We have previously shown that interleukin-1 receptor-generated ceramide induces growth arrest in smooth muscle pericytes by inhibiting an upstream kinase in the extracellular signal-regulated kinase (ERK) cascade. Here, we now report the mechanism by which ceramide inhibits ERK activity. Ceramide renders the human embryonic kidney 293 cells (HEK 293) resistant to the mitogenic actions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC-ε activities. To confirm that PKC-ε is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC-ε (ΔPKC-ε). These transfected cells respond to insulin-like growth factor I (IGF-I) with a significantly decreased ERK activity that is not further reduced by ceramide treatment. Coimmunoprecipitation studies reveal that the treatment with IGF-I induces the association of ERK with PKC-ε but not with PKC-ζ. Ceramide treatment significantly inhibits the IGF-I-induced PKC-ε interaction with bioactive phosphorylated ERK. Ceramide also inhibits IGF-I-induced PKC-ε association with Raf-1, an upstream kinase of ERK. Together, these studies demonstrate that ceramide exerts anti-mitogenic actions by limiting the ability of PKC-ε to form a signaling complex with Raf-1 and ERK.
- protein kinase C
- extracelular signal-regulated kinase
- mitogen-activated protein kinase
inflammatory cytokines, including interleukin-1 (IL-1), tumor necrosis factor-α, and interferon-γ, activate sphingomyelinases, resulting in increased cellular ceramide concentration (6, 15, 19). Ceramide is a sphingolipid-derived second messenger molecule implicated as an inducer of cellular differentiation, growth inhibition, and apoptosis (12, 13). We and others demonstrated that the inhibitory action of ceramide in cell growth involves inhibition of extracellular signal-regulated kinase (ERK) activity, a member of the mitogen-activated protein kinase (MAPK) family (7, 22). To date, however, the precise role of ceramide in inhibition of ERK activation and cell growth has not been determined. It is likely that the active site of ceramide is upstream of ERK, since ceramide does not directly regulate immunoprecipitated ERK activity in cell-free systems.
Activation of the ERK signal pathway is characterized by a cascade of protein kinases that are recruited to the plasma membrane. Specifically, GTP-dependent activation of Ras recruits Raf-1 to the plasma membrane, where it is phosphorylated and activated (34). Activated Raf-1 directly phosphorylates and activates mitogen/extracellular signal-regulated kinase (MEK), which in turn directly activates ERK. In addition to Ras, protein kinase C (PKC) has also been shown to activate the Raf-1-MEK-ERK signaling pathway (4).
In response to growth factors, such as insulin-like growth factor-I (IGF-I) and platelet-derived growth factor, PKC is activated through phospholipase C-generated diacylglycerol (DAG). At least 12 distinct isotypes of the PKC family have now been identified and subdivided into the following three classes: conventional (DAG dependent and calcium sensitive), novel (DAG dependent and calcium insensitive), and atypical (DAG independent and calcium insensitive). Among these, DAG-regulated PKC-ε, a member of the novel class of PKC isotypes, activates Raf-1 kinase (28, 31). Overexpression of active PKC-ε overcame the inhibitory effects of dominant-negative Ras, suggesting that PKC-ε-induced activation of the Raf-1-MEK-ERK signaling cascade is independent of Ras activation (4, 32). We have previously reported that the activity of PKC-ε is inhibited significantly by IL-1 treatment in rat mesangial cells (17). Furthermore, we have demonstrated that the cell-permeable ceramide analog, C6-ceramide, mimicked the effect of IL-1 to inhibit both tyrosine kinase receptor- and G protein receptor-linked mitogenesis (7, 17). Because ceramide is structurally similar to DAG, the endogenous cofactor for PKC-ε activation, it is possible that the inhibitory action of ceramide upon growth factor-induced ERK activation and subsequent cell growth inhibition may be due to the antagonistic action of ceramide displacing DAG on PKC-ε.
