1

UV-C Induces Raft-associated Acid Sphingomyelinase and JNK Activationand Translocation Independently on a Nuclear Signal*

Alexandra Charruyer,* Solène Grazide,* Christine Bezombes,* Sabina Müller,* Guy Laurent,*†

and Jean-Pierre Jaffrézou*‡

From *INSERM U563-CPTP, CHU Purpan, 31024 and †Service d' Hématologie, CHU Purpan, 31059Toulouse, France

Running Title: UV-C signaling.‡Addresse correspondence: to Jean-Pierre Jaffrézou at INSERM U563-CPTP, CHU Purpan, Toulouse,France, Tel. (33) 5 62 74 45 62; Fax (33). 5 62 74 45 58;E-Mail: jean-pierre.jaffrezou@toulouse.inserm.fr.

The initiation of UV-inducedsignaling in mammalian cells is largelyconsidered to be subsequent to DNAdamage. Several studies have also describedceramide (CER), a lipid second messenger,as a major contributor in mediating UV-induced c-jun N-terminal kinase (JNK)activation and cell death. It is demonstratedhere that UV-C irradiation of U937 cellsresults in the activation and translocation ofa Zn2+-independent acid sphingomyelinaseresulting in CER accumulation in raftmicrodomains. These CER-enriched raftsaggregate and play a functional role in JNKactivation. The observation that UV-C alsoinduced CER generation andexternalization of A-SMase and JNK inhuman platelets conclusively rules out theinvolvement of a nuclear signal generatedby DNA damage in the initiation of a UVresponse, which is generated at the plasmamembrane.

Mammalian cells respond to UV irradiationby activating a complex signaling networkwhich implies radical oxygen species (ROS)production, activation of transcription factors,and stimulation of kinases (1). Among these,the c-jun N-terminal kinase (JNK), a mainregulator of AP-1 transcription factor, isconsidered as one of the most criticalcomponents of the UV response. Indeed, theJNK/AP-1 pathway has been implicated invarious UV effects, depending on the cellularmodel, including tumor promotion (2),apoptosis (3), or cell cycle arrest (4).Therefore, it is not surprising that thecharacterization of the signal transductionpathway leading to JNK activation haveattracted a great deal of attention. From thesestudies, ceramide (CER), a lipid second

messenger, has emerged as a major contributorin mediating UV-induced JNK activation (5).Further studies have confirmed the generalfunction of CER in stress-activated JNKactivation and mediating cell death (6).

The mechanism by which CER is producedupon UV activation has been investigated.Hitherto, two main metabolic pathways havebeen identified for CER accumulation:hydrolysis from sphingomyelin (SM) throughsphingomyelinase (SMase) stimulation and denovo synthesis by CER synthase activation.The latter appears not to be involved in UV-induced CER production. Indeed, UV-A, -B,and -C induce in most cellular models SMhydrolysis due to SMase stimulation (7)although, in human keratinocytes, it has beendescribed a third, non-enzymatic mechanism ofCER formation (8). Despite somecontroversies, it appears that both neutralSMase (N-SMase) and acid SMase (A-SMase)have been implied in UV-induced CERproduction and apoptosis depending on thecellular origin and experimental conditions (9,10). However, the functional role of A-SMase,but not N-SMase, in UV-induced JNKactivation has been established (11).

Although A-SMase stimulation appears tobe a critical upstream event for JNK activation,which enzyme and how it operates followingUV irradiation has not been determined. TheA-SMase gene encodes for at least two formsof A-SMase produced by post-translationalprocessing, a lysosomal form (L-A-SMase)which is lacking in Niemann-Pick disease, anda so-called secretory form (S-A-SMase)identified by Schissel and colleagues (12). S-A-SMase targets the plasma membrane,requires exogenous Zn2+ for activity, and hasbeen involved in the cellular response toinflammatory cytokines (13). Finally, a third

JBC Papers in Press. Published on March 11, 2005 as Manuscript M412867200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

2

form of Zn2+-independent A-SMase hasrecently been identified which is present on thecell membrane surface of CD95 or CD40stimulated cells (14). Finally, it remains to bedetermined what role if any does UV-C-mediated DNA damage and hence a potentialintranuclear signaling cascade in initiating A-SMase activation.

Based on these considerations, wehypothesized that UV-irradiation may inducethe activation and the translocation of a Zn2+-independent A-SMase independently of anuclear signal, resulting in CER formation inraft microdomains, and that CER-enrichedmembrane platform formation plays a role inJNK activation.

MATERIALS AND METHODS

Drugs and Reagents - Silica gel 60 thin-layer chromatography plates were from Merck(Darmstadt, Germany). Aquasafe 300scintillation cocktail was purchased from theEG&G (Evry, France). SR33557 was kindlyprovided by Dr JM Herbert (Sanofi-Synthélabo, Toulouse, France) and maltose-binding protein (MBP)-lysenin by Dr. T.Kobayashi (Lipid Biology Laboratory, RIKEN,Saitama, Japan.) All other drugs and reagents,unless specified, were purchased from SigmaChemical Co. (St Louis, MO) or AlexisBiochemicals (Paris, France).

Cell Culture - The human myeloblastic cellline U937 obtained from the ATCC (Rockville,MD) was cultured in RPMI 1640 medium at37°C in 5% CO2. Culture medium wassupplemented with 10% heat-inactivated fetalcalf serum (FCS), 2mM glutamine, 100units/ml penicillin, and 100 µg/mlstreptomycin (all from Eurobio, les Ulis ,France). Human blood platelet concentrateswere obtained from the local blood bank(Centre Régional de Transfusion Sanguine,Toulouse, France). Normal human lymphoblastcell or Niemann-Pick disease lymphoblastMS1418 were a generous gift from Pr. TLevade (INSERM U466, CHU Rangueil,Toulouse, France).

Cell Irradiation - U937 cells wereirradiated with UV-C light (254 nm) in PBSduring 30 seconds corresponding to 30joules/m2, at a concentration of 1 million cells/ml.

Sphingomyelin Hydrolysis - SMquantitation was performed by labeling cells to

isotopic equilibrium with 0.5 µCi/ml of[methyl-3H]choline (81.0 Ci/mM, AmershamPharmacia Biotech, Orsay, France) for 48 h incomplete medium. Cells were then washed andresuspended in complete medium for kineticexperiments. Radiolabeled SM was extractedand quantified by scintillation counting aspreviously described (15, 16).

