Ceramide Directly Activates Protein Kinase C ζ to Regulate a Stress-activated Protein Kinase Signaling Complex*

We have previously shown that interleukin 1 (IL-1)-receptor-generated ceramide induces growth arrest in smooth muscle pericytes by activating an upstream kinase in the stress-activated protein kinase (SAPK) cascade. We now report the mechanism by which ceramide activates the SAPK signaling pathway in human embryonic kidney cells (HEK-293). We demonstrate that ceramide activation of protein kinase C ζ (PKCζ) mediates SAPK signal complex formation and subsequent growth suppression. Ceramide directly activates both immunoprecipitated and recombinant human PKCζ in vitro. Additionally, ceramide activates SAPK activity, which is blocked with a dominant-negative mutant of PKCζ. Co-immunoprecipitation studies reveal that ceramide induces the association of SAPK with PKCζ, but not with PKCε. In addition, ceramide treatment induces PKCζ association with phosphorylated SEK and MEKK1, elements of the SAPK signaling complex. The biological role of ceramide to induce cell cycle arrest is mimicked by overexpression of a constitutively active PKCζ. Together, these studies demonstrate that ceramide induces cell cycle arrest by enhancing the ability of PKCζ to form a signaling complex with MEKK1, SEK, and SAPK.

ceramide-activated protein kinase involved in growth control (8,9,10). Evidence suggests that PKC is directly activated by ceramide and not by diacylglycerol (DAG) (8,9,11). Several reports also relate ceramide-induced growth arrest to activation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase, but the precise mechanism remains to be elucidated (6,12,13). Because ceramide has been shown to activate PKC and SAPK in the same cell type, it has been inferred, but not proven, that PKC is a critical element in ceramide-induced SAPK activation (11). It is likely that the site of action for ceramide is upstream of SAPK, because ceramide does not directly regulate immunoprecipitated SAPK activity in a cellfree system (7). We now demonstrate that ceramide-activated PKC recruits upstream components of the SAPK cascade into a signaling complex, resulting in growth arrest.
Activation of the SAPK signaling pathway is characterized by a cascade of protein kinases, which are recruited to the plasma membrane. Specifically, GTP-dependent activation of Rac or Cdc42 leads to recruitment of MEKK1 to the plasma membrane, where it is phosphorylated and activated (14). Activated MEKK1 directly phosphorylates and activates SEK, which in turn directly activates SAPK. Activation of Rac-1 by inflammatory cytokines or ceramides has been postulated to be one mechanism to activate the SAPK cascade (14 -16). Because PKC does not directly regulate Rac-1 (17), an alternative mechanism for activation of SAPK could be ceramide-induced PKC activation of MEKK1 and/or SEK. Thus, it is hypothesized that ceramide regulation of the SAPK pathway also is dependent on direct activation of PKC.
In this study, we demonstrate that ceramide directly activates both immunoprecipitated and recombinant human PKC. Upon ceramide activation, PKC interacts with MEKK1, SEK, and SAPK to inhibit insulin-like growth factor-1 (IGF-1)-induced cell growth. Together, these findings suggest a novel role of ceramide in regulation and assembly of multi-protein signaling complexes.
are adenovirus-transformed human embryonic kidney cells of tubule epithelial origin. These cells express functional IGF receptors and are an excellent model for growth factor-induced mitogenesis as well as inflammatory cytokine-induced growth-arrest (2,18). Western blot analyses revealed that HEK-293 cells express PKC␣, ⑀, and .
Western Blot Analysis-Western blot analysis using anti-PKC antibody was performed as described previously (7). Briefly, treated HEK-293 cells were washed in ice-cold Dulbecco's phosphate-buffered saline solution and lysed in 1 ml of lysis buffer (50 mM Hepes, 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM NaVO 4 , 0.2% Nonidet P-40, 1 g/ml of leupeptin, pepstatin, and aprotinin). Cell lysates were cleared by centrifugation, and the Bio-Rad protein assay was performed to determine protein concentration. 40 g of protein lysate per sample was separated on a 10% SDS-PAGE gel and transferred to Hybond nitrocellulose membranes. The membranes were blocked in 5% nonfat milk in tris-buffered saline (TBS) for 1 h and then incubated with the primary anti-PKC antibody (1:1000 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-conjugated goat antirabbit IgG antibody (1:5000 dilution in 5% nonfat milk in TBS) for 2 h at room temperature. The membranes were then washed three times with TBS. The PKC band was visualized by ECL and quantified using laser densitometry. A similar protocol was used for detection of PCNA, MEKK1, p-SEK, and SEK expression.
