Regulation of Cyclin-Dependent Kinase 2 Activity by Ceramide

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Experimental Cell Research 261, 303–311 (2000)doi:10.1006/excr.2000.5028, available online at http://www.idealibrary.com on

Regulation of Cyclin-Dependent Kinase 2 Activity by CeramideJ. Y. Lee,1 A. E. Bielawska,2 and L. M. Obeid3

Ralph H. Johnson Veterans Administration and Medical University of South Carolina, Charleston, South Carolina 29425

Cyclin-dependent kinases have been implicated inthe inactivation of retinoblastoma (Rb) protein andcell cycle progression. Recent studies have demon-strated that the lipid molecule ceramide is able toinduce Rb hypophosphorylation leading to growth ar-rest and cellular senescence. In this study, we exam-ined the underlying mechanisms of Rb hypophosphor-ylation and cell cycle progression utilizing theantiproliferative molecule ceramide. C6-Ceramide in-

uced a G0/G1 arrest of the cell cycle in WI38 humaniploid fibroblasts. Employing immunoprecipitationinase assays, we found that ceramide specifically in-ibited cyclin-dependent kinase CDK2, with a mildffect on CDC2 and significantly less effect on CDK4.he effect of ceramide was specific such that C6-dihy-roceramide was not effective. Ceramide did not di-ectly inhibit CDK2 in vitro but caused activation of21, a major class of CDK-inhibitory proteins, and ledo a greater association of p21 to CDK2. Using purifiedrotein phosphatases, we showed that ceramide acti-ated both protein phosphatase 1 and protein phos-hatase 2A activities specific for CDK2 in vitro. Fur-her, calyculin A and okadaic acid, both potent proteinhosphatase inhibitors, together almost completelyeversed the effects of ceramide on CDK2 inhibition.aken together, these results demonstrate a dualechanism by which ceramide inhibits the cell cycle.eramide causes an increase in p21 association withDK2 and through activation of protein phosphataseselectively regulates CDK2. These events may lead toctivation of Rb protein and subsequent cell cycle ar-est. © 2000 Academic Press

INTRODUCTION

The progression of the eukaryotic cell cycle is tightlyregulated by a family of cyclin-dependent kinases

1 Present address: Children’s Hospital Oakland Research Insti-tute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609-1809.

2 Present address: Department of Biochemistry, Medical Univer-sity of South Carolina, 124 Ashley Avenue, Charleston, SC 29425.

3 To whom correspondence and reprint requests should be ad-dressed at the Department of Medicine, Medical University of SouthCarolina, P.O. Box 250779, Room 604 Strom Thurmond Bldg., 114Doughty Street, Charleston, South Carolina 29425. Fax: (843) 876-
5172. E-mail: [email protected].
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(CDKs)4 [1]. Activation of CDK requires the associationof a cyclin and phosphorylation of a conserved threo-nine residue by the CDK-activating kinase [1]. Inhibi-tion of the CDK–cyclin complexes can be achieved ei-ther by phosphorylation of conserved threonine/tyrosine residues or through binding to CDK-inhibitory subunits (CKIs) [1, 2]. p21, one of the majorclasses of CKIs, acts as a universal inhibitor for allCDKs with a preference for CDK2– and CDK4–cyclincomplexes [3–7]. Considerable effort has been directedtoward the understanding of the functions of CDKs incell cycle progression. To date, substantial evidencesuggests a G1- or S-phase role for D-type cyclinsthrough association with CDK4 and CDK6 [8]. CDC2,on the other hand, plays an essential role in mitosis byinteracting with B-type cyclin in G2/M transition [9].CDK2–cyclin E (cycE) complexes are thought to par-ticipate in the G1/S transition [10], whereas associa-tion of CDK2 with cyclin A (cycA) peaks at S phase andmay have a profound effect on proliferation [11–13].

The elucidation of the sphingomyelin cycle providedthe first clear evidence that sphingolipids function assignaling molecules in cell regulation [14, 15]. Onceactivated by extracellular agents such as tumor necro-sis factor a, interferon-g, and 1,25-dihydroxycholecal-ciferol (vitamin D3), sphingomyelinase hydrolyzessphingomyelin and forms ceramide [15–17]. Ceramidethen acts as an intracellular sensor of stress to mediatethe effects of extracellular agents on cell growth, dif-ferentiation, and programmed cell death.

