cell lysates using anti-β- orγ-tubulin antibodies and western blotting analysis with anti-S1P5 antibody. Please refer to the online version for colour images.

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Fig. 2. (continued).

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wash buffer B (10 mM HEPES pH 7, 100 mM NaCl, 0.2 mM PMSF).Beads were re-suspended in 4× sample buffer (20% (v/v) glycerol, 10%(w/v) SDS, 250 mM Tris, pH 6.5, 0.0001% bromophenol blue, 10% (v/v)β-mercaptoethanol) and boiled for 3 min. Samples were centrifugedat 21,000 g for 15 s prior to analysis by SDS-PAGE andwestern blotting.A similar method, with minor modifications, was applied forimmunoprecipitation of protein complexes from centrosomes (seebelow).

2.8. Discontinuous sucrose gradient fractionation and centrosomepreparation

Serum starved confluent cells from 4×175 cm2flasks were

combined to generate one individual sample for centrosomeisolation. Cells were incubated with serum free media containing10 µg/ml nocodazole for 2 h at 37 °C to depolarize actin andmicrotubule filaments. Where indicated, cells were incubated withS1P (5 µM), serum (10%, v/v) or both for 15 min. All subsequent stepswere performed at 4 °C. Cells were harvested in ice-cold 10× TBS(10 mM Tris pH 7.4, 150 mMNaCl) and centrifuged at 280 g for 7 min.The low speed pellet was washed with ice-cold 1× TBS with 8% (w/v)sucrose and centrifuged at 280 g for 7 min. The pellet was re-suspended using a 23 gauge needle and syringe in 1 ml of lysis buffer(1 mM HEPES, pH 7.2, 0.5% NP40, 0.5 mM MgCl2, 0.1% (v/v) β-mercaptoethanol, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/mlaprotinin, and 1mM PMSF) and samples normalised for protein. Eachsamplewas transferred to a 14ml ultracentrifuge tube towhich 13mlof lysis buffer was added. The lysate was centrifuged (SW40Ti rotor)at 250 g for 10 min to remove swollen nuclei, chromatin aggregatesand unlysed cells. The supernatant was filtered through a 50 µmnylon membrane followed by the addition of HEPES buffer andDNase1 to a final concentration of 10 mM and 2 U/ml, respectively,and incubated on ice for 30 min. The mixture was then laid over a1ml 60% sucrose cushion (10mMPIPES pH 7.2, 0.1% Triton X-100, and0.1% (v/v) β-mercaptoethanol containing 60% (w/v) sucrose) andcentrifuged at 1000 g for 30 min to sediment centrosomes onto thecushion. The upper 12 ml was removed and the remaining 2 mlcollected using a syringe and loaded onto a discontinuous sucrose

gradient consisting of 70, 50, and 40% (w/v) solutions and spun at110,000 g for 2 h. 1 ml fractions were collected from the top (topfraction=fraction 14) and each was diluted with 1 ml PIPES (10 mMpH 7.2) and mixed under rotary agitation for 15 min at 4 °C.Centrosomes were collected by centrifugation at 21,000 g for 10 min.The centrosomes were denatured in sample buffer (62.5 mM Tris,pH6.8,10% (v/v) glycerol, 2% (w/v) SDS,1.4% (v/v) β-mercaptoethanol,0.001% (w/v) bromophenol blue) and stored at −80 °C.

In experiments where protein complexes were immunoprecipi-tated from centrosomes, the centrosomeswere re-suspended in 150 µlimmunoprecipitation buffer and mixed under rotary agitation for15 min at 4 °C prior to pre-clearing (using protein G sepharose beads),immunoprecipitation using anti-GFP antibody (2 µg), washing andaddition of 4× sample buffer/boiling as described above.

2.9. Centrosomal adherent nuclei preparation

PC12 cells were fractionated using the Fraction-PREP cell fractio-nation system (Biovision, Mountain View, USA).

2.10. Sphingosine kinase assay

Cell lysates were prepared in sphingosine kinase (SK) assay buffer(20 mM Tris pH 7.4, 1 mM EDTA, 1 mM Na3VO4, 40 mM β-glycerophosphate, 1 mM NaF, 10 µg/ml soya bean trypsin inhibitor,10 µg/ml aprotinin, 1 mM PMSF, 0.5 mM 4-deoxypyridoxine, 0.007%(v/v) β-mercaptoethanol, 20% (v/v) glycerol) [15,16]. 15 µg proteinwascombined with 50 µM sphingosine, 250 µM ATP and 250 µMphosphatidylserine in a 200 µl assay in the presence of Triton X-100(0.06%) and [γ-32P] ATP (1 µCi, 44400 dpm/nmol) for 15 min at 30 °C.The resulting [32P]S1P was extracted using n-butanol, which waswashed twice using 2 M KCl, before quantification of radioactivity andcalculation of SK activity.

