activationofacidsphingomyelinasebyproteinkinase c ...april 13, 2007•volume 282•number 15 journal...

Activation of Acid Sphingomyelinase by Protein KinaseC�-mediated Phosphorylation*□S

Received for publication, October 5, 2006, and in revised form, January 23, 2007 Published, JBC Papers in Press, February 15, 2007, DOI 10.1074/jbc.M609424200

Youssef H. Zeidan and Yusuf A. Hannun1

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina,Charleston, South Carolina 29425

Although important for cellular stress signaling pathways, themolecular mechanisms of acid sphingomyelinase (ASMase)activation remain poorly understood. Previous studies showedthat treatment of MCF-7 mammary carcinoma cells with thepotent protein kinase C (PKC) agonist, phorbol 12-myristate13-acetate (PMA), induces a transient drop in sphingomyelinconcomitant with an increase in cellular ceramide levels(Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., andHannun, Y. A. (2005) J. Biol. Chem. 280, 2606–2612). Here weshow that PMA selectively activates ASMase and that ASMaseaccounts for the majority of PMA-induced ceramide. Pharma-cologic inhibition and RNA interference experiments indicatedthat the novel PKC, PKC�, is required for ASMase activation.Immunoprecipitation experiments revealed the formation of anovel PKC�-ASMase complex after PMA stimulation, andPKC� was able to phosphorylate ASMase in vitro and in cells.Using site-directed mutagenesis, we identify serine 508 as thekey residue phosphorylated in response to PMA. Phosphoryla-tion of Ser508 proved to be an indispensable step for ASMaseactivation and membrane translocation in response to PMA.The relevance of the proposed mechanism of ASMase regula-tion is further validated in a model of UV radiation. UV radia-tion also induced phosphorylation of ASMase at serine 508.Moreover, when transiently overexpressed, ASMaseS508Ablocked the ceramide formation after PMA treatment, suggest-ing a dominant negative function for this mutant. Takentogether, these results establish a novel direct biochemicalmechanism forASMase activation inwhichPKC� serves as a keyupstream kinase.

Hydrolysis of membrane sphingomyelin (SM)2 constitutes amajor route for ceramide formation in mammalian systems. In

contrast to the multistep de novo pathway of ceramide biosyn-thesis, breakdown of SM is catalyzed by a single class ofenzymes, the sphingomyelinases (SMases). Currently, theNCBI data base contains five entries for human SMases that aregrouped according to their optimal pH as acid (ASMase orSMPD1), neutral (NMase or SMPD2, -3, and -4), and alkalinesphingomyelinases (Alk-SMase or ENPP7).Acid sphingomyelinase (ASMase) was the first SMase to be

purified and cloned (2–4). Although encoded by the same geneand mRNA, two forms of the enzyme have been described: anendolysosomal form (or Zn2�-independent form) and a secre-tory form (or Zn2�-dependent form) (5). Post-translationalprotein glycosylation plays an important role in both traffickingand stability of ASMase within the lysosomal milieu (6, 7). Inaddition, cysteine 629 situated toward the C terminus ofASMase has been proposed as an important residue for regula-tion of the enzyme such that modification (mutation or dele-tion) of this residue results in 5-fold activation of the enzyme(8).In humans, mutation of the SMPD1 gene results in

Niemann-Pick disease, an autosomal recessive neurovisceraldisease of high lethality. The severity of the Niemann-Pick dis-ease phenotype correlateswell with the deficiency in the in vitroASMase activity, which has become a standard test for classifi-cation of patients with the disease. Indeed, the knock-outmouse in the SMPD1 gene recapitulates the Niemann-Pick dis-ease phenotype as described by multiple investigators (9, 10).Biologically, ASMase has been implicated in a variety of

physiological and pathophysiologic processes (reviewed inRefs. 11–14). In particular, ASMase has been shown to be acti-vated in response to various stress stimuli including 1) ligationof death receptors (tumor necrosis factor-�, CD95, andTRAIL), 2) radiation (UV-C and ionizing radiation), 3) chemo-therapeutic agents (cisplatin, doxorubicin, paclitaxel, and his-tone deacetylase inhibitors), 4) viral, bacterial, and parasiticpathogens (rhinoviruses, Neisseria gonorrhea, Staphylococcusaureus, Pseudomonas aeruginosa, and Cryptosporidium par-vum), and 5) cytokines (e.g. IL-1�). Evidence from studies con-ducted in cell culture and animal models suggested that activa-tion of ASMase by the above agents is a key step during cellulardifferentiation, growth arrest, apoptosis, and immune defensemechanisms. Although most of these studies describe rapidactivation of the enzyme (most likely via a post-translationalmodification), transcriptional change is yet another regulatorof ASMase activity. For instance, differentiation of monocyticcells to macrophages requires up-regulation of the enzyme at atranscriptional level (15). Transcription factors SP1 and AP-2

* This work was supported by NCI, National Institutes of Health (NIH), GrantNCI P01-CA97132 (to Y. A. H.). Work at the Lipidomics Core was supportedby NIH Grant C06 RR018823. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1 and Movie 1.

1 To whom correspondence should be addressed: Dept. of Biochemistry andMolecular Biology, Medical University of South Carolina, 175 Ashley Ave.,P.O. Box 250509, Charleston, SC 29425. Tel.: 843-792-9318; Fax: 843-792-4322; E-mail: [email protected].

2 The abbreviations used are: SM, sphingomyelin; SMase, sphingomyelinase;ASMase, acid sphingomyelinase; RNAi, RNA interference; SCR, scrambledRNAi; WT, wild type; PKC, protein kinase C; PMA, 12-myristate 13-acetate;FBS, phosphate-buffered saline; GFP, green fluorescent protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 15, pp. 11549 –11561, April 13, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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mediate the ASMase response during monocytic differentia-tion. Later studies demonstrated that AP-2 also up-regulatesASMase in leukemia cells treated with retinoic acid (16). Morerecently, ASMase was implicated in maintenance of calciumhomeostasis in mouse and cell culture models (17, 18). In par-ticular, cerebellar deficits in the Niemann-Pick mouse werereported to be associated with defects in the calcium transportmachinery in the endoplasmic reticulum.Thus, although the scientific literature is replete with reports

implicatingASMase in various cellular responses, the biochem-ical and molecular mechanisms of ASMase regulation remainunclear. Interestingly, recent studies suggest regulation ofASMase by redox mechanisms. For instance, the death ligandTRAIL signals ASMase activation through reactive oxygen spe-cies production (19). Additional mechanisms proposed to reg-ulate ASMase enzymatic activity include interaction with lyso-somal anionic lipids (bismonoacylglycerophosphate) andsphingolipid activator protein SAP-C (20). Nevertheless, theproximal mechanisms involved in direct regulation of ASMaseremain undefined.In a previous study, we identified a novel mechanism of reg-

ulation of ceramide formation by protein kinase C (PKC)through activation of the salvage pathway (1). In this model,treatment of MCF-7 breast cancer cells with PMA, a potentPKC agonist, resulted in a significant increase in cellular cera-mide concomitant with a drop in sphingomyelin levels. Inter-estingly, this response was blocked upon treatment with theceramide synthase inhibitor, fumonisin B1, but not an inhibitorof serine palmitoyltransferase, myriocin, thus ruling outinvolvement of the de novo pathway.In this study, we demonstrate that phorbol esters induce acti-

vation of ASMase through PKC�-dependent phosphorylation.Using site-directed mutagenesis, it is shown that PMA inducesphosphorylation of ASMase at Ser508, and this phosphorylationis indispensable for activation of the enzyme. Moreover, thisphosphorylation event appears to be required for translocationof ASMase to the plasmamembrane. The significance of Ser508phosphorylation and the role of PKC�-ASMase association arefurther confirmed in the context of agonist-driven ASMaseactivation induced by UV radiation.