Recently, ceramide has been shown to play an im-portant role in the induction of cellular senescence.Young cells treated with ceramide resembled senes-cent cells morphologically. The levels of ceramide in-creased about fourfold and the neutral sphingomyeli-nase activity also increased significantly in senescentfibroblasts [18]. In addition, ceramide inhibited AP-1activation and c-fos transcription in young fibroblasts.Numerous studies have also shown that ceramide in-duces hypophosphorylation of Rb [18, 19]. Rb, a well-known substrate for cyclin-dependent kinases [20], is

4 Abbreviations used: CDK, cyclin-dependent kinase; CAPP, cer-amide-activated protein phosphatase; GST, glutathione S-trans-ferase; PBS, phosphate-buffered saline; Rb, retinoblastoma; HDF,
human diploid fibroblasts.
0014-4827/00 $35.00Copyright © 2000 by Academic Press

All rights of reproduction in any form reserved.

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304 LEE, BIELAWSKA, AND OBEID

differentially regulated during cell cycle progression.Dephosphorylation of Rb has been correlated withgrowth inhibition in response to extracellular agentsincluding interferon-a, interleukin-6, transforminggrowth factor g, and serum withdrawal [19, 21, 22].

Despite the growing number of potential targets oferamide being found, the lack of -bona fide targets has

limited our knowledge of the regulation of ceramidefunction and the subsequent intracellular signalingevents. It has been shown that ceramide activates aprotein phosphatase (CAPP) in vitro [23]. CAPP be-longs to the PP2A heterotrimeric subfamily of theserine/threonine protein phosphatases and is potentlyinhibited by okadaic acid with an IC50 of 1–10 nM [23].Other studies have demonstrated that CAPP mediatesthe effect of ceramide on c-myc down regulation inmyeloid leukemia cells [24] and it also mediates theantiproliferative effects of ceramide in yeast [25]. Thus,CAPP may be one of the important direct cellular tar-gets of ceramide. However, the molecular mechanismsby which CAPP mediates ceramide action remain un-determined. Other putative targets include ceramide-activated protein kinase, which appears to be a kinasesuppressor of ras [26, 27], and protein kinase C z [28].

In the current study, we set out to evaluate theechanism by which ceramide induces Rb hypophos-

horylation leading to cell cycle arrest and cellularenescence. We demonstrate that C6-ceramide inhibits

CDK2 activity and leads to a G0/G1 arrest of the cellcycle and Rb hypophosphorylation. Interestingly, C6-ceramide has a marginal effect on CDK4 activity. Inaddition, we provide evidence that inhibition of CDK2activity by ceramide may be mediated by activation ofp21 protein and protein phosphatases PP1 and PP2A.Ceramide appears to exert its antiproliferative effectsthrough differential modulation of cyclins and CDKs.

MATERIALS AND METHODS

Materials. WI38 human diploid fibroblasts were obtained fromthe NIA Aging Cell Repository (Rockville, MD). Dulbecco’s modifiedEagle’s medium (DMEM) and fetal bovine serum were supplied byGIBCO Laboratories (Gaithersburg, MD). Cyclin, CDK, CKI antibod-ies, and blocking peptides were purchased from Pharmingen (SanDiego, CA) and Santa Cruz Biotechnology (Santa Cruz, CA). Anti-b-tubulin antibody was from Boehringer Mannheim (Indianapolis, IN).GST–p16 fusion proteins were obtained from Pharmingen.D-Erythro-C6-ceramide and DL-erythrodihydro-C6-ceramide weresynthesized as described [29]. [g-32P]ATP and [32P]orthophosphate

ere from Dupont NEN. Protein A–Sepharose 4B was purchasedrom Pharmacia Biotech (Piscataway, NJ). ECL detection systemas from Amersham Life Science (Arlington Heights, IL). GST–Rb

usion proteins were a generous gift from Dr. Jonothan HorowitzNorth Carolina State University). Okadaic acid and calyculin Aere purchased from CalBiochem (La Jolla, CA). Other reagentsere from Sigma (St. Louis, MO).Cell culture. WI38 HDF were maintained in Dulbecco’s modifiedagle’s medium with 4.5 g/liter glucose and 10% FBS in 5% CO2 at