2.11. DNA synthesis

[3H] thymidine (GE Healthcare, Chalfont St Giles, UK) incorpora-tion assays were performed as previously described by us [17].

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3. Results

3.1. S1P5 is localised in the centrosome of HEK 293 cells

Westernblot analysis of HEK293 cell lysateswith anti-S1P5 antibodydetected a single major protein band with Mr=42 kDa, whichcorresponds with the molecular mass of S1P5 (Fig. 1a). Immunofluor-escent analysis ofHEK293 cellswas therefore performed to establish thesub-cellular distribution of S1P5. Significant anti-S1P5 immunoreactivitywas found to be localised to a tightly focussed area adjacent to thenucleus (Fig.1a). This distribution is typical of a centrosomal localisationand indeed, S1P5 is co-localised with β-tubulin and γ-tubulin, both ofwhich were detected with specific antibodies (Fig. 1a, see yellowimmunofluorescence in merged panels). γ-tubulin is a defined markerof the centrosome and forms a ring complex which is a nucleating sitefor spindle formation. No such staining was detected when secondaryFITC- or TRITC-conjugated secondary antibodies were used alone(Fig. 1a). Antibody independent validation of the centrosomal localisa-tion of S1P5 was demonstrated by results showing that over-expressionof GFP-S1P5 inMEFresulted in significant co-localisation of recombinantS1P5 with β-tubulin in the centrosome (Fig. 1a). GFP alone did notlocalise to the centrosome but was present in both the nucleus andcytoplasm (Fig. 1a).

To confirm that the anti-S1P5 immunostaining was due to thepresence of endogenous S1P5, HEK 293 cells were treated with siRNA

Fig. 3. Centrosomal localisation of S1P5 and SK1/SK2 in MEF. (a) Western blot of MEF limmunofluorescence microscopy image demonstrating the centrosomal localisation of S1P5 (d(b)Western blot analysis of centrosomal fractions purified fromMEF probedwith anti-S1P5 antreduces S1P5 in the centrosome; (c) Immunofluorescencemicroscopy images demonstrating thantibody and FITC-conjugated secondary antibody, green) and anti-γ-tubulin (detected with r(d) Immunoprecipitation of MEF lysates using anti-S1P5 antibody and western blotting analysi

S1P5 (100 nM), which resulted in a significant knock down of 42 kDaS1P5 as assessed by western blot analysis (Fig. 1b). The treatment ofcells with siRNA S1P5 also eliminated the anti-S1P5 immunostainingin the centrosome (Fig. 1b). There was no effect on the total cellularlevels of ERK-2, indicating that there was no detrimental effect of thesiRNA treatment on cell viability (Fig. 1b). As an additional control, weused siRNA directed against S1P2, which had no effect on S1P5expression levels and did not reduce anti-S1P5 immunoreactivity inthe centrosome (Fig. 1b).

3.2. SK1 and SK2 are localised in the centrosome of HEK 293 cells

Having provided evidence that the S1P5 receptor is localised in thecentrosome, we assessed whether the sphingosine kinases, SK1 andSK2, which synthesise S1P from sphingosine are co-localised withS1P5. The localisation of these enzymes in the centrosome mightsuggest a S1P generating system that could potentially provide ligandfor the activation of S1P5. In this respect, immunofluorescence cellimaging demonstrated the presence of endogenous SK1 and SK2 inthe centrosome, which were co-localised with β- and/or γ-tubulin inHEK 293 cells (Fig. 2a, b). Moreover, γ-tubulin forms a complex withSK1 as evidenced by immunoprecipitation with respective antibodies(Fig. 2b).