In this study, we demonstrate that IGF-I treatment induces PKC-ε activation in HEK 293 cells. Upon activation, PKC-ε selectively interacts with Raf-1 and ERK. We also demonstrate that ceramide inhibits IGF-I-stimulated ERK activation and cell growth by direct inhibition of PKC-ε activation and subsequent interaction with Raf-1 and ERK. Together, these findings suggest a novel role of ceramide in modulation of the physical interactions between signaling elements in PKC/MAPK complexes.
MATERIALS AND METHODS
Human embryonic kidney 293 (HEK 293) cells were obtained from American Type Culture Collection (Rockville, MD). Anti-PKC-α, -PKC-ε, -PKC-ζ, -Raf-1, and -ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Purified, recombinant PKC-ε was obtained from Panvera (Madison, WI). 1-Oleoyl-2-acetyl-sn-glycerol (OAG) was purchased from Sigma (St. Louis, MO). DAG (1,2-diolein) was purchased from Serdary Research Laboratories (Ontario, Canada). Cell-permeable C6-ceramide, physiological C18:1-ceramide, and the inactive analog dihydro-C6-ceramide were obtained from Avanti Polar Lipids (Alabaster, AL). Human IGF-I and IL-1β recombinant proteins were purchased from GIBCO (Grand Island, NY). The enhanced chemiluminescence (ECL) detection kit and Gamma Bind Sepharose were obtained from Amersham Life Sciences (Arlington Heights, IL).
HEK 293 cell culture.
HEK 293 cells are adenovirus-transformed HEK cells of tubule epithelial origin. These cells express functional IGF receptors and are an excellent model for growth factor-induced mitogenesis and inflammatory cytokine-induced growth arrest (4). Utilizing RT-PCR protocols, we have shown that these cells express the mRNA for the type 1 form of the IL-1 receptor (data not shown). Western blot analyses revealed that HEK 293 cells express PKC-α, -ε, and -ζ.
HEK 293 cell proliferation assay.
Initially, HEK 293 cells were grown to ∼50% confluency in DMEM cell culture medium containing 10% FBS in 12-well cell culture plates. The HEK cells were then downregulated by a 48-h incubation in DMEM without FBS. The cells were pretreated with either 1 μM C6-ceramide, 1 μM dihydro-C6-ceramide, or vehicle for 1 h and then were treated with mitogens (IGF-I or OAG) for an additional 18 h. These treated HEK 293 cells were further incubated with 0.3 μCi/ml [3H]thymidine during the last 6 h of treatment. The cells were washed one time with ice-cold PBS and then were washed three times for 5–10 min with 10% TCA. The fixed cells were then solubilized in 0.3 M NaOH-0.1% SDS solution, and [3H]thymidine incorporation into acid-insoluble DNA was quantified by measuring radioactivity using a liquid scintillation counter.
Western blot analysis.
Western blot analysis using anti-PKC-ε antibody was performed as previously described (17). Briefly, HEK 293 cells were washed in ice-cold Dulbecco's PBS solution and lysed in 1 ml of ice-cold lysis buffer [20 mM HEPES, 40 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, 0.2% Nonidet P-40 (NP-40), and 1 μg/ml of leupeptin, pepstatin, and aprotinin]. Cell lysates were cleared by centrifugation, and the Bio-Rad protein assay was performed to determine protein concentration. Forty micrograms of protein lysate per sample were separated on a 10% SDS-PAGE and transferred to Hybond nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in Tris-buffered saline (TBS) for 1 h and then were incubated with the primary anti-PKC-ε antibody (1:1,000 dilution in 5% nonfat milk TBS) for 2 h at room temperature. After incubation, the membranes were washed three times with TBS for 10 min each. The blots were then incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:5,000 dilution in 5% nonfat milk in TBS) for 2 h at room temperature. The membranes were then washed three times with TBS, and PKC-ε bands were visualized by ECL and quantified using laser densitometry.