37°C in a humidified incubator. For treatment, WI38 cells were

seeded at 6 3 105 cells/plate in complete medium. After 2 days,exponentially growing cells were treated with C6-ceramide at variousconcentrations for various lengths of time. In some experiments, cellswere treated with C6-ceramide together with either calyculin A orkadaic acid or both.Cell cycle studies. Cell cycle status was monitored by propidium

odide staining and flow-cytometric analysis as described [29].Metabolic labeling and immunoprecipitation. To label phospho-

roteins, exponentially growing WI38 cells were washed once withris-buffered saline and incubated with 50 mCi/ml [32P]orthophos-

phate (9000 Ci/mmol; Dupont NEN) for 24 h in complete DMEM. Insome experiments, cells were treated with C6-ceramide at the time

hen labeling was initiated. Cells were lysed in lysis buffer (150 mMaCl, 50 mM Tris–HCl, pH 7.4, 0.5% Nonidet P-40, 5 mM NaF, 10

mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml trypsin–chymotrypsininhibitor, 5 mg/ml pepstatin A, 1 mM DTT, 1 mM Na3VO4, 1 mMPMSF). The lysates were incubated with appropriate antibody at 4°Cfollowed by protein A–Sepharose adsorption. The beads were washedand used for in vitro kinase assays (see below) or for Western blotanalysis.

Purification of GST–Rb fusion proteins. Purification was per-formed as described [30]. Briefly, Escherichia coli were transformedwith pGEX fusion plasmids, and the fusion proteins were purifiedfollowing IPTG induction and collected on glutathione-agarose beads(Sigma). Fusion proteins were harvested from 1000 ml of postinduc-tion bacterial extracts and then mixed with glutathione-agarosebeads that had been preequilibrated with NETN (20 mM Tris, pH8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing 0.5%dry milk. The glutathione-agarose beads were washed briefly and theproteins were eluted by reduced glutathione. Purified proteins wereanalyzed by SDS–PAGE and stored in aliquots until use at 270°C.

Western blot analysis. Following ceramide treatment, WI38 HDFwere harvested on ice. Proteins were fractionated on 12.5% gels bySDS–PAGE as described before [31]. The filter membrane wasblocked in 5% dry milk in PBST, incubated with various antibodies,and then developed using an ECL detection system (Amersham).

In vitro kinase assay. The protein A–Sepharose beads were re-suspended in reaction buffer (20 mM Tris–HCl, pH 7.4, 10 mMMgCl2, 10 mM unlabeled ATP, 0.2 mg/ml histone H1, and 2.5 mCi of[g-32P]ATP). The reactions were carried out at 30°C for 10 min anderminated by the addition of an equal volume of 23 sample buffer.

Proteins were separated by SDS–PAGE and visualized by autora-diography.

Phosphatase assay. WI38 HDF were labeled with 1 mCi/ml[32P]orthophosphate in serum-free, phosphate-free DMEM for 3–4 h.

ells were lysed with lysis buffer as described above. The clearedysates (50–60 mg total proteins) were immunoprecipitated with

appropriate antibody and protein A–Sepharose. The immunocom-plexes were washed and used for in vitro phosphatase assay. Phos-

hatase activity was determined as described before with modifica-ion [23]. Briefly, phosphatase activity was assayed in theephosphorylation reaction mixtures containing 50 mM Tris–HCl,H 7.4, and immunoprecipitated complexes in the absence or pres-nce of 20 mM C6-ceramide or C6-dihydroceramide. Typically, 2000-

to 4000-cpm-labeled CDK2 or CDK4 was used in the in vitro phos-phorylation. Reactions were initiated with the addition of purifiedprotein phosphatases (PP1, PP2A, PTP, or PP2B) and incubated for20 min at 30°C. The concentrations of phosphatases were 2.5 3 1025

unit/ml for PP1, 2.5 mg/ml for PP2A, 5 3 1023 unit/ml for PTP, and 13mM for PP2B per reaction. The enzymatic reaction was stopped bythe addition of 0.1 ml of 1 mM KH2PO4 in 1 N H2SO4 followed by 0.3ml of 2% ammonium molybdate and a wait of 10 min. Free 32Pi washen determined as organic extractable counts by the addition of 1 mlf toluene:isobutanol (1:1 v/v). The amount of organic extractable 32Pi

in blank assay was typically ,1% of total radioactivity. One hundred

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305REGULATION OF CDK2 BY CERAMIDE

percent phosphatase activity corresponds to the control level of de-phosphorylation of the total CDK2 or CDK4 substrates.