Metabolic Cell Labeling and Quantitationof Ceramide - Total cellular CER wasperformed by labeling cells to isotopicequilibrium with 1 µCi/ml of [9, 10-3H]palmitic acid (53.0 Ci/mmol, Amersham,Les Ulis, France) for 48 h in complete mediumas previously described (16). Cells were thenwashed and resuspended in serum-freemedium for kinetic experiments. Lipids wereextracted and resolved by thin-layerchromatography developed inchloroform/methanol/acidic acid/formicacid/water (65:30:10:4:2, by vol.) up to two-thirds of the plate and then inchloroform/methanol/acetic acid (94:5:5, byvol.). CER was scraped and quantitated byliquid scintillation spectrometry. Lipidstandards were used to identify the variousmetabolic products. Alternatively, total cellularCER quantitation was performed using E. colidiacylglycerol kinase (Amersham, UK)according to previously published procedures(17).

Sphingomyelinase Activities - Each fraction(150 µl aliquot) was assayed for the presenceof different SMase activities (18). Volumeswere adjusted to 250 µl and reactions werestarted by adding 250 µl of substrate solution.For the measurement of the A-SMase activity,this solution consisted of [14C-methylcholine]SM (54.5 mCi/mol, NENDuPont; 1x105 dpm/assay; [~1 nmol/assay]),0.1% (w/v) Triton X-100 and 10 mM EDTA in200 mM sodium acetate buffer (pH 5.0). ForN-SMase assay, the substrate solutionconsisted of [14C-methylcholine]SM (1x105

dpm/assay) and 0.1% (w/v) Triton X-100 in200 mM Tris-HCl buffer (pH 7.4) containing10 mM DTT and 10 mM MgCl2. After 2-hincubation at 37°C, reactions were terminatedby adding 300 µl of H2O and 2.5 ml ofchloroform/methanol (2 :1, v/v). Phases wereseparated by centrifugation (1000 x g, 5 min)and the amount of released radioactivephosphocholine was determined by subjecting700 µl of the upper phase to scintillationcounting.

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

3

The amount of radiolabeled substrate thatwas hydrolyzed during an assay neverexceeded 10% of the total amount of substrateadded. For calculation of the specific activitiesin total cell homogenates, values werecorrected for protein content, reaction time andspecific activity of the substrate.

Isolation of Membrane Rafts Microdomains- Raft microdomains were isolated from cellsas described (19). For each isolation, 100x106

cells were washed twice with PBS. Cells werepelleted by centrifugation, resuspended in 1 mlof ice-cold MES-buffered saline (MBS) (150mM NaCl, 25 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5), containing 1%(w/v) Triton X-100. After 30 min on ice, cellswere further homogenized by 10 strokes of aDounce homogenizer on ice. 1.5 ml of ice-coldMBS was added and 2 ml of this suspensionwas mixed with 2 ml of 80% (w/v) sucrose inMBS. This mixture was subsequently loadedunder a linear gradient consisting of 8 ml 5%to 40% (w/v) sucrose in MBS. All solutionscontained the following protease inhibitors :100 µM phenylmethylsulfonylfluoride, 1 mMEDTA and 1µM each of aprotinin, leupeptinand pepstatin A. Gradients were centrifuged ina Beckman SW 41 swinging-rotor at 39000rpm for 20 h at 4°C. Twelve fractions of 1 mleach were collected (from top to bottom),vortexed and stored at –80°C. The proteincontent of both fractions and the total initialcell suspension was measured using bovineserum albumin as standard (20). GM1 wasused as a marker of rafts (21,22). Cholesteroldepletion was performed by incubating U937cells for 30 min at 37°C in buffer A (140 mMNaCl, 5 mM KCl, 5 mM KH2PO4, 1 mMMgSO4, 10 mM Hepes, pH 6.5, 5 mMglucose, 0.2% BSA w/v, and 10 mM MβCD)(18).

Slot Blot - 20 µl of light, heavy and raftfractions was blotted onto a nitrocellulose filter(Hybond-C, Amersham, Les Ulis, France),using a slot blot apparatus from Bio-RadLaboratories (Hercules, CA). After blockingwith 10% nonfat milk in Tris-buffered saline-Tween 20 (0.1%) for 2 h, the filter wasincubated overnight at 4°C with the β−subunitof cholera-toxin, which has an affinity forGM1. The filter was then washed and boundproteins were detected by enhancedchemiluminescence (Amersham, Les Ulis,France).

FACS Analysis - Cells were irradiated with30 joules/m2 of UV-C, fixed for 10 min in 4%paraformaldehyde (PFA) (w/v) in PBS. Cellswere then washed and further incubated for 45min with a rabbit polyclonal anti-A-SMase orrabbit polyclonal anti-JNK (Santa CruzBiotechnology, Inc., CA) at 2 µg/ml. Cells werethen washed in PBS containing 1% FCS, andstained for 45 min with 7.5 µg/ml FITC-labeledgoat anti-rabbit (Jackson ImmunoResearchLaboratories, Inc., Baltimore, PA). After a finalPBS-BSA wash, cells were analyzed on afluorescence-activated cell sorter (Facscalibur,Becton-Dickinson, San Jose, CA).

Determination of ROS - Production of ROSwas detected using C2938 fluorescent probe(Molecular Probes, Paris, France). Briefly,exponentially growing cells were labeled with0.5 µM C2938 for one h and then irradiatedwith 30 joules/m2 UV-C. The cells werewashed in PBS, and cell fluorescence wasdetermined using flow cytometry on aFACScan cytometer (Beckton Dickinson, SanJose, CA).