To confirm the specificity of the antibody used for immunoprecipitation of PKC-ε, we reprobed the membranes with additional PKC isotype-specific antibodies. A band corresponding to the molecular weight of PKC-ε was observed when the membranes were probed with the antibody for PKC-ε. In contrast, bands were not observed when the membranes were probed with antibodies for α, δ, or ζ (data not shown). In some experiments, we transfected HEK 293 cells with wild-type and mutant PKC-ε constructs tagged with hemagglutinin (HA). In these experiments, the specificity of transfected PKC-ε was determined by immunoprecipitating PKC-ε using both anti-PKC-ε and anti-HA antibodies.
In vitro reconstitution activity assay for immunoprecipitated or recombinant PKC-ε.
Immunoprecipitation of PKC-ε and the subsequent reconstitution activity assay were adapted from previous methods (2, 17,23). A similar protocol using recombinant PKC-ε (50 ng) was used to verify the in vitro effects of physiological ceramide on immunoprecipitated PKC-ε. Briefly, PKC-ε was immunoprecipitated from HEK 293 lysates using 0.5 μg of polyclonal rabbit anti-PKC-ε antibody. After overnight incubation at 4°C, Gamma Bind Sepharose was added and rotated for 2 h, and the immunocomplex containing PKC-ε was pelleted by brief centrifugation. After three washes, the pellets were resuspended in kinase buffer (50 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 50 mM NaF, 1 mM NaVO4, 1 mM dithiothreitol, and 0.1% Tween 20). The in vitro kinase reaction was initiated by addition of 40 μg/ml phosphatidylserine/reaction, 10 mM MgCl2, 0.25 mM ATP (cold) and 1 μCi [γ-32P]ATP (10 mCi/mmol), and 10 μg histone IIIS as a substrate. In selected experiments, 10 μg of myelin basic protein were used as the exogenous substrate rather than histone IIIS. Specified samples were treated with DAG (1,2-diolein, 1 μM) and/or C18:1-ceramide, C6-ceramide, or dihydro-C6-ceramide (0.1–1 μM). After 20 min of incubation at 37°C, the kinase reactions were terminated by adding SDS-PAGE sample buffer and heating at 95°C for 5 min. Phosphorylated histone IIIS proteins or myelin basic protein was then separated on 12% SDS-PAGE and transferred to Hybond nitrocellulose membranes. The bands corresponding to phosphorylated histone IIIS were detected by autoradiography (Kodak X-OMAT). In contrast, the bands corresponding to phosphorylated myelin basic protein were excised and quantified by liquid scintillation analysis.
Coimmunoprecipitation of PKC with ERK or Raf-1.
Lysates from HEK 293 cells were incubated with rabbit anti-ERK2 (or anti-Raf-1) antibody for 16 h at 4°C. The next day, the goat anti-rabbit antibody conjugated to agarose was added to each sample and incubated for 2 h at 4°C. Immunocomplexes were then pelleted by brief centrifugation and washed two times in lysis buffer (20 mM HEPES, 40 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, 0.2% NP-40, and 1 μg/ml of leupeptin, pepstatin, and aprotinin). Immunoprecipitates were then heated at 95°C for 5 min in SDS-gel loading buffer and separated on 12% SDS-PAGE. Proteins were transferred to Hybond nitrocellulose membranes and probed with either anti-PKC-ε or PKC-ζ antibody (1:1,000 dilution in 5% nonfat milk in TBS). Subsequently, the membranes were incubated with the HRP-conjugated anti-rabbit IgG antibody (1:5,000), and the bands corresponding to PKC-ε or PKC-ζ were visualized by ECL. Equal loading of ERK2 was determined by reprobing the membranes with anti-ERK2 antibody.
For coimmunoprecipitation of PKC-ε with phospho-ERK (pERK), a similar protocol was performed except that pERK was immunoprecipitated using anti-pERK antibody and was transferred to nitrocellulose membranes. Subsequently, the membranes were probed with anti-PKC-ε antibody (1:1,000), and the bands corresponding to PKC-ε were visualized using ECL. Equal loading of PKC-ε protein was determined by reprobing the membranes with anti-PKC-ε antibody. We further verified that the IGF-I-stimulated interactions between PKC-ε and pERK were not the result of a basal level of contaminating pERK. We demonstrated that the increase (relative to controls) in IGF-I-stimulated pERK in lysates immunoprecipitated with PKC-ε was elevated compared with whole cell lysates (data not shown).