EXPERIMENTAL PROCEDURES

Materials—Cell culture material, including RPMI medium,fetal bovine serum (FBS), phosphate-freemedium, and dialyzedserum, were from Invitrogen. Bovine sphingomyelin, phos-phatidylserine, and diacylglycerol were from Avanti polarlipids (Alabaster, AL). [choline-methyl-14C]Sphingomyelin waskindly provided by Dr. Alicja Bielawska (Medical University ofSouth Carolina, Charleston, SC). Antibodies against PKC�(polyclonal) and LAMP-1 were from Santa Cruz Biotechnol-ogy, Inc. (Santa Cruz, CA). Monoclonal antibodies against theV5 epitope andGFPwere from Invitrogen. Anti-phosphoserine(polyclonal) was from Zymed Laboratories Inc. (San Francisco,CA). Purified ASMase was a kind gift from Dr. Gary Smith(Glaxosmithkline). The PKC antibody sampler kit was pur-chased from BD Biosciences. The endoglycosidase H digestionkit and all other materials were from Sigma.

Cell Lines and Culture Conditions—MCF-7 cells were origi-nally purchased from ATCC (Manassas, VA). Cells were main-tained in RPMI 1640 supplemented with 10% FBS at 37 °C in a5% CO2 incubator. Where indicated, cells were shifted to aphosphate-free medium. Testing for the presence of myco-plasma infections was performed routinely on a monthly basis.UV Irradiation—MCF-7 cells grown on 10-cm dishes were

coated with a thin layer of phosphate-buffered saline prior toirradiation. Radiation was performed using a GS Gene LinkerUV chamber (Bio-Rad), which emits UV-C light (�avg � 254nm) at a dose of 50 J/m2. Cells were then reincubated withRPMI medium and then collected for analysis at the indicatedtime(s) postirradiation.ASMase Peptide and Antiserum Production—Development

of ASMase antiserum was conducted at the Sigma Genosysfacility. The ASMase peptide sequence was analyzed using thePCGENE program. The program selects regions of a proteinthat are candidates for raising antibodies, based on antige-nicity, flexibility, and �-turn. Based on this analysis, the fol-lowing peptide sequence was chosen and synthesized:CVGELQAAEDRGDKV (15 mer, residues 361–374). Thissequence received the following scores: antigenicity� 2.5; flex-ibility � 1.064; �-turn � 2.4. The peptide was conjugated withkeyhole limpet hemocyanin before immunization of two rab-bits. The third bleed was affinity-purified, and specificity of theantibody was verified by enzyme-linked immunosorbent assay,Western blotting, and immunofluorescence.Site-directed Mutagenesis—Mutations of the serine residues

of ASMase were performed by PCR cloning using theQuikChange site-directedmutagenesis kit (Stratagene) accord-ing to themanufacturer’s instructions. The primers used for theS to A mutation were as follows: S149A, 5�-AGGTGTGGAG-ACGCGCAGTGCTGAG-3�; S231A, 5�-TGCCGCCGGGGT-GCTGGCCTGCCGC-3�; S248A, 5�-CTGGGGCGAATACG-CCAAGTGTGACCTGC-3�; S508A, 5�-TACTCCAGGAGC-GCTCACGTGGTCC-3�; S508E, 5�-TACTCCAGGAGCGA-ACACGTGGTCC-3�. All transformants were sequenced toverify the integrity of mutations.Immunoprecipitation and Western Blotting—Transfections

were performed according to standard protocols using Effect-ene (Qiagen). After 24 h, cells were treated as indicated in thefigure legends. Cells were then lysed in a buffer containing 20mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% Nonidet P-40, 2 mMEDTA, 5 mM sodium fluoride, 1.75 mM sodium orthovanadate,1 mM phenylmethylsulfonyl fluoride, 20 mM �-glycerol phos-phate, 10 �g/ml leupeptin, 10 �g/ml aprotinin, 1 �Mmicrocys-tin. Homogenates were centrifuged at 10,000 � g for 10 min.The supernatants were used for immunoprecipitation ofASMase via amonoclonal V5 antibody (1 �g/ml).Western blotanalysis was carried using similar procedures as describedpreviously (21).SMase Assays—In vitro enzymatic assays for acid and neutral

sphingomyelinases were performed using [choline-methyl-14C]sphingomyelin. The assays were performed as previouslyreported (21, 22).RNA Interference—Gene silencing of human ASMase and

PKC� was performed essentially according to a standard pro-tocol (21). Sequence-specific small interfering RNA reagents

Mechanism of Acid Sphingomyelinase Activation

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were purchased fromQiagen (Valencia, CA). The sequences ofthe sense and antisense small interfering RNAs are shown inTable 1. The specificity of the RNAi was verified by sequencecomparison with the human genome data base using the NIHBlast program.In Vitro Kinase Assays—In vitro phosphorylation reactions

were performed as follows: 6 �g of purified ASMase were incu-batedwith 25ng of recombinant PKC� (Calbiochem) at 30 °C ina kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 0.1 mMEGTA, 0.1 mM ATP), 0.5 �Ci of [�-32P]ATP, and where indi-cated, the buffer was supplemented with lipid-detergent mixedmicelles consisting of 100 �g/ml phosphatidylserine and 10�g/ml diacylglycerol sonicated in 0.3% Triton X-100 solution.At the indicated time points, reactions were terminated by theaddition of SDS sample buffer, and samples were boiled for 5min. Samples were electrophoresed by SDS-PAGE, and thenthe gels were dried. ASMase phosphorylation was detected byautoradiography.In Vivo Labeling of ASMase—MCF-7 cells plated in 10-cm

dishes (250 � 103 cells/plate) were transiently transfected withV5-ASMase. Cells were incubated in phosphate-free RPMImedium supplemented with 5% dialyzed FBS for 1 h. Then cellswere shifted to the labeling medium containing 0.2 mCi/ml[32P]orthophosphate and incubated for an additional 4 h.Treatment with either PMA orMe2SOwas performed in phos-phate-free medium for 1 h. ASMase was immunoprecipitatedvia the V5 epitope and loaded on SDS-PAGE. Incorporation of32P into ASMase was detected by autoradiography after geldrying.Confocal Microscopy—Approximately 5 � 105 cells were