Confocal Microscopy - Cells were irradiatedwith 30 joules/m2 of UV-C and incubated with15µg/ml of cholera toxin subunit B conjugatedto Cy5 (Molecular Probes Europe, Leiden, TheNetherlands) for 20 min. Cells were fixed for 10min in 4% PFA (w/v) in PBS and washed withPBS containing 3% bovine serum albumin(BSA) (w/v) and 1 mM Hepes (PBS-BSA).Cells were then incubated for 45 min with eithera rabbit polyclonal anti-A-SMase, mousemonoclonal anti-CER 15B4 (Alexis, Coger,Paris, France), or MBP-conjugated lyseninfollowed by mouse anti-MBP antiserum (NewEngland Biolabs, Beverly, MA) (43,44). Cellswere then washed in PBS-BSA and stained for45 min with 200 ng/ml FITC-labeled goat anti-rabbit, anti-mouse (green emission), or Cy3-labeled goat anti-mouse (red emission) (JacksonImmunoResearch laboratories, Baltimore, PA).After a final PBS-BSA wash, cells weremounted on glass coverslips with Dakomounting medium (Dako, Trappes, France).Control staining were performed with secondaryantibodies alone. Slides were examined with aCarl Zeiss LSM 510 confocal microscope (CarlZeiss, Oberkochen, Germany) using a x63 Plan-Apochromat objective (1.4 oil). An argon laserat 488 nm was used to excite FITC (emission515-540 nm), and a helium-neon laser wasfiltered at 633 or 550 nm to excite Cy5 andCy3, respectively (emission 680 and 570 nm)

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4

regulated by LSM software (Zeiss). For co-localization images were recorded inmultitracking mode and images were obtainedusing IMARIS co-localization software(bitplane, Zurich, Switzerland).

Western Blotting - Each aliquot wassubjected for 5 min at 95°C to denaturationbuffer (62.5 mM Tris pH 6.8, 10% glycerol,2% SDS, 0.04% bromophenol blue and 5%mercaptoethanol). The extracts were resolvedby electrophoresis in a 10% SDS-PAGE,transferred onto nitrocellulose membrane(Hybond-C, Amersham, Les Ulis, France),blocked with 10% nonfat milk in Tris-bufferedsaline-Tween 20 (0.1%) at 4°C for 2 h andincubated overnight at 4°C with anti-phosphoSAPK/JNK (Cell Signaling Technology,Beverly, USA) and anti-JNK (Santa CruzBiotechnology, Inc., CA) antibodies. Boundproteins were detected by enhancedchemiluminescence detection system(Amersham, Les Ulis, France).

Protein Kinase Assays - Cells extracts wereprepared by lysing cells in buffer containing 20mmol/L HEPES pH 7.4, 12 mmol/L EDTA,250 mmol/L NaCl, 1% NP-40, 2 µg/mLleupeptin, 2 µg/mL aprotinin, 1 mmol/LPMSF, 0.5 µg/mL benzamidine, and 1 mmol/LDTT. Cells extracts (150-250 µg/sample) wereimmunoprecipitated with 0.3 µg anti-JNK(Santa Cruz, Le Perray-en-Yvelines, France)for 60 minutes at 4°C. Immune complexeswere collected by incubation with protein A/Gsepharose beads (Pierce, Rockford, IL) for 60minutes at 4 °C. The beads were extensivelywashed with lysis buffer and kinase buffer (20mmol/L HEPES, pH 7.4, 1 mmol/L DTT, 25mmol/L NaCl). Kinase assays were performedfor 15 minutes at 30 °C using myelin basicprotein (MBP) (Sigma, St Louis, Mo) as asubstrate for JNK activity in 20 mmol/LHEPES pH 7.4, 10 mmol/L MgCl2, 1 mmol/LDTT and 10 µCi (γ32P) ATP (ICN, Orsay,France). Reactions were stopped with additionof 15 µL 2x SDS sample buffer, boiled for 5minutes and subjected to SDS-PAGE (9%).Phosphorylated MBP were visualized bystaining with Coomassie blue, the dried gelwas analyzed by autoradiography, and thecorresponding bands were scrapped andquantitated by scintillation counting.Statistics - The Student’s t test was performedto evaluate the statistical significance.

RESULTS

UV-C Induced CER Generation Through A-SMase but not N-SMase Activation - U937

cells were prelabeled with [9, 10-3H] palmiticacid or [methyl-3H] choline to equilibrium for48 h and then irradiated in PBS with UV-C at30 J/m2. As shown in Figure 1A, UV-Cinduced an increase in CER levels (whichwhich peaked at 5-15 min after irradiation[data not shown]). Concomitant with CERgeneration, we observed significant SMhydrolysis (Fig. 1B) which suggested theimplication of a SMase.

To determine which enzyme wasresponsible for SM hydrolysis and subsequentCER generation, we evaluated the effect ofUV-C on both N-SMase and A-SMaseactivities. We observed that UV-C did notaffect N-SMase but lead to a ~30 % increase ofa zinc-independent A-SMase activity, whichpeaked within 5-15 min after irradiation,(Figure 1C and D). To confirm this result,U937 cells were preincubated for one h withSR33557, a potent A-SMase inhibitor (23), andthen irradiated with 30 J/m2 UV-C. Asexpected, SR33557 significantly abrogatedUV-C induced A-SMase activity in a dosedependant manner (Table I, Figure 1D), andCER generation (data not shown).

UV-C Induced Activation of A-SMase inRafts - To evaluate the potential role of plasmamembrane rafts in UV-C induced A-SMaseactivation, cells were irradiated with UV-C at30 J/m2. 12 minutes post-UV-C irradiation(corresponding to the peak of UV-C triggeredA-SMase stimulation), cells were lysed in coldTriton X-100, and fractionated on a sucrosedensity gradient. We observed basal A-SMaseactivity throughout the gradient (Fig. 2A andB). Under UV-C treatment, A-SMase wasobserved to increase exclusively in raftfractions. We have previously characterizedU937 rafts whereby the Triton insolublematerial (fractions 4-6), expressed high levelsof SM which co-migrated, as shown in Fig.2C, on the density gradient with the raftmarker ganglioside GM1 (24). Again nosignificant increase in raft-associated N-SMaseactivity was observed (data not shown).

To confirm the role of rafts microdomains,we pretreated U937 cells with the cholesterol-sequestering agent methyl β-cyclodextrin(MβCD) under conditions where it is still

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

5

possible to isolate rafts but their content incholesterol is significantly reduced (~50 %decrease [data not shown; 18, 24]). As shownin Fig. 2D, the CER generation induced byUV-C was significantly decreased in MβCD-treated cells. These results strongly suggestthat raft microdomains were essential for UV-C triggered CER generation.