For coimmunoprecipitation of PKC-ε with Raf-1, a similar protocol was performed except that Raf-1 was immunoprecipitated using anti-Raf-1 antibody and was transferred to nitrocellulose membranes. Subsequently, the membranes were probed with anti-PKC-ε antibody (1:1,000), and the bands corresponding to PKC-ε were visualized using ECL. Equal loading of Raf-1 protein was determined by reprobing the membranes with anti-Raf-1 antibody.
Transfection of HEK 293 cells with either wild-type or dominant-negative PKC-ε (ΔPKC-ε) mutant constructs.
HEK 293 cells were transiently transfected with either wild-type or ΔPKC-ε constructs (a generous gift from Dr. I. Bernard Weinstein) using Superfect (Qiagen). The wild-type construct is a full-length PKC-ε in a pHACE vector, and the dominant-negative mutant construct is the same full-length PKC-ε with a point mutation in the catalytic domain at the ATP-binding site. Transfection efficiency was consistently 40–50%, as determined by green fluorescent protein cotransfection assay. Western blot analysis was performed using lysates from either wild-type or ΔPKC-ε construct-transfected HEK 293 cells to determine the expression level of pERK. pERK bands were visualized with ECL. As a control, HEK cells were transfected with empty vector. To verify that the transfections with constructs for PKC-ε did not alter protein levels of other PKC isoforms, Western analyses were performed to assess PKC-α, -ε, and -ζ expression.
Independent t-tests were used to determine the significant differences between groups. The P value of the individual components was adjusted for multiple comparisons by the Bonferroni method. The data were expressed as means ± SE. All nonparametric data were analyzed by the Kruskal-Wallis test. In those experiments where the control optical density values were set to 100%, the SE for each of these control values was reported using the nontransformed data.
Ceramide inhibits IGF-I- and OAG-stimulated HEK 293 cell growth.
To initially assess the effect of ceramide on cell growth, HEK 293 cells were treated with either IGF-I (50 ng/ml) or OAG (10−6 M), a cell-permeable mimetic of DAG, and [3H]thymidine uptake into acid-insoluble DNA was measured. As shown in Fig. 1, both IGF-I and OAG significantly increased HEK 293 cell growth by ∼150% compared with control cells. When HEK 293 cells were pretreated with C6-ceramide (1 μM), we observed a significant decrease in cell growth in response to IGF-I or OAG. Specifically, C6-ceramide inhibited IGF-I- and OAG-induced cell growth to near basal levels. In contrast, the inactive ceramide analog dihydro-C6-ceramide (1 μM) did not reduce IGF-stimulated [3H]thymidine incorporation. These results were consistent with our previous studies, which demonstrated that ceramide inhibited rat glomerular mesangial and A7r5 vascular smooth muscle cell growth induced by mitogenic stimuli (7). This inhibitory effect of C6-ceramide on HEK 293 cell growth does not appear to be caused by cell death, as C6-ceramide, at concentrations up to 100 μM, did not induce apoptotic or necrotic cell death, as assessed by lactate dehydrogenase release (unpublished data). Together, these results demonstrate that bioactive ceramide potently inhibits HEK 293 cell growth induced by OAG and IGF-I, activators of the PKC-dependent signaling pathway.
Ceramide inhibits DAG-dependent PKC-ε bioactivity.