In Vitro Reconstitution Activity Assay for Immunoprecipitated PKC-Immunoprecipitation of PKC and the subsequent reconstitution activity assay was adapted from previous methods (7,19,20). Briefly, PKC was immunoprecipitated from HEK-293 lysates using 0.5 g of polyclonal rabbit anti-PKC antibody. After overnight incubation at 4°C, goat anti-rabbit IgG agarose was added, the mixture was rotated for 2 h, and the immunocomplex containing PKC was pelleted by brief centrifugation. After three washes, the pellets were resuspended in kinase buffer (50 mM Hepes, 100 mM NaCl, 10 mM MgCl 2 , 50 mM NaF, 1 mM NaVO 4 , 1 mM dithiothreitol, 0.1% Tween 20). The in vitro kinase reaction was initiated by addition of 40 g/ml phosphatidylserine/reaction, 10 mM MgCl 2 , 0.25 mM ATP (cold), and 1 Ci of [␥-32 P]ATP (10 mCi/mmol), and 10 g of histone IIIS as a substrate. Specified samples contained DAG (1,2-diolein) and/or C 18:1 -ceramide as co-factors. After 15 min of incubation at 37°C, the kinase reaction was terminated by addition of sample buffer and heated at 95°C for 5 min. Phosphorylated Histone IIIS proteins were then separated on 12% SDS-PAGE gels and transferred to Hybond nitrocellulose membranes. The bands corresponding to phosphorylated Histone IIIS were detected by autoradiography (Kodak X-OMAT). A similar protocol utilizing recombinant PKC (50 ng) was used to verify the in vitro effects of physiological ceramide on immunoprecipitated PKC.
In Vitro Reconstitution Activity Assay for Immunoprecipitated SAPK-In vitro SAPK kinase assays were performed as described previously (6). The bioactivity of immunoprecipitated SAPK was assessed by phosphorylation of c-Jun as exogenous substrate. The phosphorylated bands were detected by autoradiography.
Co-immunoprecipitation of PKC with SAPK Cascade Signaling Components-Lysates from treated HEK-293 cells were prepared as described previously and incubated with rabbit anti-SAPK1 (or anti-MEKK1) antibody for 12 h at 4°C. Goat anti-rabbit IgG 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 twice in lysis buffer. Immunoprecipitates were combined with sample buffer and heated at 95°C for 5 min, followed by separation on 12% SDS-PAGE gels. Proteins were transferred to Hybond nitrocellulose membranes and probed with anti-PKC antibody and, in some cases, anti-PKC⑀ antibody (1:1000 dilution in 5% nonfat milk in TBS). Subsequently, the membranes were incubated with the horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000), and the bands corresponding to PKC or PKC⑀ were visualized by ECL. Equal loading of SAPK or MEKK1 was determined by reprobing the membranes with anti-SAPK or-MEKK1 antibodies.
For co-immunoprecipitation of PKC with phosphorylated SEK (p-SEK), a similar protocol was performed except that PKC was immunoprecipitated using anti-PKC antibody. Subsequent to SDS-PAGE electrophoresis and protein transfer to nitrocellulose, the membranes were probed with anti-p-SEK antibody (1:1000). The bands corresponding to p-SEK were visualized using ECL. Equal loading of PKC protein was determined by reprobing the membranes with anti-PKC antibody.