UV-C Induced A-SMase Translocation tothe Plasma Membrane Outerleaflet - We nextinvestigated the subcellular distribution of A-SMase in UV-C treated cells, UV-C induced atime-dependent recruitment of A-SMase to theouter leaflet of the cell membrane as revealedby flow cytometry analysis performed withFITC-coupled anti-A-SMase antibody on non-permeabilized cells (Fig. 3A). Confocalanalysis of A-SMase accumulation on the cellsurface was detected as early as 10 min,peaked at 12 min, after which the fluorescencesignal decreased and returned to basal levelafter 15 min (data not shown). These resultssuggested that, upon UV activation, a fractionof internal A-SMase was rapidly andtransiently externalized to the cell surface.Moreover, the temporal association betweenA-SMase stimulation and externalizationsuggested that activation was a critical eventfor enzyme relocalization. In order to clarifythis important issue, we evaluated the effect ofSR33557 on A-SMase externalization. Asshown in Fig. 3B, inhibition of the enzymeresulted in significant inhibition of itsrelocalization. Since SR33557 at 30 µMinhibits basal SMase activity by > 75 %, butdoes not lead to A-SMase degradation (23),one can rule out an artifactual event. Thisresult does suggest, however, that in order forthe enzyme to be externalized and reach theSM-enriched outermembrane (40), it must bein an active form. We next evaluated the effectof Brefeldin A on A-SMase externalization. Asshown in Fig. 3C, inhibition of the vesiculartrafficking by Brefeldin also abrogated theenzyme externalization.

UV-C Induced A-SMase Translocation toRaft Microdomains - Based on our biochemicalstudies, we hypothesized that upon UV-Cactivation, A-SMase relocalization was not arandom process, but that A-SMase wasredirected towards raft microdomains. We firstinvestigated whether UV-C activation couldinterfere with raft distribution by using a Cy3-coupled antibody directed against flotillin (datanot shown), or Cy5-coupled cholera toxin,

which specifically binds the raft componentganglioside GM1. As shown in Fig. 4A, UVirradiation induced a rapid and markedreorganization of rafts into larger platforms,producing a capping effect on the cells at 12min post-irradiation (<10 % GM1 clustering incontrols [depending on the experiment]compared to > 80 % in irradiated cells).Moreover, in irradiated cells, A-SMase co-localized with cholera toxin, suggesting thatthe enzyme translocated into raftmicrodomains (Fig. 4B). Based on theseresults, we hypothesized that CER productionpreferentially occurred at the raft level. Indeed,upon UV-C activation, CER co-localized withGM1 as shown in Figure 4C. These resultsstrongly suggested that plasma membranemicrodomains are essential constituents in UV-C-mediated A-SMase activation andexternalization. Since SM is an essentialconstituent of plasma membrane rafts, weelected to investigate SM distribution inirradiated U937 cells. As shown in Figure 4D,the SM-specific toxin lysenin (here coupled toMBP) bound uniformly to the plasmamembrane of control U937 cells, as previouslydescribed for normal fibroblasts (41).However, there appeared to be a modestredistribution after irradiation whichcolocalized with GM1 aggregation.

To further confirme the role of rafts in ourstudy, we pretreated U937 cells with MβCD.As shown in Table II, UV-C induced A-SMaseexternalization was completely inhibited inMβCD treated cells. Moreover, we observedusing confocal microscopy that both CER andGM1 redistribution in UV-C treated cells wasinhibited (as expected) in MβCD treated cells(data not shown). These results suggested thatUV-C activated an ordered signaling cascadeconsisting in A-SMase activation andrelocalization into raft microdomains, SMconsumption and CER release resulting in theformation of large CER-enriched platforms. Tofurther confirm this hypothesis, we comparedA-SMase externalization under UV-Ctreatment in normal and Niemann-Pick disease(NPD) lymphoblasts. Again, UV-C treatmentinduced significant A-SMase externalization innormal lymphoblasts which was comparable tothat observed in U937 cells, but not in NPDcells (Table II).

ROS Regulates A-SMase Activation andTranslocation to Raft Microdomains - In anattempt to characterize the signaling pathways

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

6

leading to A-SMase activation induced by UV-C, we first studied the role of ROS in UV-Csignaling. Indeed treatment of U937 cells withUV-C resulted within 5 min in a burst of H2O2

production (Fig. 5A), and stimulation of A-SMase activity (Fig. 5B), and its translocationto the outerleaflet of the plasma membrane(Fig. 5C). Moreover, inhibiting UV-C inducedROS production by PDTC abolished both A-SMase activation (Fig. 6A) and membraneexternalization (Fig. 6B).

JNK is activated and Relocalized into RaftsFractions Following UV-C Irradiation - CERhas been described to trigger distinctintracellular signaling pathways, including thestimulation of JNK (5). Therefore, weevaluated whether UV-C could activate thispathway through CER production. Theseexperiments showed that, in U937 cells,treatment with UV-C resulted in rapid (as earlyas 10 min) and prolonged (up to 24 h) JNKphosphorylation (Fig. 7A). Furthermore, theinhibition of A-SMase, by pretreatment withSR33557 (data not shown) or desipramine,another potent A-SMase inhibitor (23),blocked UV-C induced JNK kinase activityusing MBP as substrate (Fig. 7B). Moreover,we observed that under UV-C treatment, JNKand a fraction of P-JNK was redistributedtowards raft microdomains (Fig. 7C), thistranslocation was also inhibited bydesipramine and MβCD (data not shown). Byconfocal microscopy, we clearly observed P-JNK externalization in UV-C treated cells, aportion of which colocalized with GM1 (Fig.7D). These results suggested that SM-derivedCER, resulting in CER-enriched rafts,mediated the stimulatory effect of UV-C onthese signaling kinases.

UV-C Induced CER Production and JNKActivation is not Dependent on a NuclearSignal - It has been suggested that cellsignaling induced by UV-damaged DNA in thenucleus is rapidly transferred to the cytosol,leading to downstream events (25, 26). Inorder to determine if a nucleus is necessary forUV-C induced CER generation, we attemptedto prepare U937 cytoplasts. However,karyophilic staining revealed that enucleationefficiency was < 70%, making it impossible tointerpret a clear result. Therefore, we elected toirradiate platelets with UV-C. As shown in Fig.8 A and B, exposure to UV-C induced CERgeneration and A-SMase translocation to theexternal leaflet of the plasma membrane

similar to that in observed in intact U937 cells.Furthermore, since platelets also express JNK(42), we were able through flow cytometryanalysis to demonstrate JNK externalization inUV-C treated platelets (Fig. 8C). Hence aspreviously described by Devary and colleagues(27), UV-C induced JNK activation wasobserved to be independent of a nucleus. Theseexperiments conclusively rule out theinvolvement of a nuclear signal generated byDNA damage in the production of CER.