The fact that ceramide potently inhibited IGF-I- and OAG-stimulated HEK 293 cell growth strongly suggests a possible inhibitory role of ceramide in PKC-ε activation, since IGF-I-induced mitogenesis is predominantly transduced through PKC-ε in a DAG-dependent manner (30). Therefore, we examined the ability of ceramide to directly and acutely affect the bioactivity of immunoprecipitated PKC-ε by performing in vitro reconstitution activity assays. The immunocomplexes were treated with physiological DAG (1,2-diolein) and/or physiological ceramide (C18:1), and bioactivity was assessed by resolving radiolabeled phosphorylation of histone IIIS. As shown in Fig. 2, the bioactivity of PKC-ε in DAG-treated immunoprecipitates was significantly increased (3-fold) compared with the control immunocomplexes without DAG treatment. This result was consistent with previous findings demonstrating that DAG is required for PKC-ε activation (17). When DAG-treated immunocomplexes were challenged with the addition of C18:1-ceramide, the bioactivity of PKC-ε was decreased significantly. To confirm these findings, additional experiments quantified phosphorylation of an alternative PKC substrate, myelin basic protein, by liquid scintillation analysis (Table 1). Again, with the use of immunoprecipitated PKC-ε, physiological ceramide significantly inhibited DAG-stimulated phosphorylation of exogenous substrate. In contrast, the inactive ceramide analog dihydro-C6-ceramide did not inhibit DAG-stimulated PKC-ε activity. Together, these results further suggest an apparent reciprocal relationship between bioactive ceramide and DAG for PKC-ε bioactivity.
To further verify the inhibitory actions of physiological C18:1-ceramide on immunoprecipitated PKC-ε, we performed direct in vitro kinase assays using purified, recombinant PKC-ε (Fig.3). Supporting the immunoprecipitated PKC-ε kinase assay, physiological ceramide significantly reduced DAG-stimulated recombinant PKC-ε activity. In addition, the cell-permeable C6-ceramide mimicked the effect of physiological ceramide to diminish DAG-stimulated PKC-ε activity. Again, the inactive cell-permeable ceramide analog dihydro-C6-ceramide had no significant effect on either basal or DAG-stimulated PKC-ε activity. These studies, utilizing recombinant PKC protein, confirm that bioactive ceramides directly inhibit DAG-activated PKC-ε activity.
Ceramide does not change PKC-ε expression.
To determine whether the inhibitory actions of ceramide on PKC-ε activity are also a consequence of altered protein expression, we examined the PKC-ε protein expression level by performing Western blot analysis using anti-PKC-ε antibody. As shown in Fig.4, when HEK 293 cells were treated with C6-ceramide, the protein expression level of PKC-ε was not altered compared with the control cells without ceramide treatment. These results demonstrated that ceramide treatment of HEK 293 cells does not alter PKC-ε protein expression levels. Furthermore, these results suggest that the inhibitory actions of ceramide may involve a direct inactivation of PKC-ε and not downregulation of protein expression.
PKC-ε is a necessary component for ceramide inhibition of ERK activity.
To further confirm that ceramide may exert its cell growth inhibitory actions through inactivation of PKC-ε, we examined the effects of C6-ceramide in HEK 293 cells overexpressing dominant-negative PKC-ε (ΔPKC-ε). Because the activation of ERK is required for IGF-I-induced mitogenesis, we initially investigated the involvement of PKC-ε in the ERK cascade through the use of wild-type and ΔPKC-ε mutants. In data not shown, transfection with wild-type and dominant-negative constructs resulted in equal expression of PKC-ε. However, both of these constructs had a higher level of PKC-ε expression compared with empty vector controls. In contrast to PKC-ε, the cellular levels of other PKC isoforms, including α and ζ, did not change as a result of transfection with any of the cDNA constructs.
As shown in Fig. 5, HEK 293 cells overexpressing the wild-type PKC-ε showed a significant increase in ERK bioactivity, as assessed by pERK, in response to IGF-I treatment. However, HEK 293 cells overexpressing the ΔPKC-ε showed a significant reduction of IGF-induced ERK bioactivity by ∼50%, which correlated with the determined transfection efficiency. When cells were treated with cell-permeable ceramide in the presence of IGF, the bioactivity of ERK was reduced substantially in HEK 293 cells overexpressing wild-type PKC-ε, whereas the bioactivity of ERK in ΔPKC-ε-overexpressing HEK 293 cells was not changed significantly. Furthermore, dihydro-C6-ceramide-treated wild-type PKC-ε- or ΔPKC-ε-expressing cells did not manifest a decreased pERK expression in the presence of IGF-I. These results demonstrate that PKC-ε is a necessary signaling component for modulating ceramide-mediated inhibition of ERK bioactivity. In addition, these results suggest a role of ceramide in modulation of PKC-ε interaction with elements of the ERK signaling cascade.