Additional evidence to support the centrosomal localisation of SK1was obtained using stable over-expressing GFP-SK1 HEK 293 cells

ysate using anti-S1P5 antibody demonstrating expression of S1P5. Also shown is anetected with anti-S1P5 antibody and FITC-conjugated secondary antibody, green) in MEF;ibody demonstrating that S1P (5 µM) but not serum (10%, v/v) treatment ofMEF for 15mine centrosomal localisation of endogenous SK1 and SK2 (detectedwith anti-SK1 or anti-SK2espective anti-tubulin antibodies and TRITC-conjugated secondary antibody, red) in MEF;s with anti-SK1 antibody. Please refer to the online version for colour images.

Fig. 4.Regulationof the centrosomal localisationof S1P5 inPC12,MEFandHEK293cells. (a)Immunofluorescence microscopy image demonstrating the centrosomal localisation ofendogenous S1P5 (detected with anti-S1P5 antibody and FITC-conjugated secondaryantibody, green; nuclei are stained with DAPI (blue)) in PC12 cells. Also shown is westernblot analysis of PC12 cells using anti-S1P5 antibody; (b) Western blot analysis (using anti-S1P5 antibody) of isolated nuclei with adherent centrosomes (NC) and high-speed pelletcontaining theplasmamembrane (P) demonstrating that S1P (5 µM) butnot serum (10%, v/v) treatment of PC12 for 10 min reduces S1P5 levels in centrosome adherent nuclei;(c) Immunofluorescence microscopy images demonstrating the effect of leptomycinB (5ng/ml, 20h) treatmentofHEK293 cells on the centrosomal/nuclear localisationof S1P5(detected with anti-S1P5 antibody and FITC-conjugated secondary antibody, green; nucleiare stainedwith anti-PCNAantibodyand TRITC-conjugated secondaryantibody, red). Scalebar=50 µm. Please refer to the online version for colour images.

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(~50% transfectants). Western blot analysis and activity measure-ments of cell lysates confirmed the over-expression of GFP-SK1 in HEK293 cells (Fig. 2c). Over-expressed GFP-SK1 exhibited a diffusecytoplasmic localisation and was concentrated in centrosomes,where it co-localised with γ-tubulin, detected using immunofluores-cence with anti-GFP antibody (Fig. 2c). We have previously demon-strated that SK1 contains a PA binding domain in the C-terminalregion of the protein [15]. Mutant enzyme that was deficient in PAbinding (GFP-(PA-)-SK1 (Ktistakis et al. unpublished) is catalyticallyinactive (Fig. 2c). Stable expression of GFP-(PA-)-SK1 mutant was alsolocalised in the centrosome of HEK 293 cells (Fig. 2c) and can thereforebe used as a potential dominant negative to interrogate thecentrosomal function of endogenous SK1 and to characterise interac-tion with other regulatory proteins. GFP-SK1 and GFP-(PA-)-SK1 wasalso co-localised with endogenous S1P5 in the centrosome (Fig. 2d).GFP-SK1 (Fig. 2e) and GFP-(PA-)-SK1 (data not shown) was isolated incentrosomal fractions (identified by the presence of γ-tubulin) thathad been purified from nocodazole-treated HEK 293 cells (seeMaterials and methods). These centrosomal fractions also containedendogenous S1P5 (Fig. 2e). The molecular mass of the S1P5 receptor inthese fractions was 42 kDa indicating that the native full length S1P5receptor is localised in the centrosome. Immunoprecipitation analysisusing centrosomal fractions (prepared from HEK 293 cells over-expressing GFP-(PA-)-SK1) and anti-GFP antibody, demonstrated thatγ-tubulin and 42 kDa S1P5 can be co-immunoprecipitated with GFP-(PA-)-SK1, suggesting that these proteins might be arranged in acomplex in the centrosome (Fig. 2f). No GFP-SK1 or S1P5 was detectedin immunoprecipitates when anti-GFP antibody was omitted from theimmunoprecipitation procedure. The amount of γ-tubulin was alsosignificantly reduced by omission of anti-GFP antibody (Fig. 2f).Identical results were obtained withWT GFP-SK1 (data not shown). Inaddition, we found that endogenous centrosomal S1P5 appears toform a complex with endogenous γ-tubulin, as assessed by immuno-precipitation analysis of HEK 293 cell lysates using anti-γ tubulinantibody (Fig. 2g).