DISCUSSION

In this study, we show that UV-Cirradiation induced a rapid and transientincrease in cellular CER concentration. CERproduction correlated with the stimulation ofan A-SMase, whereas N-SMase activity wasunaffected. Moreover, SR 33557 anddesipramine, both inhibitors of A-SMase (23),prevented not only A-SMase stimulation butalso CER generation. These results suggestthat an A-SMase plays a critical role in UV-Cmediating SM cycle activation. Characterizedas a Zn2+-independent A-SMase, which haspreviously been shown to present on the cellmembrane surface of CD95 or CD40stimulated cells (14). Gulbins and co-workershave described this enzyme as mainly residinginside secretory vesicles, and that, uponstimulation, at least a fraction of the enzyme israpidly translocated to the cell surface incluster-like structures concomitant withextracellularly oriented CER (identified usingthe anti-ceramide 15B4 mAb [28]). Thespecificities of the anti-A-SMase and anti-CERantibodies has previously been described (45),and we confirmed this by western blot analysis(normal versus NPD lymphoblasts) and thinlayer chromatography immunostaining onU937 cells (data not shown). Our studyrevealed striking similarities between the A-SMase involved in UV-C response and thatdescribed by Gulbins and colleagues. Indeed,not only was UV-induced A-SMasestimulation found to be Zn2+-independant, butalso UV-C irradiation resulted in an increase inA-SMase localized on the outer leaflet of thecell surface. This finding suggested that, uponUV-C stimulation, A-SMase translocated fromthe cytoplasm to the external leaflet of theplasma membrane probably by intracellularvesicles, as this event was found to bebrefeldin sensitive (29). Since, we were unable

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

7

to detect any modification of the totalintracellular distribution of A-SMase byconfocal microscopy following UV-Cirradiation, only a very small sub-populationfraction of the enzyme was implicated.

Little is known about the regulation of A-SMase in the context of stress response. ROShave been described as one of the majorsignaling components of the UV response (30,31). For this reason, we investigated whetherROS could be involved in the stimulationand/or the translocation of the enzyme. Ourstudy shows that the antioxydant PDTC, aROS scavenger, not only inhibited A-SMasestimulation but also prevented its translocationto the cell surface. Indeed, UV-induced ROSgeneration could be localized in raftmicrodomains (30). Of course, one can not ruleout the possibility that UV-C inducedcholesterol peroxidation, however UV-C didnot induce ROS generation in NPD cells (datanot shown). Hence our study not only showsthat ROS operated upstream A-SMase but alsosuggests that stimulation of the enzyme isrequired for its translocation to the cell surface.This hypothesis is supported by the fact thatSR33557, an inhibitor of A-SMase activity,also abrogated its relocalization to the cellsurface.

This study also shows that UV-C inducedsignificant redistribution of GM1, suggesting amajor reorganization of raft microdomains.Indeed, we found that non irradiated cellsdisplayed dispersed GM1 distribution at thesurface of the cell whereas, upon UVactivation, GM1 concentrated to one pole ofthe cell in a majority of cells. One couldspeculate that UV-C induces the aggregation ofraft components leading to the formation of amajor polarized signaling microdomain.Indeed, a model has been suggested in whichthe TCR-associated signaling machineryinitiates raft aggregation by promoting F-actinreorganization, permitting full activation of thetyrosine phosphorylation cascade,reorganization of the actin cytoskeleton andsustained cell signaling and activation (33, 34).It has been proposed that it is the consumptionof SM in rafts by A-SMase which results in theformation of large CER-enriched membranedomains which, in turn, could facilitaterecruitment and activation of signalingmolecules (35, 36). Using the SM-specifictoxin lysenin, however, we did not observe adecrease a cell membrane labeling, but rather a

slight redistribution of SM towards GM1enriched regions. Further studies aimed atquantifying raft-associated SM, includingcomparing SM levels in the inner versus outerleaflet are needed to confirm raft-associatedSM-hydrolysis.

Finally, our study questions theinvolvement of a nuclear signal generated byUV-induced DNA damage in the induction ofA-SMase activation and CER generation.Indeed it is generally postulated that UVresponse occurs by induction of a nuclearsignaling cascade by damaged DNA (25).However, Karin and colleagues proposed thatUV response (such as JNK activation) waslikely to be initiated at or near the plasmamembrane through alterations at the cellsurface leading to receptor clustering (27, 37).In our study, we also observed UV-C-inducedJNK activation in U937 cells, as well as itstranslocation to the outer surface of raftmicrodomains. Our results are furthermoreconsistent with the hypothesis of Karin andcolleagues, as we observed that the initiation ofUV cell response (i.e., CER generation, A-SMase and JNK activation and externalizationto the external plasma membrane) wassimilarly observed in platelets. A recent study,also proposed that mitochondrial CERgeneration was implicated in UV signaling(38), perhaps as a consequence ofmitochondrial DNA damage (39). In the fieldof cell response to UV light, our observationsshould provide new investigative paths intoUV-induced photoaging, immunosuppression,and photocarcinogenesis.

ACKNOWLEDGEMENTS

This work was supported by la LigueNationale Contre le Cancer and les ComitésDépartementaux du Gers, de l’Aveyron, et dela Haute-Garonne (J.P.J.). A.C. and C. B. arethe recipient of a grant from l’Association pourla Recherche contre le Cancer and S. G. fromla Fondation pour la Recherche Médicale. Wethank Pr. Thierry Levade (INSERM U466,CHU Rangueil, Toulouse) for his help duringthe course of this study.