Ceramide inhibits PKC-ε-ERK interaction.
Because PKC-ε activation has been shown to be an upstream kinase of ERK activation (4, 32), we next performed coimmunoprecipitation assays to determine whether ceramide can inhibit the ability of PKC-ε to interact with ERK, resulting in a decreased ERK activity. Because ceramide has also been shown to activate PKC-ζ (21), we also investigated whether ceramide regulates PKC-ζ-ERK interaction. As shown in Fig.6, HEK 293 cells treated with IGF-I specifically increased PKC-ε, but not PKC-ζ, association with ERK2. C6-ceramide treatment abrogated this IGF-I-induced interaction between ERK and PKC-ε. C6-ceramide had no significant effect by itself on any of these interactions. These results demonstrate that ceramide specifically prevents the IGF-I-induced interaction between PKC-ε and ERK2.
Ceramide inhibits PKC-ε association with pERK.
It is possible that only activated PKC-ε may recruit and activate ERK through phosphorylation. Therefore, the inactivation of PKC-ε may result in blocked ERK recruitment and subsequent activation. To determine whether ceramide specifically blocks PKC-ε interaction with pERK, we performed coimmunoprecipitation assays between PKC-ε and pERK. As shown in Fig. 7, IGF-I treatment significantly increased (6-fold) the association of PKC-ε with pERK in HEK 293 cells. C6-ceramide, but not dihydro-C6-ceramide, pretreatment led to a significant reduction in the IGF-I-stimulated PKC-ε association with pERK. Changes in pERK association with activated PKC-ε most likely reflect the specific interactions between PKC-ε and pERK, as equal levels of PKC-ε were observed in the immunoprecipitates from all treatments. These results clearly demonstrate that bioactive ceramide specifically inhibits PKC-ε interaction with pERK.
Ceramide inhibits PKC-ε interaction with Raf-1.
We demonstrated that ceramide blocked ERK activation via selective inhibition of PKC-ε activity. However, it is not clear whether the selective inhibitory action of ceramide on ERK activity is dependent on Raf-1, an upstream kinase of ERK. Both Raf-1-dependent and -independent activation of ERK by PKCs have been demonstrated by others. Therefore, we next examined if Raf-1 kinase is coimmunoprecipitated with PKC-ε in HEK 293 cells treated with IGF-I. As shown in Fig. 8, we observed a strong association of PKC-ε with Raf-1 in response to IGF-I treatment. This result was consistent with other reports (5) that demonstrated PKC-ε association with Raf-1 and that the association was increased with growth factor treatment. In contrast, HEK 293 cells treated with either C6-ceramide or IL-1β, a receptor-mediated inducer of ceramide formation (6), did not induce association of PKC-ε with Raf-1. In fact, IGF-I-stimulated Raf-1 association with PKC-ε was inhibited significantly by pretreatment with C6-ceramide or IL-1, demonstrating that ceramide potently inhibits IGF-I-stimulated PKC-ε interaction with Raf-1. Together these data support our conclusion that direct inhibition of PKC-ε by ceramide inhibits the interaction between PKC-ε and upstream elements of the ERK cascade.
The field of signal transduction is now embracing the concept of signaling complexes in which the assembly and interactions of multiple kinases in large-scale aggregates determines the specificity and selectivity of cellular responses. The role of scaffolding and/or adapter proteins such as MEK partner-1, Jun-interacting protein, kinase suppressor of Ras (KSR), and 14–3-3 proteins to assemble these signaling aggregates is only recently being appreciated (9, 27,29, 33). Adding to this orchestrated complexity, we now elucidate a novel mechanism by which the sphingolipid metabolite ceramide can negatively modulate cellular responses by antagonizing the protein-protein interaction of kinases involved in mitogenic signaling pathways.