Transfection of HEK-293 Cells with Either Wild-type (WT), Dominant-negative PKC, or Constitutively Active PKC Constructs-HEK-293 cells were transiently transfected with either WT or mutant PKC constructs (a generous gift from Dr. J. Moscat) using Superfect (Qiagen). The wild-type construct is a full-length PKC in a pCDNA3 expression vector. The dominant-negative mutant construct is a kinasedefective mutant that contains a point mutation in the catalytic domain. The constitutively active PKC construct is the catalytic domain without the regulatory domain. Transfection efficiency was consistently ϳ40% as determined by green fluorescence protein co-transfection assay. Cells expressing the wild-type and dominant-negative mutant constructs were subjected to in vitro kinase assays to assess SAPK bioactivity, as described above. In addition, lysates from wildtype or constitutively active PKC-expressing cells were subjected to Western blot analysis to assess proliferating cell nuclear antigen (PCNA) expression. To verify that the transfections with constructs for PKC did not alter protein levels of other PKC isoforms, Western analyses were performed and PKC␣ or PKC⑀ expression did not change (data not shown).
Statistical Analysis-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 mean Ϯ S.E. All non-parametric data were analyzed by the Kruskal-Wallis test. In those experiments where the control optical density values were set to 100%, the S.E. for each of these control values was reported utilizing the non-transformed data.

Ceramide Directly Stimulates PKC Bioactivity-Ceramide
has been shown to activate the SAPK cascade. However, the mechanism of activation has not been clearly defined. Because PKC has one cysteine-rich domain (CRD), which is suggested to interact with ceramide, but not diacylglycerol (DAG) (21), we hypothesize that ceramide-activated PKC regulates the SAPK cascade. Therefore, the ability of ceramide to directly and acutely affect the bioactivity of immunoprecipitated PKC from HEK-293 cells was assessed by performing in vitro reconstitution activity assays. The immunocomplexes were treated with physiological ceramide (C 18:1 -ceramide) and/or physiological DAG (1,2-diolein) for 15 min. Bioactivity was assessed by resolving radiolabeled phosphorylation of Histone IIIS. As shown in Fig. 1, A and B, the bioactivity of immunoprecipitated PKC was significantly increased (3-fold) by ceramide but not DAG treatment. When C 18:1 -ceramide-treated immunocomplexes were challenged with the addition of DAG, the bioactivity of PKC was significantly decreased. These results suggest an apparent reciprocal relationship between ceramide and DAG for PKC bioactivity.
To verify the stimulatory actions of physiological C 18:1 -ceramide on immunoprecipitated PKC, we performed direct in vitro kinase activity assay using purified, recombinant PKC (Fig. 1C). Consistent with the immunoprecipitated PKC kinase assay, physiological ceramide significantly activated recombinant PKC activity. Similar to results obtained in the immunoprecipitated PKC kinase assay, DAG treatment had no effect by itself but was able to reduce C 18:1 -ceramide-induced PKC activity. In addition, cell-permeable C 6 -ceramide mimicked the effect of physiological C 18:1 -ceramide to stimulate PKC activity in the cell-free assays (data not shown). In support of the specificity of the actions of ceramide on PKC, the inactive cell-permeable ceramide analogue, dihydro-C 6 -ceramide, had no effect on PKC activity. These studies, utilizing both immunoprecipitated and recombinant human PKC protein, confirm the hypothesis that bioactive ceramides directly activate PKC activity.
Ceramide Does Not Change PKC Expression-In addition to directly activating PKC, ceramide could also increase PKC activity by inducing PKC protein expression. We examined the PKC protein expression level by performing Western blot analysis using anti-PKC antibody. As shown in Fig. 2, when cycling (10% FBS) or non-cycling (serum-deprived, SD) HEK-293 cells were treated with cell-permeable C 6 -ceramide for 24 h, the protein expression level of PKC was not altered compared with the control cells without C 6 -ceramide treatment. These results demonstrate that ceramide regulates HEK-293 cells as a consequence of a direct activation of PKC and not up-regulation of PKC protein expression.
PKC Is a Necessary Component for Ceramide Activation of SAPK Activity-Because we demonstrated that ceramide directly activates PKC, we next examined whether ceramideinduced SAPK activity is dependent on PKC. As shown in Fig.  3, HEK-293 cells overexpressing the WT PKC construct resulted in an increase in C 6 -ceramide-stimulated immunoprecipitated SAPK bioactivity. In contrast, C 6 -ceramide-induced SAPK activity was blocked with a dominant-negative mutant of PKC. These results suggest that PKC is a necessary signaling component for modulating ceramide-mediated activation of SAPK bioactivity.