REFERENCES

1. Karin, M. (1998) Ann N Y Acad Sci 851,139-146

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

8

2. Barthelman, M., Chen, W., Gensler, H.L.,Huang, C., Dong, Z., and Bowden, G.T.(1998) Cancer Res 58, 711-716

3. Zanke, B.W., Boudreau, K., Rubie, E.,Winnett, E., Tibbles, L.A., Zon, L.,Kyriakis, J., Liu, F.F., and Woodgett,J.R. (1996) Curr Biol 6, 606-613

4. Goss, V.L., Cross, J.V., Ma, K., Qian, Y.,Mola, P.W., and Templeton, D.J. (2003)Cell Signal 15, 709-718

5. Verheij, M., Bose, R., Lin, X.H., Yao, B.,Jarvis, W.D, Grant, S., Birrer, M.J.,Szabo, E., Zon, L.I., Kyriakis, J.M.,Haimovitz-Friedman, A., Fuks, Z., andKolesnick, R.N.(1996) Nature 380, 75-79

6. Basu, S., and Kolesnick, R. (1998)Oncogene 17, 3277-3285

7. Zhang, Y., Mattjus, P., Schmid, P.C., Dong,Z., Zhong, S., Ma, W.Y., Brown, R.E.,Bode, A.M., Schmid, H.H., and Dong, Z.(2001) J. Biol. Chem 276, 11775-11782

8. Grether-Beck, S., Bonizzi, G., Schmitt-Brenden, H., Felsner, I., Timmer, A., Sies,H., Johnson, J.P., Piette, J., Krutmann, J.(2000) EMBO J 19, 5793-5800

9. Chatterjee, M., and Wu, S. (2001) Mol CellBiochem 219, 21-27

10. Magnoni, C., Euclidi, E., Benassi, L.,Bertazzoni, G., Cossarizza, A., Seidenari,S., and Giannetti, A. (2002) Toxicol InVitro 16, 349-355

11. Huang, C., Ma, W., Ding, M., Bowden,G.T., and Dong, Z. (1997) J Biol Chem272, 27753-27757

12. Schissel, S.L., Keesler, G.A., Schuchman,E.H., Williams, K.J., Tabas, I.(1998) JBiol Chem 273, 18250-18259

13. Schissel, S.L., Jiang, X., Tweedie-Hardman, J., Jeong, T., Camejo, E.H.,Najib, J., Rapp, J.H., Williams, K.J., andTabas, I. (1998) J Biol Chem 273, 2738-2746

14. Gulbins, E., and Grassme, H. (2002)Biochim Biophys Acta 1585, 139-145

15. Andrieu, N., Salvayre, R., and Levade, T.(1994) Biochem. J 303, 341-345

16. Jaffrézou, J.P., Levade, T., Bettaieb, A.,Andrieu, N., Bezombes, C., Maestre, N.,Vermeersch, S., Rousse, A., and Laurent,G. (1996) EMBO J 15, 2417-2424

17. Van Veldhoven, P.P., Matthews, T.J.,Bolognesi, D.P., and Bell, R.M. (1992)Biochem Biophys Res Commun 187, 209-216

18. Bodin, S., Giuriato, S., Ragab, J., Humbel,B.M., Viala, C., Vieu, C., Chap, H., andPayrastre, B. (2001) Biochemistry 40,15290-15299

19. Lisanti, M.P., Tang, Z., Scherer, P.E., andSargiacomo, M. (1995) Methods Enzymol250, 655-668

20. Smith, P.K., Krohn, R.I., Hermanson, G.T.,Mallia, A.K., Gartner, F.H., Provenzano,M.D., Fujimoto, E.K., Goeke, N.M.,Olson, B.J., and Klenk, D.C. (1985) AnalBiochem 150, 76-85

21. Solomon, S., Masilamani, M., Rajendran,L., Bastmeyer, M., Stuermer, C.A., andIllges, H. (2002) Immunobiology 205,108-119

22. Nichols, B.J. (2003) GM1-containing lipidrafts are depleted within clathrin-coatedpits. Curr. Biol 13, 686-690

23. Jaffrézou, J.P., Chen, G., Duran, G.E.,Muller, C., Bordier, C., Laurent, G., Sikic,B.I., and Levade, T. (1995) BiochimBiophys Acta 1266, 1-8

24. Grazide, S., Maestre, N., Veldman, R.J.,Bezombes, C., Maddens, S., Levade, T.,Laurent, G., and Jaffrézou, J.P. (2002)FASEB J 16, 1685-1687

25. Radler-Pohl, A., Sachsenmaier, C., Gebel,S., Auer, H.P., Bruder, J.T., Rapp, U.,Angel, P., Rahmsdorf, H.J., and Herrlich,P . (1993) EMBO J 12, 1005-1012

26. Matsumura, Y., and Ananthaswamy, H.N.(2004) Toxicol Appl Pharmacol 195, 298-308

27. Devary, Y., Rosette, C., DiDonato, J.A.,and Karin, M. (1993) Science 261, 1442-1445

28. Grassme, H., Bock, J., Kun, J., Gulbins E.(2002) J Biol Chem 277, 30289-30299

29. de Gassart, A., Geminard, C., Fevrier, B.,Raposo, G., and Vidal, M. (2003) Blood102, 4336-4344

30. Chang, H., Oehrl, W., Elsner, P., andThiele, J.J. (2003) Free Radic Res 37,655-663

31. Herrling, T., Fuchs, J., Rehberg, J., andGroth, N. (2003) Free Radic Biol Med 35,59-67

32. Gniadecki, R., Christoffersen, N., andWulf, H.C. (2002) J. Invest Dermatol 118,582-58

33. Cremesti, A.E., Goni, F.M., and Kolesnick,R.(2002) FEBS Lett 531, 47-53

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

9

34. Gulbins, E., Dreschers, S., Wilker, B., andGrassme, H. (2004) J Mol Med 82, 357-363

35. Galjart, N., and Perez, F. (2003) Curr.Opin.Cell.Biol 15, 48-53

36. Rajendran, L., Masilamani, M., Solomon,S., Tikkanen, R., Stuermer, C.A., Plattner,H., and Illges, H.(2003) Proc Natl AcadSci U S A 100, 8241-8246

37. Rosette, C., and Karin, M. (1996) Science274, 1194-1197

38. Dai, Q., Liu, J., Chen, J., Durrant, D.,McIntyre, T.M., and Lee, R.M. (2004)Oncogene 23, 3650-3658

39. Birch-Machin, M.A. (2000) Clin ExpDermatol 25, 141-146

40. Bettaieb, A., Record, M., Come, M.G.,Bras, A.C., Chap, H., Laurent, G.,Jaffrézou, J.P. (1996) Blood 88,1465-72

41. Yamaji, A., Sekizawa, Y., Kazuo Emoto,K., Sakuraba, H., Keizo Inoue, K.,Kobayashi, H., and Masato Umeda, M.(1998) J Biol Chem 273, 5300-5306