seeded to a 2-cm poly-L-lysine-coated confocal plate (MatTekCorp.). After treatment, cells were fixed with 4% paraformalde-hyde for 10 min and permeabilized with methanol for 5 min.Blockingwas performed in 2.5%FBS solution. Primary antibod-ies were diluted in a solution of 1.5% FBS, 0.15% saponin andincubated for 3 h. The sampleswere thenwashedwith 1.5% FBSsolution three times. This was followed by incubation with sec-ondary antibodies for 1 h at room temperature. Samples werestored at 4 °C until image acquisition. Images were acquiredusing a Zeiss laser-scanning confocal microscope (LSM 510).Excitation wavelengths 488, 543, and 633 were used. Imageswere acquired at the equatorial plane of monolayer cells guidedbyDRAQ5 (Alexis) nuclear stain. For in vivo imaging, cells werefitted in a special chamber under controlled temperature andCO2 settings.Mass Spectroscopy for Ceramide and Sphingomyelin—Sphin-

golipid analysis was performed using electrospray ionization/tandem mass spectrometry on a Thermo Finnigan TSQ 7000triple quadrupole mass spectrometer, operating in a multiplereaction-monitoring positive ionization mode. This methodhas been recently described (23).Plasmid Constructs and Overexpression—Original ASMase

cDNA was a kind gift from Dr. Ed Schuchman (Mount SinaiSchool of Medicine). The cDNA (2.37 kb) encoding humanASMase was excised from the original PSVK3 plasmid usingEcoRI and EcoRV restriction enzymes. After amplification byconventional PCR, the cDNA was ligated into pEF6/V5-His-TOPO vector (Invitrogen). The V5 epitope is situated at the

carboxyl terminus of ASMase. Complementary DNA for PKC�was ligated into pEGFP-C3 vector (Clontech). The restrictionsites used were XhoI and KpnI. The GFP epitope is situated atthe amino terminus of PKC�. The integrity of the new plasmidswas verified by DNA sequencing. For transient overexpressionexperiments, endotoxin-free plasmids were transfected intoMCF-7 cells using Effectene (Qiagen) according to the manu-facturer’s recommendations.Real Time PCR Analysis (Reverse Transcription-PCR/Quan-

titative PCR)—DNA-free messenger RNA from MCF-7 cellswas isolated using the Qiagen minikit for mRNA extraction.Complementary DNA was synthesized from 5 �g of total RNAusing the reverse transcriptase kit from Promega (Madison,WI). Real time reverse transcription-PCRwas performed on aniCycler system from Bio-Rad. The sample real time reversetranscription-PCR reaction volumewas 25�l, including 12.5�lof SYBR Green PCR reagents (Qiagen, Valencia, CA), 7.5 �l ofcDNA template, and 5 �l of the diluted forward and reverseprimers (3 �M). First steps of reverse transcription-PCR were 2min at 50 °C followed by a 10-min hold at 95 °C. Cycles (n� 40)consisted of a 15-smelt at 95 °C, followed by a 1-min annealing/extension at 60 °C. The final step was a 60 °C incubation for 1min. Reactions were performed in triplicate. The threshold forcycle of threshold (Ct) analysis of all samples was set at 0.15relative fluorescence units. The data were normalized to levelsof an internal control gene, �-actin. The reverse transcriptionprimers used in this study were as follows: �-actin (forward,5�-ATTGGCAATGAGCGGTTCC-3�; reverse, 5�-GGTAGT-TTCGTGGATGCCACA-3�) and ASMase (forward, 5�-TGG-CTCTATGAAGCGATGGC-3�; reverse, 5�-TTGAGAGAGA-TGAGGCGGAGAC).Deglycosylation with Endoglycosidase H—Treatment of

immunoprecipitated ASMase with endoglycosidase H (Sigma)was performed according to the instructions of the manufac-turer. Briefly, samples were boiled in denaturation buffer con-taining 50 mM �-mercaptoethanol and 0.1% SDS for 5 min.Samples were cooled on ice for another 5 min. Then endogly-cosidase was added (0.01 units/sample), and the reaction wascarried at 37 °C for 12 h in a reaction buffer consisting of 50mMsodium phosphate, pH 5.5.Statistical Analysis—Mann-Whitney or Student’s t tests

were performed between control and treated states and/orbetween treatment and treatment plus RNAi-mediated inhibi-tion states in a minimum of three independent experiments. Ap value of 0.05 or less is considered as statistically significantand marked in the figures with an asterisk.

RESULTS

PMA Induces Activation and Membrane Translocation ofASMase—Previously, we reported that PMA treatment inducesan elevation in ceramide levels that persisted after blocking thede novo pathway with myriocin but was inhibited by fumonisinB1, suggesting operation of the salvage pathway (resynthesis ofceramide from sphingosine derived from the breakdown ofcomplex sphingolipids but not from de novo synthesis) (1). Theconcomitant drop in sphingomyelin levels induced by PMAtreatment, therefore, prompted us to investigate the potentialinvolvement of SMases. As shown in Fig. 1A, treatment of



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MCF-7 cells with 100 nM PMA resulted in transient activationofASMase, with themaximum in vitro activity detected after 60min (Fig. 1A). Since diacylglycerol has been suggested to acti-vate ASMase directly (24), the effect of direct addition of PMAinto the enzyme reaction buffer (post-cell lysis) was evaluated;however, PMA failed to activate ASMase directly (data notshown), suggesting that the stimulation process was indirect.Moreover, no change in total cellular neutral SMase activitywas detected within 60 min of PMA treatment (Fig. 1A).Next, we examined whether activation of ASMase was asso-

ciated with a change in its subcellular localization. For thesestudies, a novel polyclonal antibody that recognizes ASMasewas employed. After affinity purification of third bleed serum,the specificity of the antibody was tested.Western blot analysisof liver homogenates derived from WT and ASMase�/� micerevealed that the new antibody detected the 72-kDa form ofASMase (supplemental Fig. 1A). Moreover, the antibodyproved useful for immunofluorescence applications, as indi-

cated by colocalization of its signal with the lysosomal markerLAMP-1 (lysosomal-associated membrane protein 1) (supple-mental Fig. 1B).As expected, under basal conditions, most of cellular

ASMase was concentrated within acidic compartments, as evi-denced by colocalization with the acidophilic dye lysotrackerred. There was no change in ASMase staining acutely afterPMA treatment (5 min). However, confocal microscopy imag-ing revealed translocation of ASMase from endolysosomes tothe plasmamembrane after sustained PMA treatment (100 nM,60 min) (Fig. 1B). Therefore, these data demonstrate that thestimulatory effect of PMA on ASMase is associated with relo-cation of the enzyme to the plasma membrane.