3.3. Regulation of the centrosomal localisation of S1P5 by S1P in MEF andPC12 cells

We found that there was significant constitutive centrosomallocalisation of S1P5 in HEK 293 cells. We therefore, looked in other celltypes to establish whether there was (a) a centrosomal localisation ofS1P5 and (b) a mechanism by which trafficking to the centrosomemight be regulated by extrinsic factors. For this purpose we used MEFand PC12 cells. Western blot analysis of MEF probed with anti-S1P5antibody detected one major band with Mr=42 kDa (Fig. 3a)corresponding to S1P5 and which was localised in the centrosomedetected by immunofluorescence. We therefore prepared centro-somes by discontinuous sucrose density gradient centrifugation andaddressed directly the question of how trafficking is regulated. In thisrespect, we found that stimulation of MEFwith serum had no effect onthe centrosomal localisation of S1P5 (Fig. 3b). However, there was anapparent reduction in the amount of centrosomal S1P5 in MEF treatedwith S1P or serum plus S1P (Fig. 3b). MEF exhibit similarities with HEK293 cells, in as much as there is marked centrosomal localisation ofendogenous SK1 and SK2 (Fig. 3c), and that endogenous S1P5 and SK1appear to form a complex as evidenced by immunoprecipitationanalysis with anti-S1P5 antibody (Fig. 3d).

We also found that the S1P5 receptor was localised in thecentrosome of PC12 cells (Fig. 4a). Western blot analysis of PC12 celllysates with anti-S1P5 antibody detected a single immunoreactivepolypeptide with Mr=42 kDa (Fig. 4a). During the course of thesestudies, we found that purified nuclei (N) contain adherent centro-somes, as judged by western blotting of isolated nuclei with anti-γtubulin antibody (data not shown). We therefore used isolated nucleias a convenient and simple method for assessing the amount of S1P5

localised in the centrosome adherent nuclei (NC). This allowedcomparison with the presence of S1P5 in the pellet (P, containingplasma membrane) fractions. In this regard, we found that thetreatment of PC12 cells with serum had no apparent effect on thedistribution of S1P5 between nuclear adherent centrosomes andmembranes, while treatment of cells with S1P markedly reduced thecentrosomal localisation of S1P5, while increasing the amount ofreceptor in membranes (Fig. 4b).

We also considered the possibility that S1P5 might traffic betweenthe nucleus and centrosome. This was based upon the fact that humanS1P5 contains a nuclear localisation sequence in the third cytoplasmicloop. Indeed, S1P5 is localised to the nucleus, upon treatment of HEK293 cells with leptomycin B, which inhibits nuclear export of proteins(Fig. 4c). Significant amounts of endogenous receptor remained in thecentrosome suggesting that it was not merely sequestered to thenucleus to inhibit the centrosomal function of the receptor.

3.4. Localisation of S1P5 and SK1 in dividing cells

A key question is what happens to S1P5 and SK1 during celldivision. We noticed when examining cells in culture, even underquiescent conditions, that some cells were undergoing mitosis andthat endogenous S1P5 receptor and SK1 were present in duplicatedcentrosomes (e.g. in HEK 293 cells and MEF, Fig. 5). Indeed, both S1P5

Fig. 6. Effect of over-expression of catalytically active and inactive form of SK1 on DNAsynthesis. Histograms showing the effect of GFP-SK1 or catalytically inactive GFP-(PA-)-SK1 on basal and serum-stimulated (10% v/v, 20 h) DNA synthesis in HEK 293 cells andMEF. Open bars are control cells, filled bars are serum-stimulated cells. ⁎pb0.05 serumversus unstimulated control in vector transfected cells; ⁎⁎pb0.05 versus relevantvector-transfected, unstimulated or serum-stimulated sample indicating significantlydecreased basal and serum-stimulated DNA synthesis for WT and (PA-)-SK1 cellscompared with basal and serum stimulated DNA synthesis in vector transfected cells. Inall cases, charcoal stripped serum was used to ensure removal of the potential effect ofserum borne S1P.

Fig. 5. Localisation of S1P5 and SK1 during mitosis of HEK 293 cells and MEF. Upper panel -immunofluorescence microscopy images demonstrating the presence of S1P5 (detectedwith anti-S1P5 antibody and FITC-conjugated secondary antibody, green) in duplicated centrosomes (detected with anti-γ-tubulin antibody and TRITC-conjugated secondaryantibody, red) during mitosis of HEK 293 cells and MEF. Lower panel—immunofluorescence microscopy images demonstrating the presence of SK1 (detected with anti-SK1 antibodyand FITC-conjugated secondary antibody, green) in duplicated centrosomes during mitosis of MEF. Nuclei were stained with DAPI (blue). Scale bar=50 µm. Please refer to the onlineversion for colour images.