RESULTS

Inhibition of CDK2 activity in WI38 HDF followingceramide treatment. Since cyclin-dependent kinasesCDC2, CDK2, and CDK4 phosphorylate the Rb protein[32–34], and previous studies have demonstrated thatceramide induces Rb hypophosphorylation [18, 19], wewondered whether treatment by ceramide would mod-ulate the activity of cyclin-dependent kinases. Previousstudies have demonstrated that histone H1 is a rela-tively poor substrate for CDK4–cyclinD (cycD) andCDK6–cyclinD complexes, which preferentially phos-phorylate Rb protein [35–38] through physical interac-tion of Rb protein and D-type cyclins [8, 39]. Our re-sults also showed that CDK2–cyclinA and CDK2–cyclinE complexes preferred histone H1, whereasCDK4–cyclinD complexes phosphorylate Rb proteinmuch better than histone H1 (data not shown). Theactivities of both CDK2 and CDC2 were determined byhistone phosphorylation following immunoprecipita-tion with specific antibodies, whereas CDK4 activitywas determined by immunoprecipitation with anti-CDK4 antibody and analyzed using GST–Rb fusionprotein as an exogenous substrate. We treated WI38HDF with C6-ceramide at various concentrations forvarious lengths of time. Figure 1a shows that ceramideinhibited CDK2 kinase activity significantly. The effectof ceramide on CDC2 was observed only at a relativelyhigh concentration (Fig. 1b). In contrast, CDK4 (Fig.1c) kinase activity was not affected significantly byceramide. The intensity of the Coomassie BrilliantBlue staining indicated that the amounts of proteinloaded in each lane were approximately equal (data notshown). In addition, immunoprecipitation–Westernblotting analysis demonstrated that the amount ofCDK2 protein was not changed significantly by cer-amide (Fig. 1d). Since CDK4 activity was less promi-nent than that of CDK2, we elected to determine if theactivity in the immunoprecipitation was specificallydue to CDK4. We used GST–p16 and a CDK4 peptidein the GST–Rb kinase assay. Figure 1e (top) showsthat p16 (1 mg) was able to inhibit the CDK4 activity inthe GST–Rb kinase assay. Moreover, CDK4 kinaseactivity was significantly inhibited in the presence ofblocking peptide (.10-fold excess peptide antigen),

hereas ceramide did not affect the CDK4 activityFig. 1e, bottom). These studies demonstrate that cer-mide specifically inhibited CDK2 activity with a mildffect on CDC2 but significantly less effect on CDK4ctivity.The inhibition of CDK2 was observed at ceramide

oncentration as low as 3 mM with maximal effects
reached at a ceramide concentration of 10 mM (Fig. 1a).
After 12 h of treatment, C6-ceramide (5 mM) signifi-antly inhibited CDK2 activity. By 24 h of treatment,DK2 activity was almost completely abolished (Fig.f). Thus, the inhibitory effect of ceramide on CDK2ctivity is concentration and time dependent.Cell cycle arrest at G1/S and G2/M phases in WI38DF by ceramide. In order to understand the under-

ying mechanisms by which ceramide regulates Rb pro-ein hypophosphorylation, and the role of ceramide inell cycle progression, exponentially growing WI38DF were treated with various concentrations of C6-

ceramide and analyzed by flow cytometric analysis ofDNA synthesis. Twenty-four hours following exposureto C6-ceramide, a marked decrease in the proportion ofcells in S phase and an increase in the G0/G1 phase ofthe cell cycle was observed at a ceramide concentrationof 3 mM with maximal effects reached at a concentra-tion of 10 mM (Fig. 2). Interestingly, ceramide concen-trations of 10 mM or higher arrested WI38 HDF at bothG0/G1 and G2/M phases of cell cycle (Fig. 2). The effectof ceramide on cell cycle was not observed before 12 htreatment. These results from WI38 HDF were in ac-cordance with results from previous studies in othercell types that ceramide induces cell cycle arrest [29,40]. The antiproliferative effects of ceramide on cellcycle progression mimic what is observed in senescentfibroblasts. Since the inhibition of CDK2 activity oc-curred at an earlier time than the time course for thedetection of cell cycle arrest, it is likely that alterationof CDK2 activity accounts for the subsequent cell cyclearrest. The inhibitory effect of ceramide on CDK2 ac-tivity may lock Rb in a dephosphorylated form andthus lead to cell cycle arrest.