FIGURE 1. Transient activation of ASMase after treatment with PMA.MCF-7 cells were seeded in 100-mm dishes (5 � 105 cells/plate). Then cellswere treated with either PMA (0.1 �M) or organic solvent (Me2SO (DMSO)) forthe indicated time course prior to collection and analysis of SMase activity asdescribed under “Experimental Procedures.” A, in vitro SMase (ASMase (black)and neutral SMase (NSMase) (gray)) activity as detected in lysates (100 �geach) derived from cells treated with PMA. B, subcellular localization ofASMase as detected by indirect immunofluorescence. Cells plated on poly-L-lysine-coated 2-cm dishes (50 – 60% confluence) were treated with eitherMe2SO or PMA (0.1 �M; 5 and 60 min). Staining for ASMase was detected in thegreen channel (Alexa Fluor 488), lysotracker red is detected in the red (rhoda-mine) channel, and the third channel represents an overlay of the two. Scalebar, 10 �m. Results shown are representative of three independent experi-ments � S.E.

FIGURE 2. Effect of ASMase knockdown on ceramide formation post PMAtreatment. A, quantitative PCR analysis of ASMase after transfection ofASMase RNAi or control (SCR) sequences. Transfection was carried at 60%confluence using Oligofectamine (Invitrogen), and cells were incubated for48 h. Then cells were collected for quantitative PCR analysis as indicatedunder “Experimental Procedures.” The 21-bp sequences used are illustratedin Table 1. �-Actin was used as internal control, and results were normalizedto the amount of �-actin. B, in vitro ASMase activity after knockdown usingRNAi technology. Cells were transfected with a 10 nM concentration of eitherSCR or ASMase RNAi (sequence 1) for 48 or 72 h. Lysates (100 �g) from eachtreatment group were subject to an ASMase activity assay. C, effect of ASMaseknockdown on PMA-induced ceramide formation. MCF-7 cells plated at 60%confluence were transfected with either ASMase RNAi or SCR sequence for48 h. Consequently, cells were treated with either PMA (100 nM, 1 h) or Me2SO.The different treatment groups were subjected to mass spectrometric analy-sis as indicated under “Experimental Procedures.” Ceramide measurementswere normalized to total phospholipids. Quantitative PCR measurements areaverages of two experiments. Ceramide and SMase activity results were per-formed in triplicate. Shown are averages � S.E. (p � 0.05). ns, not significant;CN, control.



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Requirement for ASMase in PMA-induced Ceramide For-mation—The contribution ofASMase to the observed accumu-lation of ceramide following PMA treatment was consequentlyinvestigated. Although tricyclic amines (and other closelyrelated compounds) have been routinely used to blockASMase,our recent data showed that the action of these compounds isnot restricted to ASMase (21). Therefore, knockdown ofASMase was attempted using two different RNAi sequences.Real time PCR analysis was employed to analyze levels ofASMase mRNA (with �-actin as internal control) 48 h aftertransfection of two different RNAi sequences. As shown in Fig.2A, successful knockdown of ASMase mRNA was achievedwith one of the two RNAi sequences (sequence 1) at a concen-tration as low as 5 nM. Knockdown of ASMase was further con-firmed by in vitro ASMase enzymatic assay. Transfection ofsequence 1 (5 nM) resulted in about 70% reduction in totalASMase activity observed at 48 and 72 h post-transfection (Fig.2B). Therefore, cells were transfected with either scrambled(SCR) or ASMase RNAi (5 nM for 48 h) prior to treatment withPMA. Mass spectrometric analysis showed a doubling in totalcellular ceramide levels in response to PMA in cells pretreatedwith SCR sequence. This response was significantly blocked incells where ASMase was knocked down (Fig. 2C). Takentogether, these results suggest that ASMase is required forPMA-induced ceramide formation in the salvage pathway.Role of PKC� in Activation of ASMase—Phorbol esters signal

primarily through proteins with C1 domains, of which the clas-sical and novel PKCs are the most studied. Therefore, to inves-tigate the possible involvement of PKCs inASMase activation, apanel of pharmacologic inhibitors that block different classes ofPKCs was employed. In vitro SMase assays showed that pre-treatment with either bisindoleilamide, an inhibitor of classicaland novel PKCs, or rottlerin, an inhibitor of novel PKC�,blocked the ASMase activation in response to PMA. However,the classical PKC inhibitor GO 6976 did not significantly affectASMase activity (Fig. 3A). Based on the above results and onpublished studies on the role of PKC� in mediating stressresponses, we hypothesized that the novel PKC� may mediateactivation of ASMase. RNAi knockdown of PKC�was thereforeemployed. Transfection of a specific PKC� RNAi sequence(Table 1) at a dose of 5 nM for 48 h resulted in substantialdown-regulation of PKC� at the protein level without influenc-

FIGURE 3. Involvement of PKC� in ASMase activation. A, MCF-7 cells werepretreated with the PKC inhibitor bisindoleilamide (Bis) (1�M), classical PKC inhib-itor GO 6976 (3 �M), or the novel PKC inhibitor rottlerin (10 �M) for 1 h. Then cellswere stimulated with PMA for an additional 1 h prior to cell collection and lysis.

SMase assay was conducted in an acidic buffer (pH 4.5), and results are nor-malized to protein levels. B, knockdown of PKC� using RNA interference. Cellswere transfected with a 5 nM concentration of either SCR or PKC�-specificRNAi for 48 h. Cells were collected and lysed, and 30 �g of lysates from eachsample were loaded to 10% SDS gels. PKC�, -�, -�, -�, and - were detected at78, 75, 79, 90, and 68 kDa, respectively, using specific polyclonal and mono-clonal antibodies. C, effect of PKC� knockdown on ASMase activity. Aftertransfection of either SCR or PKC� RNAi sequences (48 h, 5 nM), cells weretreated with PMA (100 nM, 1 h). After cell lysis, 100 �g of each lysate were usedfor determination of ASMase activity. D, cells were treated as indicated in C.After Bligh and Dyer lipid extraction, ceramide levels were determined bymass spectroscopy. Results are normalized to total phospholipids. Shown areaverages of three independent measurements of SMase activity and cera-mide levels � S.E. (p � 0.05). ns, not significant. E, cells were double trans-fected with V5-ASMase and PKC� RNAi (or SCR) for 48 h. After treatment witheither PMA (100 nM) or Me2SO for 48 h, cells were stained with a V5 mono-clonal antibody (green channel). Nuclei were visualized via DRAQ-5 nuclearstain (red). Results shown are representative of images obtained in three inde-pendent experiments. CN, control.