The mechanism by which growth factors and their receptors regulate the assembly of kinase signaling complexes between PKCs and the elements of the ERK cascade is still unclear. However, the fact that Raf-1 is activated by PKC-ε suggests that PKC-ε may directly phosphorylate Raf-1. Supporting this, Ueffing et al. (31) demonstrated that PKC-ε and Raf-1 coimmunoprecipitate from PKC-ε-transformed NIH/3T3 cells, indicating that PKC-ε may activate Raf-1 through direct protein-protein interactions. Our study clearly demonstrates the novel role for lipid-derived second messengers to modulate these protein-protein interactions, as ceramide inactivation of PKC-ε limits interactions of PKC-ε with Raf-1 and ERK.
Our previous studies showed that ether-linked diglyceride species competitively bound to the DAG-binding site on PKC-δ and -ε without activating the kinase (17). Because ceramide structurally resembles DAG, it is possible that ceramide competes against DAG for the putative DAG-binding site. Ceramide could also bind to a secondary ceramide-binding site, rendering the PKC-ε insensitive to activation by DAG. Alternatively, when ceramide is bound to PKC-ε, it may hinder PKC-ε from interacting with other proteins, such as Raf-1 and ERK. Whether ceramide directly competes with DAG for the putative C-1-lipid-binding motif within PKC-ε is not clear at the present time. In fact, a radioiodinated photoaffinity-labeled ceramide analog was unable to directly interact with immunoprecipitated nonactivated PKC-ε (11). Regardless of the mechanism, our finding of ceramide-induced inactivation of immunoprecipitated and recombinant PKC-ε is supported by previous studies that demonstrated that ceramide treatment induced the translocation of PKC-δ and -ε from the plasma membrane to the cytosol (14, 26), an event consistent with inactivation. Ceramide has also been shown to inhibit PKC-α activity (16), perhaps in a similar mechanism to PKC-ε inactivation by ceramide.
Our data indicate that one mechanism by which ceramide decreases ERK activity is via direct inhibition of PKC-ε and the subsequent inability to form a signaling complex with Raf-1 and ERK. Other studies have postulated alternative mechanisms by which ceramide regulates the Raf-1/ERK cascade. Ceramide has been shown to bind to c-Raf (24) and KSR (34). Ceramide binding to Raf-1 leads to sequestration of Raf-1 into inactive Ras-Raf-1 complexes (22). Moreover, KSR has been shown to bind to and functionally inactivate MEK1 (8, 35). All of these studies are consistent with decreased ERK activity. Finally, a recent study by Basu et al. (1) has shown that downstream targets, such as BAD, convert the normally promitogenic ERK cascade into a ceramide-dependent proapoptotic signal pathway. Thus ceramide may regulate several mechanisms to inhibit ERK-mediated proliferation.
The finding that ceramide-induced cell growth inhibition is a consequence of inactivated PKC-ε clearly suggests the critical role of PKC-ε in mitogenesis. Downregulation of PKC-ε has been shown to inhibit the G1/S transition in vascular smooth muscle cells, an event consistent with IL-1-induced growth arrest (18,25). Furthermore, overexpression of PKC-ε induced tumorigenicity in fibroblasts (3, 20) and enhanced nerve growth factor-induced phosphorylation of ERK in PC-12 pheochromocytoma cells (10). Taken together, the role of ceramide to selectively limit interactions between PKC-ε and Raf-1/ERK may illustrate one mechanism by which a proinflammatory response can be maintained in the absence of cell growth. This novel role of ceramide to regulate protein-protein interactions, including the PKC-ε-Raf-1-ERK interactions, is an attractive hypothesis by which inflammatory cytokine-induced ceramide formation may inhibit cellular proliferation in models of nonproliferative inflammatory renal diseases.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53715.
Address for reprint requests and other correspondence: M. Kester, The Pennsylvania State Univ., The Milton S. Hershey Medical Center, Dept. of Pharmacology, PO Box 850, Hershey, PA 17033 (E-mail:).
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- Copyright © 2001 the American Physiological Society