42. Bugaud, F., Nadal-Wollbold, F., Levy-Toledano, S., Rosa, J.P., and Bryckaert,M. (1999) Blood 94, 3800-3805

43. Kiyokawa, E., Makino, A., Ishii, K.,Otsuka, N., Yamaji-Hasegawa, A., andKobayashi, T. (2004) Biochemistry 43,9766-9773

44. Nakai, Y., Sakurai, Y., Yamaji, A., Asou,H., Umeda, M., Uyemura, K., and Itoh,K.J. (2000) Neurosci Res 62, 521-529

45. Grassme, H., Jekle, A., Riehle, A.,Schwarz, H., Berger, J., Sandhoff, K.,Kolesnick, R., and Gulbins, E. R.(2001) J Biol Chem 276, 20589-20596

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

10

FIGURE LEGENDS

FIG. 1. Effect of UV-C on intracellularCER levels and SMase activity. U937 cellswere prelabeled with [9, 10-3H] palmitic acid(A) or [methyl-3H] choline (B) to equilibriumfor 48 h and then treated with 30 J/m2 UV-C.CER and SM levels were quantitated at varioustime points as described under “Materials andMethods”. Results are mean of peak CERgeneration and SM hydrolysis (5-15 min) incontrol ( ) and UV-C treated ( ) cellsobtained from three independent experiments.N-SMase (C) and A-SMase (D) activities werequantitated as described under “Materials andMethods”. Results in C are representative of 3independent experiments performed intriplicate ± SD. In D, U937 cells werepreincubated ( ) or not ( ) for one h with30µM of SR33557 before irradiation. Non-irradiated control A-SMase values ( ) were3654±306 dpm and 1094±56 dpm in theabsence and in the presence of SR33557,respectively. Results are mean of peak A-SMase activation obtained from 3 independentexperiments. *p< 0,01.

FIG. 2. UV-C induced activation of A-SMase in raft microdomains. U937 cellswere either untreated ( ) or treated ( ) withUV-C at 30 J/m2. 12 min post-irradiation, cellswere lysed in cold Triton X-100 andfractionated on a sucrose density gradient.Aliquots were collected and analyzed for A-SMase activity. Enzyme activities present ineach fraction are expressed against the aliquot-associated protein levels (A). bars, raftfractions. Results are representative of 3independent experiments performed intriplicate. *p< 0,01. B. A-SMase activities areexpressed compared against the amount ofproteins in each fraction, light (pooledfractions 1-3), rafts (4-6) and heavy (7-10).Results are mean of triplicate determinations ofa representative experiment (one of threeindependent experiments). *p< 0,01. (C)Rafts (pooled fractions 4-6) were identified bythe ganglioside GM1 which is enriched in thismicrodomain and not in the light fractions (1-3) and heavy fractions (7-10). Results arerepresentative of 3 independent experiments.(D) U937 cells were prelabeled with [9, 10-3H]

palmitic acid to equilibrium for 48 hours andthen cholesterol depleted using MβCD ( ) asdescribed under “Materials and Methods”, orleft as is ( ). CER levels were quantitated afterUV-C irradiation, results are representative of3 independent experiments. *p< 0,05. Non-irradiated control A-SMase values were4966±86 dpm and 3970±175 dpm in theabsence and in the presence of MβCD,respectively.

FIG. 3. UV-C induces A-SMaseexternalization to the outer leaflet of theplasma membrane. U937 cells were treatedwith UV-C at 30 J/m2. 12 min post-irradiation,flow cytometry analysis of unpermeabilizedcells was performed on UV-C irradiated U937cells (A), cells pretreated for one h with 30µMSR33557 (B), or 12 h with 10 µg/ml brefeldinA (C). (dotted arrow), stained FITC anti-rabbit control; (thin and thick arrows), FITC-rabbit anti-A-SMase of nonirradiated andirradiated cells, respectively. Results arerepresentative of three independentexperiments.

FIG. 4. UV-C induced A-SMasetranslocation to raft microdomains. U937cells were irradiated or not with UV-C at 30J/m2. 12 min post-irradiation, cells wereanalyzed by confocal microscopy usingcholera toxin subunit B conjugates with Cy5(blue emission) (A - D), rabbit FITC/anti-A-SMase (B), rabbit FITC/anti-CER (C) (greenemission), or rabbit Cy3/anti-MBP-lysenin(red emission) (D). Turquoise and purple areFITC/Cy5 and Cy3/Cy5 merge, respectively.Result are representative of three independentexperiments.

FIG. 5. UV-C induced ROS generation andH2O2 induced A-SMase translocation andactivation. (A) U937 cells were loaded withC2938, a fluorescent probe, then irradiatedwith 30 J/m2 UV-C and ROS production wasthen analyzed at different time points by flowcytometry. (B) U937 cells were treated with1mM of H2O2 for various time points and A-SMase activity was quantitated. Results aremean of three independent experiments. *p<0,05. (C) Flow cytometry analysis ofunpermeabilized cells pretreated with 1mM ofH2O2 using FITC-rabbit anti-A-SMase. (thinarrow), stained FITC anti-rabbit control;(dotted arrow) untreated cells; (dashed and

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11

thick arrows), 12 and 14 min post H2O2treatment, respectively. Results arerepresentative of three independentexperiments.

FIG. 6. PDTC inhibited UV-C induced A-SMase activation and externalization. U937cells were pretreated ( ) or not ( ) with 100µM PDTC for 30 min, and then irradiated withUV-C at 30 J/m2. 12 min post-irradiation, A-SMase activity was quantitated and comparedto nonirradiated cells ( ) (A). Results aremean of A-SMase activation obtained fromthree independent experiments ± SD. *p< 0,01.Flow cytometry analysis of unpermeabilizedcells was also performed after UV-Cirradiation (B). stained FITC anti-rabbitcontrol, (thin arrow); FITC-rabbit anti-A-SMase of nonirradiated cells (dotted arrow)and 10 and 12 min after UV-C treatment(dashed and thick arrows, respectively).Results are representative of three independentexperiments.