Ceramide Augments PKC⅐SAPK Interaction-To further define the mechanism by which ceramide activates PKC leading to SAPK complex formation, we next examined whether PKC associates with SAPK. Therefore, to document if ceramide induces a potential interaction between PKC and SAPK, we performed co-immunoprecipitation assays. As shown in Fig. 4, HEK-293 cells treated with C 6 -ceramide specifically increased PKC association with SAPK1. Because ceramide induces the translocation of PKC⑀ from the plasma membrane to the cytosol, an event consistent with inactivation (22), we also investigated if ceramide regulates PKC⑀⅐SAPK interaction as a negative control. Ceramide did not augment an association between PKC⑀ and SAPK. These results demonstrate that ceramide specifically induces an interaction between PKC and SAPK. . Control experiments showed that histone phosphorylation was dependent on exogenous phosphatidylserine (PS) (data not shown). For the immunoprecipitated assays, phosphorylated histone IIIS protein was resolved on a 12% SDS-PAGE gel electrophoresis, visualized by autoradiography, and quantified by laser densitometry. For the recombinant protein assays, the phosphorylated histone bands were excised and quantified by liquid scintillation counting. For both immunoprecipitated and recombinant PKC, C 18:1 -ceramide directly stimulated PKC activity. DAG itself had no effect on either immunoprecipitated or recombinant PKC but significantly reduced C 18:1 -ceramide-induced PKC activity. Results similar to C 18:1 -ceramide were observed for the cell-permeable C 6 -ceramide (data not shown). For the recombinant PKC assay, the inactive ceramide analogue, DH-C 6 -ceramide, did not increase PKC activity. A, a representative autoradiogram for the immunoprecipitated PKC assay. B, the quantification of the immunoprecipitated PKC bioactivity. C, the quantification of the recombinant PKC assays (mean Ϯ S.E.; n ϭ 3-4). Star, significantly different from vehicle control; p Ͻ 0.05, paired t-test. Sun, combination treatment is significantly different from individual treatment alone; p Ͻ 0.05, paired t-test. IGF-1 induces DAG generation, which we have previously shown to inhibit ceramide-activated PKC. Thus we investigated whether IGF-1 could diminish ceramide-induced PKC⅐SAPK interactions. Our studies document that IGF-1, in contrast to ceramide, does not induce PKC association with SAPK. In fact, ceramide-induced association between SAPK and PKC was diminished. These data further support the specificity of ceramide-activated PKC to form SAPK signaling complexes.
Ceramide Induces PKC Association with p-SEK and MEKK1-Our results imply a role for ceramide to modulate PKC interactions with upstream elements of the SAPK signaling cascade. Activated SEK is the immediate upstream dual specificity kinase that phosphorylates SAPK on threonine and tyrosine residues. It is possible that bioactive PKC may recruit and activate SEK through phosphorylation on Ser-219 and Thr-223. Therefore, we next investigated the ability of ceramide to induce an association between PKC and bioactive (phosphorylated)-SEK (p-SEK) by co-immunoprecipitation assays. As shown in Fig. 5, A and B, C 6 -ceramide or IL-1 treatment significantly increased (5-fold) the association of PKC with p-SEK in HEK-293 cells. Consistent with the effect of IGF-1 on ceramide-induced PKC⅐SAPK interaction, IGF-1 treatment also reduced both C 6 -ceramide-and IL-1-induced association of PKC with p-SEK. These results clearly demonstrate that ceramide regulates the activity of PKC and its interaction with p-SEK.
To further define the upstream SAPK signaling elements regulated by ceramide-activated PKC, we also investigated whether ceramide induces an association between PKC and MEKK1. As shown in Fig. 5, C and D, we observed a strong association of PKC with MEKK1 in response to ceramide or IL-1 treatment. Again, IGF-1 reduced both C 6 -ceramide-and IL-1-induced PKC⅐MEKK1 interactions. These results strongly suggest that the stimulatory action of ceramide on SAPK activation are a result of activated PKC interacting with MEKK1 as well as SEK.