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ing total cellular levels of other detected PKC isoforms (Fig. 3B).Interestingly, knockdown of PKC� blocked activation ofASMase after PMA treatment but had no effect on basal enzy-matic activity (Fig. 3C). In further support of these results, massspectrometric analysis revealed a proportional drop in cera-mide levels after PKC� knockdown (Fig. 3D). Additionally,knockdown of PKC� blocked the PMA-inducedASMase trans-location to the plasma membrane (Fig. 3E). Therefore, theseresults indicate that PKC� lies upstream of ASMase activation.Early Translocation of PKC� to the Plasma Membrane Fol-

lowed by Partial Localization to the Lysosome—Similar to mostother members of the PKC family, translocation of PKC� fromthe cytosol to the plasma membrane is a hallmark of its activa-tion. However, it is worth mentioning that additional sites ofPKC� translocation have been recently reported, includingmitochondria (25, 26), Golgi (27), and the nuclear envelope (28,29). Using confocal microscopy imaging of live cells, time-de-pendent changes in the subcellular localization of PKC� wereexamined after PMA treatment. To that end, MCF-7 cells weretransiently transfectedwithGFP-tagged PKC� and labeledwiththe acidophilic dye lysotracker red. Initially (t � 0), PKC�adopted a diffuse cytosolic pattern, whereas lysotracker redconcentrated mainly within compartments of different sizes,consistent with endolysosomes. These compartments were notonly undergoing fission and fusion but also in continuous con-tact with the plasma membrane (see supplemental material formovie). As expected andwithin 1min of PMA treatment, PKC�translocated to the plasmamembrane alongwith clearing of theGFP signal from the cytosol (Fig. 4). In some instances, trans-location of PKC� was seen as early as 30 s after PMA addition.This localization pattern persisted up to 15 min. By 20 min,PKC� was seen to (a) internalize within endocytic compart-ments that colocalized with lysotracker red and (b) adopt aconcomitant perinuclear distribution (Fig. 4 and supplementaldata). It should be also noted that the intensity of the lyso-tracker red signal was preserved up to 1 h, suggesting that PMAtreatment does not profoundly influence lysosomal integrity.Therefore, the above results raised the possibility that inaddition to the plasma membrane, PMA treatment inducesredistribution of a portion of PKC� to ASMase proximal sites,such as endolysosomes.Formation of a PKC�-ASMase Complex upon PMA Treat-

ment—Given that PMA treatment induced ASMase activa-tion as well as PKC� relocation to endolysosomes withincomparable time frames, it became important to determinewhether there is direct or indirect protein-protein interac-tion between PKC� and ASMase. To that end, ASMase wasimmunoprecipitated from lysates of cells treated with eitherMe2SO or PMA. The isolated immune complexes were thentested for the presence of PKC� using a polyclonal antibody.Although PKC� was absent (or below detection level) in

complexes derived from cells treated with a short course ofPMA (5 min), PKC� coimmunoprecipitated with ASMaseafter 60 min of PMA treatment (Fig. 5A), corresponding tothe time frame in which maximal ASMase activation wasdetected. These results were further corroborated by tran-sient overexpression of PKC� (GFP-tagged) and ASMase(V5-tagged). Association of PKC� and ASMase was againobserved to be dependent on PMA stimulation (Fig. 5B). Thesubcellular site of PKC�-ASMase association was furtherexamined by immunofluorescence. Consistent with the pre-vious result (Fig. 4, third panel), within 30 min of PMA stim-ulation, ASMase and PKC� co-stained in a perinuclear sub-set of lysosomes, and only minimal ASMase translocationwas observed (Fig. 5C). Taken together, these results suggestthat sustained stimulation of PKC� with PMA induces itstranslocation to endolysosomes and further association withASMase.Phosphorylation of ASMase in Vitro by PKC�—The above

results prompted us to investigate whether ASMase is a poten-tial substrate for PKC�. To that end, an in vitrophosphorylationassay was performed using recombinant PKC� and ASMaseproteins. The purity of the recombinant proteins was evaluatedby Coomassie Blue gel staining, which revealed only the majorbands for ASMase at 72 kDa and PKC� at 78 kDa (data not

FIGURE 4. PMA induces early plasma membrane translocation of PKC�followed by lysosomal internalization. Cells were plated on 2-cm glass bot-tom dishes (5 � 104 cells/dish). After transient transfection with GFP-PKC�,cells were incubated for 24 h for overexpression. Lysotracker red dye wasadded 30 min prior to PMA stimulation. Cells were fitted inside an in vivoimaging chamber with controlled CO2 and temperature settings. Imageswere acquired at the nuclear plane at a frequency of 4 scans/min over a timeframe of 30 min. Shown are representative images of the in vivo imagingexperiment with the corresponding time after PMA stimulation indicated.The complete movie is provided as supplemental data. Scale bar, 20 �m.

TABLE 1RNAi target sequences for specific protein knockdown

Target protein RNAi target sequence (21 bases)hASMase (sequence1) AAC TCC TTT GGA TGG GCC TGG

hASMase (sequence2) AAG GTT ACA TCG CAT AGT GCC

PKC� AAC GAC AAG ATC ATC GGC AGA



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shown). In the absence of diacylglycerol and PS, only minorincorporation of 32P into ASMase could be detected (Fig. 6,top). However, the addition of diacylglycerol (10 �g/ml) and PS(100�g/ml) resulted inASMase phosphorylationwithin 10minof coincubation with PKC� and was further enhanced after 60min (Fig. 6, bottom).

PKC� Phosphorylates ASMase atSer508—Based on the data above, itbecame important to investigatewhether ASMase is regulated byphosphorylation and to determine ifthere are specific phosphorylationsites that respond to PKC�. Thehuman ASMase primary sequencewas analyzed using the NetPhosphosphorylation prediction algo-rithm. Four potential serine phos-phorylation residues were found tobe conserved in mouse, rat, andCaenorhabditis elegans homologsof ASMase. Interestingly, two ofthese sites were also reported to bemutated in Niemann-Pick diseasepatients (30). To investigate phos-phorylation of ASMase at thesesites, a site-directed mutagenesisapproach was employed, whereeach of the candidate serines wasmutated to alanine. After transienttransfection into MCF-7 cells, theactivity, expression, and localizationof all four mutants were determinedand compared with wild type(WT) ASMase. In vitro SMaseassays and Western blot analysisrevealed similar basal activitiesand degree of expression amongthe mutant andWTASMases (Fig.7A). Furthermore, an endolysoso-mal staining pattern was observedfor these mutants and confirmedby partial colocalization withLAMP-1 (Fig. 7B).Next, the response of the four

Ser 3 Ala mutants to agonist-driven activation of ASMase wasexamined. Three of the mutantsexhibited a response comparablewith that of WT ASMase uponstimulationwith PMA (100 nM, 1 h).In contrast, mutation of serine 508to alanine abrogated PMA-inducedASMase activation (Fig. 8A). Serinephosphorylation of WT and thefour Ser 3 Ala ASMase mutantswas studied next. As shown in Fig.8B, although PMA treatmentinduced serine phosphorylation of

WT and three of the mutants, ASMaseS508A failed to show anychange (Fig. 8B). Interestingly, a basal phosphoserine signalcould be detected and remained unchanged in all mutants.Next, 32P metabolic labeling studies were employed to cor-

roborate these results. However, there was a major drawbackfor this approach, since initially a very intense signal was