Because ceramide had a mild effect on CDC2 and didnot affect CDK4 activities significantly, we carried outour subsequent studies focusing on CDK2 activity witha ceramide concentration of 6 mM. To define the spec-ificity of ceramide’s effect on CDK2 activity, we com-pared the effects of C6-dihydroceramide, a lipid mole-cule structurally similar to C6-ceramide. The CDK2activity was significantly inhibited by C6-ceramide,whereas C6-dihydroceramide was not effective (Fig. 3).Therefore, the effect of ceramide on inhibition of CDK2activities was specific.

To substantiate the effect of ceramide on cell cycleprogression, we synchronized WI38 cells by serum de-privation followed by serum stimulation. Serum with-drawal arrested cells in the G0/G1 phase of the cellcycle, and stimulation with serum enabled cells to syn-chronously enter S phase (data not shown). Concomi-tantly, serum withdrawal inhibited CDK2 activity andstimulation with serum restored the kinase activity(Fig. 4). C6-Ceramide, when added at the same timewith serum to the previously deprived cells, arrested
cells at G0/G1 phase and inhibited CDK2 activity sig-

Wconcentrations of C6-ceramide for 24 h. The activities of both CDK2

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nificantly at concentrations of 15 and 20 mM (Fig. 4).These results suggest that ceramide mimics the effectof serum withdrawal.

Mechanism of ceramide inhibition on CDK2 activity.To determine the mechanisms by which ceramide reg-ulates CDK activity, we first examined whether C6-ceramide directly affects CDK activity using an in vitrokinase assay. Lysates were first immunoprecipitatedwith anti-CDK2 antibody and the immunocomplex wasincubated with kinase reaction mixture in the absenceand presence of various concentrations of C6-ceramide.As shown in Fig. 5, C6-ceramide had no effect on CDK2activity in vitro. Therefore, these results indicate thatceramide regulates CDK2 activity indirectly.

We then examined whether the inhibition of CDK2activity by ceramide was attributed to alteration ofCDK and/or cyclin proteins. Since ceramide has a pro-found inhibitory effect on S phase of cell cycle, and theassociation of cycA and CDK2 peaks at S phase,whereas the association of cycE and CDK2 peaks at theG1/S transition [13], we focused on the expression ofcycA, cycE, and CDK2 proteins. Western blot analyseswere carried out on lysates from control and ceramide-treated cells. The protein level of both CDK2 and cycEdid not change significantly (Figs. 6a and 6c). However,the amount of cycA showed a small but significantdecrease (Fig. 6b). It is not known what the impact ofthis change in cycA protein is on CDK2 activity.

One mode of regulation of CDK activity is throughCDK inhibitory subunits, which bind to CDK–cyclincomplexes and inhibit kinase activity [1, 2]. To deter-mine whether the inhibition of CDK2 activity wascaused by activation of CKIs, Western blot analyseswere carried out. The results showed that p21 proteinlevels were increased two- to threefold following cer-amide treatment (Fig. 7a). Immunoprecipitation–Western blotting demonstrated that p21 protein is as-sociated with CDK2 and that treatment with ceramideresulted in a higher level (approximately twofold) ofp21 protein associated with CDK2 (Fig. 7b). Ceramide,however, did not affect p27 or p16 proteins signifi-cantly (data not shown). These results suggest thatceramide may lead to activation of p21 protein which

(a) and CDC2 (b) were determined by histone phosphorylation fol-lowing immunoprecipitation with CDK2 and CDC2 antibodies, re-spectively. CDK4 (c) activity was determined by using GST–Rbfusion protein as an exogenous substrate following immunoprecipi-tation with CDK4 antibody. Effects of ceramide on the levels ofCDK2 protein in WI38 cells were determined (d). CDK4 activity wasdetermined in the absence or presence of GST–p16 protein or block-ing CDK4 peptide (e). WI38 cells were treated with 5 mM C6-cer-mide for various lengths of time (f). Proteins were separated byDS–PAGE on a 10% gel [31]. Histone H1 and GST–Rb are indicatedith an arrow on the right. Results are representative of four differ-

FIG. 1. Inhibition of CDK2 activity by C6-ceramide in WI38 cells.I38 cells were treated either with ethanol vehicle or with various

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subsequently inhibits CDK2 activity through a greaterphysical association.