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and SK1 could be clearly observed associated with the spindlestructures that radiate from the centrosome poles and which co-stained with γ-tubulin (Fig. 5).

3.5. Effect of catalytically inactive SK1 on DNA synthesis in MEF and HEK293 cells

The effect of SK1 on DNA synthesis was assessed based on therationale that the centrosomal cycle and replication is closely coupledwith DNA synthesis. For this purpose we used MEF and HEK 293 cellsin which WT GFP-SK1 or catalytically inactive GFP-(PA-)-SK1 wasexpressed. Over-expression of WT GFP-SK1 or catalytically inactiveGFP-(PA-)-SK1 reduced DNA synthesis (Fig. 6). This reduction in DNAsynthesis with the WT and PA binding deficient kinase inactive SK1mutant was correlated with the centrosomal localisation of theseproteins in HEK 293 cells (see Fig. 2c) and suggests a possible role forS1P in desensitising centrosomal S1P5 receptor action with WT SK1and perturbation of endogenous SK1 function by competition forcentrosomal binding sites with inactive GFP-(PA-)-SK1.

4. Discussion

We have demonstrated for the first time that S1P5 is localised inthe centrosome of HEK 293 cells, PC12 cells and MEF. This raises thepossibility of a role for S1P5 as a GEF in the regulation of G-protein-dependent spindle formation andmitosis. Until now, the predominantview is that GEF activity in the centrosome is uniquely regulated byRIC-8, which operates outside the GPCR paradigm. RIC-8 interactswith GPR1/2 GoLocomotifs to regulate Gα. In this regard, GPR1/2 maypresent Gβγ free Gα.GDP to RIC-8, thereby enabling co-operativefunctioning. However, in addition to Gα.GTP functioning as an activespecies in regulating spindle organization, a role for Gβγ is alsoevident [9,10]. Moreover, RIC-8 is unable to induce G-proteinheterotrimeric subunit dissociation. Therefore, an initial round ofGEF activity may be required in order to induce G-protein dissociation.

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Indeed, Roychowdhury and colleagues have recently shown thatheterotrimeric G protein dissociation is a pre-requisite for functionalcoupling between Giα1/Gβγ and tubulin/microtubule organization invitro [12]. These findings therefore suggest the requirement for a GPCRGEF activity or an unidentified GEF or a Gβγ dissociating factor.Therefore, the localisation of S1P5 in the centrosome identifies thisreceptor as a potential GEF for the first round of GDP-GTP exchange inGi. Indeed, the demonstration that S1P5 forms a complex with γ-tubulin supports, albeit indirectly, a role in nucleating microtubulesrequired for spindle formation. The localisation of S1P5 in thecentrosome raises the question of how this receptor could be switchedon to promote GEF activity. In this regard, we demonstrate here thatboth SK1 and SK2, the enzymes that catalyse the formation of S1Pfrom sphingosine, are also localised in the centrosome and that SK1forms a complex with S1P5 and γ-tubulin. This suggests that a selfgenerating system for S1P production might maintain centrosomalS1P5 receptor signaling to promote mitosis.

We have also demonstrated that the amount of S1P5 in thecentrosome is reduced, while membrane S1P5 is increased bytreatment of cells with S1P. These findings suggest that the traffickingof S1P5 to the centrosome might be limiting for any potential effect ofintracellular S1P in the centrosome. Therefore, it is possible that S1Pbinding to S1P5 traps the receptor at the plasma membrane or divertsit to a different endocytic signaling route involving recycling to theplasma membrane or lysosomal degradation. Interestingly, the cellsurface S1P5 receptor has been implicated in inhibiting proliferationvia a tyrosine phosphatase-dependent pathway [18]. Therefore, thefunctioning of cell surface and centrosomal S1P5 might be opposingand regulated in a concerted manner.

Human S1P5 also contains a nuclear localisation sequence in itsthird cytoplasmic loop, suggesting that it might be localised to thenucleus. Indeed, others have reported nuclear localisation of a relatedS1P receptor termed S1P1 in T-cells [19]. In the current study we havedemonstrated that treatment of cells with leptomycin B increases thenuclear localisation of S1P5, although S1P5 is still localised in thecentrosome. These data might suggest shuttling of S1P5 between thenucleus and centrosome as has been observed for other proteins suchas NUMA. However, it is also possible that the localisation of S1P5 incentrosome and nucleus are mutually exclusive and that S1P5 trafficsto the nucleus via a distinct route and from an alternative plasma-membrane pool of S1P5.