FIGURE 5. Association of ASMase and PKC� after PMA treatment. A, immunoprecipitation (IP) of endoge-nous ASMase and PKC�. Cells grown on 10-cm plates were treated with either vehicle (Me2SO) or PMA (100 nM)for 5 or 60 min. After cell lysis, ASMase was immunoprecipitated overnight from the different samples using ahomemade polyclonal antibody. Representative samples of the immune complexes and supernatants wereloaded on 10% SDS gels. The presence of PKC� was checked by Western blotting. B, immunoprecipitation ofoverexpressed PKC� and ASMase. MCF-7 cells transiently overexpressing PKC� and ASMase were treated withPMA (100 nM) for 1 h. PKC� (via GFP tag) or ASMase (via V5 epitope) was immunoprecipitated, separated onSDS-PAGE, and subjected to Western blot for V5-ASMase or GFP-PKC�. Total cellular lysates were also sepa-rated by SDS-PAGE and analyzed for levels of ASMase and PKC� overexpression (bottom). Blots shown arerepresentative of those obtained in three independent experiments. C, cells were plated on 2-cm glass bottomdishes (5 � 104 cells/dish). After treatment with either PMA (100 nM) or Me2SO (DMSO) for 30 min, cells wereco-stained using polyclonal antibodies for ASMase (red channel) and PKC� (green channel), as described under“Experimental Procedures.” Results shown are representative of images obtained in at least three independentexperiments.



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detected by autoradiography for both WT and ASMaseS508Athat masked any differences in peptide phosphorylation. Thissignal has been examined previously and found to be due tophosphomannose glycosylation of asparagine residues ofASMase (7). Therefore, digestion with endoglycosidase H wasemployed to remove this phosphomannose. Indeed, endogly-cosidase H abrogated this signal (Fig. 3C), supporting the pre-vious findings that the 32P labeling was preferentially takingplace at mannose 6-phosphate (7). A concomitant decrease inmolecular mass of about 10 kDa was also detected. In line withprevious studies, basal phosphorylation of the ASMase peptidecore was still observed after deglycosylation (31). Importantly,the addition of PMA induced a significant increase in the phos-phorylation of WT ASMase but not ASMaseS508A (Fig. 8C).Moreover, ClustalW analysis of ASMase sequence revealedthat Ser508, located within the catalytic domain, is conserved inhuman, mouse, and monkey homologs (Fig. 8D). Therefore,these results corroborate the phosphorylation of ASMase inresponse to PMA and provide strong evidence that Ser508 isinvolved in this phosphorylation.Next, the role of PKC� in PMA-induced ASMase phospho-

rylationwas investigated. Knockdownof PKC�was successfullyachieved (more than 80%) via PKC�-specific RNAi oligonu-cleotides. Phosphorylation of ASMase in response to PMAwasdetermined 48 h after transfection of either PKC� RNAi orcontrol sequence (SCR). As shown in Fig. 8E, loss of PKC�impeded the ability of PMA to induce ASMase serine phospho-rylation. Therefore, these findings provide genetic evidence forthe requirement of PKC� in the ASMase response to PMA.

UVLight Triggers the PKC�-ASMase Pathway—Based on theabove biochemical studies, it became important to determine ifthe phosphorylation of Ser508 is required for stress-inducedactivation of ASMase. To this end, MCF-7 cells overexpressingWTASMase or ASMaseS508A were exposed toUV-C light radi-ation (50 J/m2), a previously established ASMase stimulant(32–34). Phosphorylation of ASMase was examined 10 minafter UV exposure, a time where maximal ASMase activity wasdetected. In line with previous results, basal serine phosphoryl-ation could be detected in untreated cells overexpressing WTASMase and ASMaseS508A. However, enhanced serine phos-phorylation, comparable with the one previously observed withPMA treatment (positive control), was detected inWTASMasebut not ASMaseS508A overexpressor following UV exposure(Fig. 9). These results demonstrate that phosphorylation ofASMase at Ser508 is induced by stress stimuli as represented byUV light.ASMaseS508A Acts as a Dominant Negative ASMase—Several

recent reports described that activation ofASMase is associatedwith its translocation from the endolysosomal compartment tothe plasma membrane (35). Since the above results indicated asimilar translocation process after PMA treatment, it becameimportant to investigate whether ASMase translocation to theplasma membrane is dependent on Ser508 phosphorylation. Tothat end, MCF-7 cells plated on poly-L-lysine-coated confocaldishes were transiently transfected with either WT or S508AASMase. Although a similar distribution pattern was observedunder basal conditions, both PMA and UV light stimulationinduced membrane association of only WT and notASMaseS508A (Fig. 10A). Therefore, these findings suggest thatphosphorylation of Ser508 is also required for ASMase translo-cation to the plasma membrane.The significance of Ser508 phosphorylation in the response of

MCF-7 cells to PMA was further pursued. Cells transientlyoverexpressing either WT or ASMaseS508A were treated withPMA (1 h). After lipid extraction, samples were subjected tomass spectrometric analysis of different ceramide species. Nomajor differences in the different ceramides were seen underbasal conditions. However, upon PMA treatment, a majorincrease in C16-ceramide and a minor one in C24-ceramidewere detected only inWT- and not ASMaseS508A-overexpress-ing cells (Fig. 10B). Therefore, these results demonstrate thatphosphorylation of Ser508 is required for agonist-induced acti-vation of ASMase and subsequent ceramide generation.

DISCUSSION

ASMase is a well characterized lipid phospholipase with sug-gested roles in cellular stress responses, yet its biochemical andmolecular mechanisms of regulation remain largely unknown.The results from the present study show that activation ofASMase proceeds through phosphorylation at Ser508. In addi-tion to its activation, translocation of ASMase from the endoly-sosomal compartment to the plasma membrane was found tobe dependent on this phosphorylation step. The study also indi-cates that PKC� is an important kinase that could functionupstream of ASMase in cell signaling pathways.A major conclusion from this study relates to the role of

ASMase in the regulated salvage pathway of ceramide accumu-

FIGURE 6. In vitro phosphorylation of recombinant ASMase by PKC�. Invitro phosphorylation of purified ASMase (72 kDa) with human recombinantPKC� was carried out as described under “Experimental Procedures.” Thetime courses of the phosphorylation of ASMase were detected by autoradiog-raphy after 48 h of film exposure. The experiment was performed either in theabsence (top) or presence of PKC lipid activators (diacylglycerol (10 �g/ml)and PS (100 �g/ml)) (bottom). The same experiment was performed twicewith similar results obtained.