Since cyclin binding and phosphorylation of a con-served threonine residue by the CDK activating kinaseare necessary to activate CDK [1], we next consideredif ceramide, added to the cells, would modulate phos-phorylation of CDK2. WI38 HDF were labeled with

FIG. 2. Effects of C6-ceramide on the cell cycle distribution in WIf C6-ceramide for 24 h. Cells were analyzed as described under Mateells; b, cells treated with 3 mM C6-ceramide; c, cells treated with 6 mre representative of four independent experiments.

FIG. 3. Specificity of inhibition of CDK2 activity by ceramide.I38 cells were treated with equimolar concentrations of C6-cer-

amide and C6-dihydroceramide. In vitro kinase assay was performed
s in Fig. 1. Results are representative of two separate experiments.
[a-32P]orthophosphate in the absence and presence ofC6-ceramide. Lysates were prepared from both controland treated cells and immunoprecipitated with anti-CDK2 antibody. After extensive washing of the immu-nocomplex, the pellets were solubilized with Laemmli[31] buffer and analyzed by SDS–PAGE. Clearly, cer-amide altered phosphorylation of CDK2 but notCDC25A (Fig. 8). These results suggest that phosphor-ylation of CDK2 may represent a target for ceramideaction.

Ceramide has been demonstrated to activate a pro-

ells. WI38 cells were grown and treated with various concentrationss and Methods. (a–d) Quantitative measure of FACS data (a, controlC6-ceramide; and d, cells treated with 10 mM C6-ceramide). Results

FIG. 4. Effects of C6-ceramide on CDK2 activity in synchronizedWI38 cells. Exponentially growing cells (Exp) were serum deprived(SD) for 48 h and restimulated with serum in the absence or presenceof indicated concentrations of C6-ceramide. In vitro kinase assay wasdone essentially the same as in Fig. 1. Results are representative of

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tein phosphatase (CAPP) in vitro [23]. It is not clearwhether CAPP may mediate the cellular action of cer-amide. To determine the role of CAPP in ceramidesignaling, we investigated the ability of ceramide toactivate CAPP, which may lead to inactivation ofCDK2 through dephosphorylation. To evaluate the roleof phosphatase on ceramide signaling, WI38 HDF werelabeled with [a-32P]orthophosphate. Following immu-

oprecipitation with either anti-CDK2 or anti-CDK4ntiserum, phosphatase assay was done with purifiedhosphatase PP1 or PP2A in the presence of C6-cer-

amide using labeled immunocomplex as the substrate.C6-Ceramide (20 mM) was able to activate both PP1(Fig. 9a) and PP2A (Fig. 9b) phosphatase, regulatingCDK2. C6-Dihydroceramide was not effective (data notshown). In addition, C6-ceramide did not activate PP2B(Fig. 9b). Furthermore, CDK4 appeared not to be apotential substrate for either PP1 or PP2A and C6-ceramide was unable to induce dephosphorylation ofCDK4 or increase its activity (Fig. 9c). Thus, differen-tial regulation by phosphorylation may be one of thecontributing factors in the differential responses ofCDK activities.

In order to test the involvement of protein phospha-tases PP1 and PP2A in the regulation of CDK2 activ-ity, we treated WI38 HDF with either of the phospha-tase inhibitors calyculin A or okadaic acid in thepresence of C6-ceramide; they showed modest protec-ive effects. However, when cells were treated withoth calyculin A and okadaic acid together, they wereble to reverse the effects of C6-ceramide on CDK2

inhibition almost completely (Fig. 10). Therefore, inhi-bition of CDK2 by ceramide may be partially mediatedby phosphatases 1 and 2A.

DISCUSSION

In this study, we demonstrate that ceramide inhibitsCDK2 activity and leads to an arrest at G0/G1 phase ofthe cell cycle. The effect of ceramide is concentration

FIG. 5. In vitro effects of ceramide on CDK2 activity. CDK2proteins immunocomplexed to protein A–Sepharose from untreatedWI38 cells were incubated with kinase reaction mixture in the ab-sence or presence of various concentrations of C6-ceramide. In vitro

inase assay was performed as in Fig. 1. Results are representativef two different experiments.

and time dependent and specific in that the closely

related dihydroceramide is ineffective. Importantly,our data show that ceramide inhibits CDK2 activitysignificantly with a mild effect on CDC2 but is lesseffective on CDK4 activities. CDK2 does not appear tobe the direct target for ceramide. Furthermore, alter-ation of CDK2 activity may be due to activation of p21and protein phosphatase such as CAPP. These resultssuggest that antiproliferative molecules (e.g., cer-amide) exert their inhibitory effects on cell growth bymodulating the balance of phosphorylation and de-phosphorylation and by regulating CDK inhibitorysubunits.