Our findings raise an important question as to how S1P5 canmove tothe centrosome and bemaintained within a hydrophobic environment.In this regard, Hannun and colleagues have recently demonstrated anovel endosomal route of PKCα trafficking to the centrosome [20]. Itremains to bedeterminedwhether S1P5 canuse a similar route. In termsof maintenance of S1P5 in the centrosome, this would require a lipidenvironment. Indeed, fluorescent analogues of sphingomyelin, acomponent of lipid rafts [21] and a precursor of sphingosine, havebeen reported to translocate to the centrosome and co-localise withcentrioles [22]. When the microtubule function was disrupted in cellsusing nocodazole, the fluorescently labeled sphingomyelin analogue nolonger localised to the centrosome but remained in lipid vesicles in thecytoplasm, suggesting that the lipid may have been transported alongmicrotubules to the centrosome [22]. The presence of sphingomyelinand ceramide [23] in the centrosome not only suggests the presence oflipid rafts capable of supporting S1P5 signaling as part of a vesiclestructure but also the potential for the generation of sphingosine in thecentrosome which could then be phosphorylated by SK1/2 in order toactivate the S1P5 receptor. In addition it has also been reported that thelipid raft micro-domain-associated protein reggie-1/flotillin-2 is loca-lised in the centrosome in peripheral blood mononuclear cells [24].Interestingly, it has also been reported that Gβγ can promote thepolymerisation of microtubules in vitro in a prenylation dependentmanner [10]. Thus, the prenyl group on Gβγ might facilitate anchorageto a possible lipid membrane in the centrosome.

Notably, both isoforms of SK were detected in the centrosome,which raises the possibility of redundancy of function. Indeed, partialredundancy does exist for SK1 and SK2 since SK1−/− or SK2−/− mice arephenotypically normal whereas elimination of both genes is embryo-nic lethal [25,26]. However, SK1 has been implicated in regulating cellproliferation and is activated by many different agonists such asantigen, platelet derived growth factor, nerve growth factor andtumour necrosis factor α via post-translational modification whereasSK2, at the endoplasmic reticulum, has been implicated in apoptosis(reviewed by [27,28]). Mutation of critical amino-acids in the BH3domain of SK2 prevents its interaction with anti-apoptotic Bcl-xL andreduces SK2-induced apoptosis, suggesting that sequestration of Bcl-xL by SK2 prevents its anti-apoptotic function [29]. However, the sub-cellular localisation of SK and, therefore, the site of S1P productionmight determine functional role. For example, targeting SK1 to theendoplasmic reticulum resulted in apoptosis [30] whereasmutation ofthe nuclear localisation sequence of SK2 prevented its nuclear entryand inhibition of DNA synthesis [31]. A number of regulatorymechanisms affecting SK1 re-distribution have been identifiedincluding phosphorylation by ERK-1/2, association with calcium/calmodulin and interaction with phosphatidylserine and PA ([15] andreviewed by [27,28]). Additionally, SK1 can be activated by protein–protein interaction, e.g. with the monomeric GTPase eEF1A [32],which is involved in protein synthesis, actin bundling, cell cycleregulation and apoptosis. Similarly, SK2 is activated by eEF1A [32] andcan be phosphorylated by ERK-1 [33].

In conclusion, the findings of the current study provide the firstevidence for a potential centrosomal function of the S1P5 receptor,SK1 and/or SK2. Therefore, S1P5 might function as a GEF for the Gi

heterotrimer to produce Giα-GTP and Gβγ subunits in thecentrosome. S1P5 might therefore function to initiate G-protein-dependent regulation of tubulin polymerisation/de-polymerisationdynamics and microtubule formation required for mitosis. S1P isunusual in that it exhibits both extracellular and intracellularactions, the latter related to mobilization of ER-dependent calciumpools and regulation of cell proliferation [34,35]. Although theformal identification of S1P receptors has provided a physiologicalcontext for the action of extracellular S1P, the identification ofintracellular S1P targets has been lacking. We therefore proposethat centrosomal S1P5 might be an intracellular target of S1P,although this requires further experimentation.

Acknowledgement

This work was supported by BBSRC (BBS/S/K/2004/11257) and TheSynergy Fund.

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