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lation. It was shown previously that the ceramide response toPMA stimulation requires ceramide synthase activity (inhib-ited by fumonisin B1) but not de novo synthesis (not inhib-

ited by myriocin). Using a meta-bolic labeling approach, the samestudy indicated that the ceramideresponse originated from break-down of complex sphingolipids andnot from de novo synthesis (1).Therefore, these results indicatedthe operation of the “salvage path-way,” whereby complex sphingolip-ids are broken down to ceramideand then to sphingosine, which isthen reacylated to ceramide.In preliminary results, it was

found that PMA stimulated break-down of SM; therefore, it becameimportant to determine the enzymeresponsible for the initiation of thesalvage pathway. The results fromthis study implicate ASMase in theceramide response to PMA. This issupported by two lines of evidence.First, RNAi targeting of ASMasesignificantly attenuated ceramideaccumulation in response to PMA.Second, the dominant negativeASMaseS508A mutant also inhibitedthe PMA response. Therefore, inaddition to the classical de novoand SM breakdown pathways,these results highlight a third“hybrid” salvage pathway of cera-mide formation.Interestingly, this salvage path-

way, by combining ASMase acti-vation with fumonisin-inhibitableceramide formation, may explainpreviously seemingly contradictoryresults. For example, the chemo-therapeutic agents fenretinide andanandamide were shown to induceceramide production through acti-vation ofASMase (36, 37).However,other reports showed that ceramidegenerated by these agents can beblocked by fumonisin B1 treatment(38, 39). In some cases, there weredifferent results reported for thesame cell line. Although radiationof MOLT-4 cells induced break-down of sphingomyelin to ceramidethrough activation of ASMase, itwas shown in a recent study that thisceramide response can be blockedby fumonisin in the same cell line(40, 41). Also, a previous study

showed an important role for ceramide synthase in the PMAresponse of LNCaP prostate cancer cells (42); however, theeffects on SM turnover were not investigated.

FIGURE 7. Characterization of ASMase serine to alanine mutants. A, expression and activity of ASMaseSer3 Ala mutants. Cells grown on 10-cm dishes (60% confluence) were transiently transfected with WT ormutant ASMase plasmids (V5 tagged). After 24 h, cells were collected and lysed, and lysates were used forASMase activity assays. Alternatively, lysates were electrophoresed by SDS-PAGE, and ASMase overexpressionwas detected by Western blotting (via V5 epitope). B, localization of ASMase mutants. MCF-7 cells transientlyoverexpressing the indicated ASMase mutants (V5-tagged) were analyzed by confocal microscopy. ASMasewas visualized by staining for V5 epitope (green channel), and LAMP-1 (red channel) was used as a lysosomalmarker. Nuclei were visualized via DRAQ-5 nuclear stain (blue). Scale bar, 5 �m. Data are expressed as mean �S.E. from three SMase activity assays.



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The effects of PKCagonists on sphingolipidmetabolismhavebeen studied previously. In GH3 pituitary cells, it was shownthat diacylglycerol but not phorbol esters directly trigger SMbreakdown independent of PKC (24). However, using the same

metabolic labeling approach with radioactive sphingomyelin asin the aforementioned study, Tettamanti et al. (43) came to adifferent conclusion. The authors found that treatment of thehuman neuroblastoma cell line SH-SY5Y with PMA inducesSM breakdown, a critical step in the differentiation of this cellline. In agreement with the latter study, we previously reportedthat stimulation of theMCF-7 breast cancer cell line with PMAtriggered the sphingomyelin breakdown pathway (1). In addi-

FIGURE 8. Role of Ser508 in ASMase activation. Cells transiently overexpressingWT ASMase or the indicated Ser3Ala mutants were treated with either vehicle(Me2SO) or PMA for 1 h. A, lysates (100 �g each) were used for the ASMase enzy-matic assay described under “Experimental Procedures.” B, ASMase was immu-noprecipitated (IP) (via V5 epitope) from each sample. Immunoprecipitates wereelectrophoresed by SDS-PAGE and probed using anti-phosphoserine antibody.C, in vivo phosphorylation of WT ASMase and ASMaseS508A. Cells transiently trans-fected with either WT ASMase or ASMaseS508A were labeled with [32P]orthophos-phate for 1 h, followed by treatment with PMA for 1 h. After immunoprecipitationwith a V5 monoclonal antibody, ASMase was deglycosylated by overnight incu-bation with endoglycosidase H (Endo H) (except the first lane sample). D, ClustalWalgorithm sequence alignment of ASMase sequences from different organisms inthe region of Ser508 of human ASMase. E, cells overexpressing WT-ASMase weretransfected with PKC� RNAi or SCR (5 nM) for 48 h. ASMase was immunoprecipi-tated 10 min after treatment with PMA (100 nM, 1 h), using a V5 monoclonalantibody. Serine phosphorylation was evaluated by immunoblotting using aphosphoserine polyclonal antibody. Data are from one of three independentexperiments with similar results (p � 0.05). ns, not significant. CN, control.

FIGURE 9. Role of Ser508 in UV light-induced ASMase activation. MCF-7cells overexpressing WT-ASMase or ASMaseS508A were seeded in 100-mmdishes (5 � 105 cells/plate). ASMase was immunoprecipitated (IP) 10 min afterexposure to UV light or treatment with PMA (100 nM, 1 h), using a V5 mono-clonal antibody. Serine phosphorylation was evaluated by immunoblottingusing a phosphoserine polyclonal antibody. Blots shown are representativeof at least three independent experiments. CN, control.

FIGURE 10. ASMaseS508A displays dominant negative properties.A, ASMaseS508Afails to translocate to the plasma membrane. Cells transientlytransfected with WT ASMase or ASMaseS508A were analyzed by confocalmicroscopy prior to and after treatment with PMA for 1 h or 10 min afterexposure to UV light (� � 254 nm, 50 J/m2). ASMase was stained using a V5antibody (green channel), and nuclei were visualized using a nuclear dye (redchannel). Scale bar, 5 �m. B, overexpression of ASMaseS508A blocks ceramideformation after PMA treatment. Cells transiently overexpressing WT ASMaseor ASMase S508A were treated with either Me2SO or PMA for 1 h. Lipids wereextracted according to the Bligh and Dyer method, and ceramide specieswere analyzed by mass spectroscopy. Ceramide measurements areexpressed as mean � S.E. from three experiments (p � 0.05). ns, not signifi-cant. Measurements were normalized to total cellular phospholipids. CN,control.