FIG. 6. Expression of CDK2 (a), cyclin A (b), cyclin E (c), andtubulin as loading control (d) in WI38 cells following C6-ceramidereatment. WI38 cells were treated with C6-ceramide for 24 h. Pro-

teins were separated by SDS–PAGE. Results are representative of
three different experiments.

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The effects of ceramide on cell cycle progression werepreviously demonstrated in Molt-4 cells [29]. Serum de-privation leads to a significant elevation of ceramide lev-els through activation of a magnesium-dependent neu-tral sphingomyelinase. Ceramide is shown to induce asubstantial cell cycle arrest accompanied by apoptosis.

FIG. 7. Expression of p21 protein (a) and association of p21 withDK2 (b) following the treatment with ceramide. WI38 cells were

reated with C6-ceramide (various concentrations) for 24 h. Proteinsere either separated by SDS–PAGE and Western blot analyses wereone using anti-p21 antibody (a) or immunoprecipitated by CDK2 an-ibody followed by Western blot analyses using anti-p21 antibody (b).esults are representative of two to three different experiments.

Exogenous ceramide mimics the effect of serum with-

experiments.

drawal on the regulation of cell cycle progression. Ourcurrent study using WI38 cells reinforced the significance

FIG. 9. Activation of both PP1 and PP2A by C6-ceramide in vitro.WI38 cells were labeled with [32P]orthophosphate in serum-free,phosphate-free DMEM for 3–4 h. Lysates were prepared and immu-noprecipitated with CDK2 (a, b) or CDK4 (c) antibodies followed byprotein A–Sepharose. Phosphatase activity was determined as de-scribed under Materials and Methods using 32P-labeled CDK2 orCDK4 as the substrates. Results are representative of four differentexperiments.

FIG. 8. Effects of C6-ceramide on CDK2 phosphorylation. WI38ells were labeled with [32P]orthophosphate for 24 h in complete

DMEM. In some experiments, cells were treated with C6-ceramide atindicated concentrations for 24 h. The cleared lysates were preparedand immunoprecipitated by CDK2 (a) or CDC25A (b) antibodies,followed by SDS–PAGE. Results are representative of two separate

of ceramide action on multiple cell types.


Another study using NIH 3T3 fibroblasts overex-pressing insulin-like growth factor-1 receptors re-ported that an inhibitor of glucosylceramide synthase,PDMP, had profound effects on cell cycle progressionand cell-dependent kinases [40]. PDMP inhibited cellcycle progression at both G1/S and G2/M and decreasedp34cdc2 and CDK2 kinase activities. C2-Ceramide mim-icked the effect of PDMP on cell cycle progression. Inour system, C2-ceramide does not have a significanteffect on cell cycle progression in the presence of se-rum. Since C6-ceramide is effective in the presence ofserum, we have therefore chosen C6-ceramide in thestudies of WI38 HDF to minimize the effect of serumdeprivation. In addition, C8-ceramide has a similarinhibitory effect on CDK2 activity (data not shown).

One major mechanism for regulating CDK activity isthrough the interaction of CDK inhibitory proteins.Presumably, most CKIs directly bind to CDK–cyclincomplexes and inhibit kinase activity [1, 2]. We havefound that treatment of WI38 fibroblasts with C6-cer-amide leads to the induction of p21 protein and en-hancement of association of p21 with CDK2 (Fig. 7).These data are consistent with early reports that cer-amide treatment leads to the induction of the CDKinhibitor p21 [41, 42]. These results suggest that cer-amide can affect CDK activity by modulating CDKinhibitory subunits.