Ceramide-induced Growth Arrest Is Dependent on PKC-
The critical role of PKC in ceramide-and IL-1-mediated inhibition of cell growth was assessed in HEK-293 cells transiently transfected with either wild-type (WT) or constitutively active PKC. Cell cycle arrest was evaluated by proliferating cell nuclear antigen (PCNA) expression (Fig. 6). PCNA expression is used as a marker of cells entering the cell cycle at early G 1 and S phases. In cells transfected with the WT PKC construct, both IL-1 and its second messenger, ceramide, significantly reduced IGF-1-induced PCNA expression. This inhibitory effect of C 6 -ceramide on HEK-293 cell growth does not appear to be caused by necrosis as C 6 -ceramide, at concentrations up to 100 M, did not induce lactate dehydrogenase release (data not shown). In contrast to C 6 -ceramide, the inactive cell-permeable ceramide analogue, dihydro-C 6 -ceramide, did not have an inhibitory effect on either basal or IGF-1-induced PCNA expression. Most importantly, the mitogenic effect of IGF-1 was reduced in cells overexpressing the constitutively active PKC. Compared with wild-type PKC-expressing cells, a similar pattern of PCNA expression was observed in non-transfected or mock transfected cells (data not shown). Together, these re- sults suggest that PKC is necessary for IL-1 and/or ceramideinduced cell cycle arrest. Moreover, these data imply that PKC⅐SAPK complex formation is required for ceramide-induced growth arrest. DISCUSSION The concept of signaling complex formation determining the specificity and selectivity of cellular responses is an exciting new area in the field of cellular communication. Recent studies have shown that scaffold/adapter proteins serve to assemble multiple signaling proteins together in large scale aggregates. The importance of these scaffolding proteins, such as Juninteracting protein-1, kinase suppressor of Ras, and 14-3-3, in signal transduction pathways is only recently being appreciated (23)(24)(25)(26). Adding to this orchestrated regulation, we now elucidate a novel mechanism by which the sphingolipid metabolite, ceramide, can regulate protein-protein interactions between PKC and elements of the SAPK cascade, culminating in cell cycle arrest.
We demonstrate that ceramide selectively augments a signal complex formation of PKC with MEKK1, SEK, and SAPK. The fact that our co-immunoprecipitation experiments were performed in the presence of a non-ionic detergent (0.2% Nonidet P-40) suggests that direct protein-protein interactions are occurring as a consequence of ceramide activation of PKC, and presumably not by ceramide promoting hydrophobic associations of these proteins at the membrane. Based on this evidence, it is suggested that direct binding and activation of PKC by ceramide leads to recruitment and activation of upstream elements in the SAPK cascade resulting in cell cycle arrest. Other studies have postulated alternative mechanisms by which ceramide regulates the SAPK cascade. Ceramide has been shown to activate small molecular weight G-proteins that may couple inflammatory cytokine receptors with the SAPK cascade (14,15). Specifically, ceramide stimulates Rac-1, as well as Vav, a guanine nucleotide exchange factor for Rac (27). Another SAPK regulatory mechanism may involve ceramide activation of PP1 and PP2A phosphatases (28). Even though overexpression of a dual specificity threonine/tyrosine phosphatase, M3/6, diminishes ceramide-activated SAPK (29), other studies demonstrate that ceramide activates SAPK under conditions where ceramide also activates protein phosphatases (6,12). The role of ceramide-activated phosphatases to modulate ceramide-dependent PKC⅐SAPK interactions is of potential interest. Thus, ceramide may regulate several mechanisms mediating SAPK-induced cell cycle arrest.