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tion to the difference in cell lines used, it isworth indicating thatthese studies employed different time frames of PMA stim-ulation (1, 12, and 24 h) as well as different methods for SMdetection (mass spectroscopy versus metabolic labeling).Taken together, it is likely that the effects of PKC agonists onthe sphingomyelin/ceramide pathway vary according to the cellline and pattern of PKC expression.Another major conclusion from this study emanates from

defining a molecular/biochemical mechanism for activation ofASMase. Phosphorylation assays and site-directedmutagenesisrevealed that phosphorylation of ASMase at Ser508 is essentialfor activation of the enzyme. Moreover, the results show thatthis phosphorylation is required for translocation of ASMase tothe plasmamembrane. Indeed, this finding potentially explainsprevious reports describing ASMase translocation to theplasma membrane. In response to UV radiation and Fas ligand(CD 95), ASMase has been reported to externalize to choleratoxin-positive membrane domains (so-called “lipid rafts”) (32,33, 44). This process seemed to require the integrity of vesiculartrafficking in addition to microtubules and actin cytoskeleton.Hence, it is tempting to speculate that phosphorylation ofASMase facilitates its interaction with membrane transportand/or docking machinery. This hypothesis is further sup-ported by several studies documenting a central role for PKC�in stress responses elicited by UV radiation and Fas ligand (45–47). However, further studies are required in order to identifyspecific mechanisms by which the phosphorylated form ofASMase translocates to the plasma membrane.Although determining the exact mechanism by which PKC�

interactswithASMase requires further study, it is clear that thiskinase is critical for ASMase activation at least in response to

PMA and UV light. This conclusionmay begin to tie in previous resultson stress signaling involving bothPKC� and ASMase. First, genera-tion of reactive oxygen species hasbeen recently shown to be an oblig-atory step for ASMase activation byUV radiation and TRAIL (19, 33).Interestingly, there are severalreports highlighting the role ofPKC� both as a key target and effec-tor during oxidative stress (48). Sec-ond, the PKC��/� mouse shows aphenotype of suppressed apoptosisin response to tumor necrosis fac-tor-�, UV, and �-radiation (49).Importantly, the aforementionedstress agents signal ceramide gener-ation primarily through ASMaseactivation; this raises the question ofwhether the PKC��/� mouse has adefective sphingomyelin/ceramidestress signaling pathway. Althoughthe results shown in this study indi-cate that PKC� can associate andphosphorylate ASMase in cells andin vitro, we cannot rule out the

potential involvement of other kinases at this point.The identification of key substrates for PKC� has emerged

as an important goal in deciphering mechanisms of action ofthis kinase. Although activation of PKC� has been associatedwith growth arrest and in some cases initiation of an apopto-tic cascade, only few PKC� substrates have been identified.For instance, PKC�-dependent phosphorylation of phos-pholipid scramblase 1 (plasma membrane) (50), phospho-lipid scramblase 3 (mitochondrion) (51), and LaminB(nucleus) (52) are critical events during certain apoptoticcascades. Given the involvement of PKC� in apoptotic path-ways, perhaps it is not surprising that ASMase, which in turnis critical in several cellular stress responses, is a substrateand an effector of PKC�.

The novel PKC� and the sphingolipid ceramide are wellestablished as intermediates in signal transduction pathways,and recent studies have begun to explore the cross-talk betweenthe two (see commentary by Grant and Spiegel (53)). Saito andco-workers (27) showed that ceramide induces translocation ofPKC� to the Golgi apparatus in HeLa cells. In a later study, theauthors reported that ceramide activation of Src kinase inducesphosphorylation of PKC� inside the Golgi and that this processis indispensable for ceramide-mediated apoptosis (54). Fur-thermore, de novo synthesis of ceramide caused cytochrome crelease by targeting PKC� to the mitochondrion in prostatecancer cells treated with DNA-damaging agents (55). The cur-rent results further extend this area of research to show thatPKC� can also function upstreamof ceramide generation throughregulation ofASMase.With this inmind, one can hypothesize theexistence of a positive feedback loop between activation of PKC�and downstream ceramide formation.

FIGURE 11. Hypothetical model for ASMase regulation. The model depicts PKC� as a central kinase upstreamof ASMase activation. After translocating to the plasma membrane, PKC� is differentially trafficked to endoly-sosomes, causing ASMase phosphorylation at Ser508. The phosphorylated form of ASMase (p-ASMase) is con-sequently targeted to the plasma membrane, where it hydrolyzes membrane SM to ceramide.



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The results from this study also raise intriguing questions asto the subcellular site(s) of PKC� and ASMase interaction. Inaddition to the plasma membrane, PKC� has been reported torelocate to other compartments, such as the mitochondrion,the Golgi complex, and the nuclear envelope (48). For the firsttime, we show that sustained activation of PKC�, which inducesan early plasma membrane association, is followed by its relo-cation to endolysosomes. It is more likely that the lysosomalpool of PKC� rather than the plasma membrane one is criticalfor ASMase activation. First, the time course of activation andtranslocation of both PKC� andASMase appears to favor initialinteraction prior to translocation of ASMase to the plasmamembrane. Second, inhibition of phosphorylation of ASMasein the S508A mutant abrogated translocation to the plasmamembrane, demonstrating that regulation by PKC� precedesthis translocation and is required for it. Finally, topologically, aprotein-protein interaction at the plasma membrane seemsunfavored, since ASMase and PKC� are expected to reside onopposite membrane leaflets (external and internal, respec-tively). However, membrane fusion and formation of multive-sicular endolysosomal structures may allow for interaction ofPKC� and ASMase (Fig. 11).

How does Ser508 phosphorylation induce ASMase activa-tion? Although answering this question is rather difficult in thecurrent absence of a crystal structure for ASMase, three spec-ulations can be made. First, given that Ser508 lies within thecatalytic domain, it is possible that this may directly influencecatalysis. Second, it is possible that Ser508 phosphorylationmayincrease the affinity of the enzyme to one of its previouslyreported cofactors: Zn2� (56), the lipid (bismonoacylglycero-phosphate), and/or the protein activator SAP-C (20). Finally,this phosphorylationmay change the pH profile of the enzyme,allowing it to act at the neutral pH of the plasma membrane.Further studies are required to investigate these possibilities.In conclusion, the findings from this study disclose a novel bio-

chemical mechanism of ASMase activation through phosphoryl-ation of the enzyme by PKC�. This phosphorylation appears to beessential for activationaswell as translocationof theenzyme to theplasma membrane. Mass spectrometric measurements of cera-midespeciesandconfocalmicroscopyanalysisof subcellular local-ization showed that the S508Amutant of ASMase exhibits domi-nant negative properties. Based on the results of this study, ahypothetical model for ASMase regulation is illustrated in Fig 11.It is hoped that the delineation of thismechanismofASMase acti-vationwill advance the study of signaling and regulatory pathwaysinvolving this enzyme.

Acknowledgments—We thank the Lipidomics Core Facility (Drs.Jacek Bielawski and Alicja Bielawska) at the Medical University ofSouth Carolina. We are grateful to Dr. Besim Ogretmen for carefulreading of the manuscript and Dr. Edward Schuchman (Mount SinaiSchool of Medicine) for sharing ASMase cDNA.

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Youssef H. Zeidan and Yusuf A. HannunPhosphorylation

-mediatedδActivation of Acid Sphingomyelinase by Protein Kinase C

doi: 10.1074/jbc.M609424200 originally published online February 15, 20072007, 282:11549-11561.J. Biol. Chem.

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activationofacidsphingomyelinasebyproteinkinase c ...april 13, 2007•volume 282•number 15 journal...

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