Our observations suggest that ceramide may be animportant modulator in orchestrating cell cycle progres-sion. The biological significance of differential regulationof CDKs by C6-ceramide depends on the contribution ofeach downstream effector pathway. Regulation of eachCDK may have a different impact on the phosphorylationof Rb protein and its biochemical functions. Recent stud-ies by Zarkowska and co-workers [43] have demonstratedthat Rb protein is differentially phosphorylated by differ-ent G1/S CDKs. Notably, CDK2–cyclinA, CDK2–cy-clinE, and CDK4–cyclinD1 show distinct phosphoryla-tion sites of Rb protein. Thus activation of CDK2–cyclinEand CDK4–cyclinD may complement each other for theprogression into S phase. Differential regulation of CDKsby ceramide provides an additional mode of regulation incontrolling Rb protein phosphorylation and cell cycle pro-

FIG. 10. Okadaic acid and calyculin A partially block the effectsof ceramide on CDK2 activity. WI38 cells were treated with okadaicacid alone, calyculin A alone, or both in the absence or presence ofC6-ceramide (6 mM). The in vitro kinase assay was performed as inFig. 1. Results are representative of three independent experiments.

gression.

Previously, we showed that ceramide induced Rb hy-pophosphorylation in WI38 HDF [18]. In the currentstudy we also found that cycA and cycE kinase activitieswere significantly inhibited by C6-ceramide (data notshown), whereas cycD3 kinase activity was not affectedby C6-ceramide (data not shown). Therefore, ceramidemay exert its antiproliferative effects on G1/S transitionby locking Rb protein in a dephosphorylated state.

Protein phosphorylation is regulated by protein ki-nases as well as protein phosphatases. Activation ofCDK requires cyclin binding and phosphorylation atthe Thr160/161 residue. Conversely, inhibition ofCDK–cyclin complexes can be achieved by removal ofcyclin or through dephosphorylation of the Thr160/161residue. The importance of the Thr160/161 dephos-phorylation and the regulation of this dephosphoryla-tion are largely unknown. Considerable effort has beendirected toward CDK-activating kinase. Since CDK-activating kinase activity does not appear to be cellcycle regulated, it is not likely to be the limiting factorfor the regulation of Thr160/161 and cell growth [1]. Itis not clear at present whether ceramide exerts itsinhibitory effects on CDK2 through dephosphorylationof the Thr160/161 residue or whether CDK-activatingkinase is a cellular target of ceramide.

Human cyclin-dependent kinase interactor 1 (Cdi1)is a novel type of protein phosphatase with dual spec-ificity that interacts with cyclin-dependent kinases [44,45]. Human Cdi1 or KAP has been shown to dephos-phorylate Thr160 in human CDK2 [46]. In addition,protein phosphatase 2A can dephosphorylate Thr160in vitro in the absence or presence of cyclin [46]. Phos-phorylation at Thr14 and Tyr15 residues in humanCDC2 and CDK2 presents another mode of CDK reg-ulation. Phosphorylation/dephosphorylation of Thr14and Tyr15 is particularly important in the regulationof CDC2 at mitosis. The major protein phosphatasethat dephosphorylates both Thr14 and Tyr15 is CDC25with dual specificity. It has been postulated that PP1and PP2A may act on different targets to preventCDC2 activation [47].

Early studies have demonstrated that ceramide ac-tivates CAPP in vitro [23]. CAPP belongs to the PP2Aheterotrimeric subfamily of the serine/threonine pro-tein phosphatase. In the current study, we found thatC6-ceramide was able to activate both PP1 and PP2Aactivities using CDK2 as a substrate. Furthermore,calyculin A and okadaic acid partially reversed theeffect of C6-ceramide on CDK2 inhibition. These stud-ies suggest that the activation of both PP1 and PP2Aby ceramide may partially account for subsequent in-tracellular events such as inhibition of CDK2 activity.Whether PP2A may act to inhibit Thr160/161 phos-phorylation, whereas PP1 may act to prevent CDC25activation, remains to be elucidated. Our results do not
rule out the possibility that other phosphatases acti-

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vated by ceramide or other signaling molecules alsoparticipate in the regulation of CDK2 activity and Rbhypophosphorylation. Future studies should addresshow the relevant protein phosphatases are activatedby ceramide and how these phosphatases regulate cellcycle progression.

Owing to the complexity of sphingolipid species anddifferent enzymatic pathways in metabolism, it is highlylikely that the cross-talk between the proliferation andthe antiproliferation transduction pathways may influ-ence cell growth. Studies on the interactions betweenlipids and different signaling pathways would help todefine the specific role of ceramide in cell cycle progres-sion and to gain insight on the basics of cell growth.

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Regulation of Cyclin-Dependent Kinase 2 Activity by Ceramide