The role of ceramide binding to, and activating, PKC is still somewhat controversial. Our studies, using both immunoprecipitated and recombinant human PKC, clearly demonstrate that ceramide, but not dihydroceramide, directly induces PKC bioactivity. Supporting our findings, ceramide has been shown to bind to PKC as determined by kinetic analyses and in vitro phosphorylation studies (9,30). In contrast, a radioiodinated photoaffinity-labeled ceramide analogue was unable to directly interact with immunoprecipitated PKC (31). These apparent contradictions in the literature may be due to structural differences in the ceramide analogues. Alternatively, the ability of ceramide to interact with the cysteine-rich lipid-binding domain (CRD) of immunoprecipitated PKC could be altered by co-immunoprecipitating proteins, such as Par-4, that also interact with this domain (21). It has been proposed that the single CRD of PKC may interact with ceramide, but not DAG (6,32). This may be due to the lack of a second CRD, which is observed in conventional and novel PKC classes, or to subtle differences in the loop structure of the PKC CRD that respond to the unique polar regions of ceramide (21). Although DAG does not directly activate PKC, it is suggested that DAG selectively inhibits PKC activity by antagonizing ceramide binding at the CRD. The fatty acyl groups of DAG may block this putative ceramide-binding domain in an analogous fashion to arachidonic acid blocking ceramide binding to PKC (9).
In our studies, we found that the actions of ceramide on PKC⅐SAPK interactions were inhibited by IGF-1 treatment. This somewhat surprising finding has several possible explanations. IGF-1-generated DAG may compete with ceramide at the putative ceramide binding site on PKC (21) or activate other PKC isotypes linked to mitogenesis. In addition, IGF-1 has also been shown to stimulate PKC through a phosphatidylinositol 3-kinase-dependent mechanism (33). This alternate mechanism to stimulate PKC may couple PKC to other mitogen-activated protein kinases (MAPK), such as extracellular signal-regulated kinases (ERK), which are more closely linked to mitogenesis. The role of sphingolipid-and polyphosphoinositide-derived second messengers to differentially regulate PKC⅐MAPK signaling complexes is an attractive theory that is beyond the focus of the current studies. Alternatively, ceramide may become phosphorylated by IGF-1 treatment, generating FIG. 4. Ceramide enhances PKC interaction with SAPK1. Protein interactions between select PKC isoforms and SAPK were assessed by Western analyses utilizing anti-PKC or -PKC⑀ antibodies on SAPK1 immunoprecipitates from HEK-293 cells. PKC isoforms, co-immunoprecipitated with anti-SAPK1 antibody, were detected with enhanced chemiluminescence and quantified by laser densitometry. HEK-293 cell monolayers in 12-well plates were made quiescent in Dulbecco's modified Eagle's medium supplemented with 0.5% FBS for 24 h. The monolayers were then treated with vehicle control (0.01% Me 2 SO), IGF-1 (50 ng/ml), C 6 -ceramide (10 Ϫ6 M), or IGF plus C 6 -ceramide for 5 min. The basal interaction between SAPK⅐PKC (2.19 Ϯ 0.21 arbitrary units) was slightly more than the interaction between SAPK⅐PKC⑀ (1.45 Ϯ 0.33). C 6 -ceramide enhanced interactions of PKC, but not PKC⑀, with the SAPK1 pathway. Equal loading of the samples was assessed by reprobing the blots with anti-SAPK1 antibodies. A, representative Western blots. B, the quantification of the PKC bands (mean Ϯ S.E.; n ϭ 3). Star, significantly different from vehicle control; p Ͻ 0.01, paired t-test. Sun, combination treatment is significantly different from individual treatment alone; p Ͻ 0.005, paired t-test.
ceramide-1-phosphate, a pro-mitogenic lipid (34). Another possibility suggests that IGF-1 treatment induces the activation of ceramidase, an enzyme that catalyzes the deacylation of ceramide to form the pro-mitogenic lipids, sphingosine or sphingosine-1-phosphate (35). Regardless of mechanism, IGF-1 cotreatment reduces the actions of inflammatory cytokines or ceramides to activate SAPK activity. However, this compensatory mechanism does not supersede the ability of ceramides to induce cell cycle arrest.