FIG. 7. UV-C induced JNK activation andexternalization. U937 cells were irradiated ornot with UV-C at 30 J/m2. (A), at differenttime points cells were immunoblotted withantibodies against P-JNK and anti-JNK(loading control). (B), similarly, U937 cellswere pretreated or not with 10 µM A-SMaseinhibitor desipramine for 1 h, afterwhich JNKactivity was determined by MBPphosphorylation as described under “Materialsand Methods” in control and irradiated cells(12 min). (C), Raft fractions were isolatedfrom irradiated cells, and blotted for JNK andP-JNK. Results are representative of threeindependent experiments. (D), 12 min post-irradiation, unpermeabilized cells were alsoanalyzed by confocal microscopy usingcholera toxin subunit B conjugates with Cy5(blue emission) and rabbit FITC/anti-P-JNK(red emission). Turquoise emission isFITC/Cy5 merge, . Result are representative ofthree independent experiments.

FIG. 8. UV-C induces CER generation andA-SMase and JNK translocation to theexternal plasma membrane of humanplatelets. Platelets (1x 109/per time point)were treated with UV-C at 30 J/m2. (A), CERlevels were quantitated using E. colidiacylglycerol kinase. Results are mean of 3independent experiments ± SD. *p< 0,01. (B

and C), Flow cytometry was performed 12min post-irradiation on unpermeabilized cellsusing FITC-rabbit anti-A-SMase (B) or FITC-rabbit anti-JNK (C). (thin arrow), stained FITCanti-rabbit control; (thin arrow), non irradiatedcells; (thick arrow), UV-C treated cells.Results are representative of 3 independentexperiments.

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

12

TABLE I

Effect of SR33557 on UV-C induced A-SMase externalization

U937 cells were preincubated or not with SR33557 during one h at 37°C. Cells were thenirradiated with UV-C at 30 joules/m2. 12 min after UV-C, A-SMase externalization was determined byFACS analysis on non permeabilized cells using FITC-rabbit anti-A-SMase.

SR33557 Fluorescencea

µM %

None 167±15d

1 171±86 116±9

10 107±1130 96±7

a Results are expressed in % of total cell surface fluorescence compared to nonirradiated controls andmean of 3 independent experiments ± SD

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

13

TABLE II

Effect of cholesterol depletion and A-SMase deficiency on UV-C induced A-SMase externalization

% Fluorescencea

U937 cellsb lymphoblastsc

- MβCD + MβCD Normal NPD type A

142±15d 90±13e 154±18 100±11e

a Cells were irradiated with UV-C at 30 joules/m2. 12 min after UV-C, A-SMase externalization wasdetermined by FACS cytometry on non permeabilized cells using FITC-rabbit anti-A-SMase.bU937 cells were pretreated or not with MβCD as described under Materials and Methods, followedby UV-C irradiation.cNormal and NPD type A (cell line MS1418) EBV-transformed lymphoid cells.dResults are expressed in % of total cell surface fluorescence compared to untreated controls and meanof 3 independent experiments ± SD.e p< 0,02

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

14

90

100

110

120

130

140

150

160

[3H]-

cera

mid

e(%

of

contr

ols

)

60

70

80

90

100

110

[3H]-

sphin

gom

yelin

(% o

f contr

ols

)

Figure 1 A and BCharruyer et al.

A

B

*

*

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

15

80

90

100

110

120

130

140

150

Neutr

al SM

ase a

cti

vit

y(%

of

contr

ols

)

0 5 10 15 20

Time post irradiation (min)

100

150

200

250

300

A-S

Mase a

ctiv

ity

(% o

f contr

ol)

C

D

Figure 1 C and DCharruyer et al.

*

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

16

0

50

100

150

200

Acid

SM

ase a

cti

vit

y(p

mol/

h.m

g f

racti

on p

rote

in)

1 2 3 4 5 6 7 8 91011

Fractions

B

C

0

100

200

300

Acid

SM

ase a

cti

vit

y(%

of

contr

ol)

LIGHT RAFT HEAVY

LIGHT HEAVYRAFT

CT

UV-C

Figure 2 A-CCharruyer et al.

A

GM1

*

**

*

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

17

Charruyer et al. Figure 2 D

50

100

150

200

250

0 5 10 15 20

Time post irradiation (min)

** *

D

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

18

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

19

AIrradiatedNonirradiated

Charruyer et al. Figure 4 A

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

20

FITC-anti ASMase

Cy5-Cholera toxin

FITC/Cy5

Charruyer et al. Figure 4 B

B IrradiatedNonirradiated

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

21

Irradiated

FITC-anti CER

Cy5-Cholera toxin

FITC/Cy5

NonirradiatedC

Charruyer et al. Figure 4 C

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

22

Irradiated

Cy3-anti MBP-lysenin

Cy5-Cholera toxin

Cy3/Cy5

Nonirradiated

D

Charruyer et al. Figure 4 D

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

23

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

24

*

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

25

JNK

+-

P-JNK

UV-C

0 0,5 3 6 24 hrs

A

Figure 7 A-CCharruyer et al.

C

B

P-MBP

UV-C

desipramine ---- +

+++

JNK

0 0,5 1 12 min

P-JNK

JNK

P-JNK

JNK

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

26

Irradiated

FITC-anti P-JNK

Cy5-Cholera toxin

FITC/Cy5

Nonirradiated

D

Figure 7 DCharruyer et al.

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

27

0

200

400

600

800

1000

[3H]-

cera

mid

e(%

of

contr

ols

)

0 10 20 30 40

Time post irradiation

Figure 8 A and BCharruyer et al.

B

A

60

50

40

30

20

10

0

Co

un

ts

10 0 101 102 103 104

FITC-anti-ASMase

*

*

*

**

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

28

100 101 102 103 104

FITC-anti-JNK

C

Figure 8CCharruyer et al.

60.

50.

40.

30.

20.

10.

0 by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from

and Jean-Pierre JaffrezouAlexandra Charruyer, Solène Gradize, Christine Bezombes, Sabina Muller, Guy Laurent

translocation independently on a nuclear signalUV-C induces raft-associated acid Sphingomyelinase and JNK activation and

published online March 11, 2005J. Biol. Chem. 

  10.1074/jbc.M412867200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on April 9, 2018

http://ww

w.jbc.org/

Dow

nloaded from