Our finding that ceramide-induced cell growth inhibition is a consequence of activated PKC coupling to elements of the SAPK cascade clearly suggests the critical role that specific PKC⅐MAPK signaling complexes play in cell cycle arrest. This is particularly intriguing, because PKC can be activated by both mitogenic and anti-mitogenic stimuli. Thus, the ability of activated PKC to interact with distinct MAPK signaling elements could explain the contradictory actions of PKC as a regulator of cell growth. For example, it was initially documented that PKC is required for maturation of Xenopus oocytes and for DNA synthesis in fibroblasts (36). Interactions FIG. 5. Ceramide induces PKC association with p-SEK and MEKK1. HEK-293 cell monolayers in 6-well plates were used to assess PKC interaction with bioactive phospho-SEK (p-SEK) (A and B) as well as MEKK1 (C and D). Cells were treated with C 6 -ceramide (C 6 -Cer, 1 M), IL-1 (20 ng/ml), and/or IGF-1 (50 ng/ml) for 5-10 min. To assess PKC⅐p-SEK interactions, the lysed cells were immunoprecipitated with the anti-PKC antibody and immunoblotted with an antibody that detects phosphorylated SEK. To assess PKC⅐MEKK1 interactions, the lysed cells were immunoprecipitated with an anti-MEKK1 antibody and immunoblotted with the anti-PKC antibody. Reprobing the blots with the appropriate antibody (anti-PKC for A and anti-MEKK1 for C) assessed equal loading. Positive and negative controls included whole cell lysates and cell-free samples, respectively (data not shown). Both C 6 -ceramide and IL-1 treatment led to a significant interaction between PKC and p-SEK as well as MEKK1, which was reduced by co-treatment with IGF-1. A and C, representative Western blots of three such FIG. 6. Ceramide-induced cell cycle arrest is dependent on PKC. HEK-293 cells were transfected with either wild-type or constitutively active PKC constructs. After transfection, cells were treated with ceramide (1 M), IL-1 (20 ng/ml), or dihydro-C 6 -ceramide (DH, 1 M) with or without IGF-1 (50 ng/ml) for 18 h. Western analysis was performed on the cell lysates to assess PCNA expression. Both C 6ceramide and IL-1 treatment significantly reduced IGF-induced PCNA expression in wild-type transfected cells. The specificity of the actions of ceramide was confirmed by the lack of an effect with dihydro-C 6 -ceramide. The constitutively active PKC-expressing cells mimicked the actions of C 6 -ceramide and IL-1 to induce cell cycle arrest in wild-type transfected cells. A, representative Western blots. B, the quantification of the PCNA bands (mean Ϯ S.E.; n ϭ 4). Star, significantly different from vehicle control; p Ͻ 0.05, paired t-test. Sun, significantly different from IGF-stimulated condition; p Ͻ 0.05, paired t-test. experiments each. B and D, the quantification of p-SEK and PKC protein levels in the complex, respectively (mean Ϯ S.E.; n ϭ 3). Star, significantly different from vehicle control; p Ͻ 0.01, paired t-test. Sun, combination treatment is significantly different from individual treatment alone; p Ͻ 0.05, paired t-test.
between PKC and the pro-mitogenic ERK cascades have been suggested, as a dominant-negative mutant of PKC suppressed activation of MEK and ERK by TNF␣ (37). However, recent studies suggest a growth inhibitory role for PKC. NIH-3T3 fibroblasts transfected with wild-type PKC are not tumorigenic (38). In fact, PKC has been reported to actually suppress the neoplastic transformation of fibroblasts mediated by the v-raf oncogene (39) and does not activate Raf-1 activity, an upstream kinase in the ERK cascade in vitro (40,41). These observations offer an explanation for inhibition of ERK bioactivity by ceramide (6,7,12,42). Thus, the ability of ceramide to preferentially couple activated PKC to upstream elements in the SAPK cascade, and not the ERK cascade, could be one possible mechanism for inducing cell cycle growth arrest.
We have shown that ceramide induces cell cycle arrest via selective interactions of PKC with elements of the SAPK cascade. Moreover, these interactions are modulated by the ability of ceramide to bind to and directly activate PKC. The role of ceramide to selectively induce PKC⅐SAPK complex formation may illustrate one mechanism by which a pro-inflammatory response can lead to cell growth arrest. This critical role of ceramide to directly activate PKC and regulate MEKK1⅐SEK⅐SAPK interactions is a novel hypothesis by which inflammatory cytokine receptor-induced ceramide formation may limit cellular proliferation in models of non-proliferative immunological renal diseases.