Ceramide Recruits and Activates Protein Kinase C ζ (PKCζ) within Structured Membrane Microdomains*

We have previously demonstrated that hexanoyl-d-erythro-sphingosine (C6-ceramide), an anti-mitogenic cell-permeable lipid metabolite, limited vascular smooth muscle growth by abrogating trauma-induced Akt activity in a stretch injury model of neointimal hyperplasia. Furthermore, ceramide selectively and directly activated protein kinase C ζ (PKCζ) to suppress Akt-dependent mitogenesis. To further analyze the interaction between ceramide and PKCζ, the ability of ceramide to localize within highly structured lipid microdomains (rafts) and activate PKCζ was investigated. Using rat aorta vascular smooth muscle cells (A7r5), we now demonstrate that C6-ceramide treatment results in an increased localization and phosphorylation of PKCζ within caveolin-enriched lipid microdomians to inactivate Akt. In addition, ceramide specifically reduced the association of PKCζ with 14-3-3, a scaffold protein localized to less structured regions within membranes. Pharmacological disruption of highly structured lipid microdomains resulted in abrogation of ceramide-activated, PKCζ-dependent Akt inactivation, whereas molecular strategies suggest that ceramide-dependent PKCζ phosphorylation of Akt3 at Ser34 was necessary for ceramide-induced vascular smooth muscle cell growth arrest. Taken together, these data demonstrate that structured membrane microdomains are necessary for ceramide-induced activation of PKCζ and resultant diminished Akt activity, leading to vascular smooth muscle cell growth arrest.

A common response to inflammation or stress is the formation of ceramide, an anti-mitogenic, pro-apoptotic lipid metabolite. Ceramide suppresses vascular smooth muscle (VSM) 4 cell mitogenesis in vitro and in vivo (1,2). The initial mechanisms by which ceramide leads to suppression of mitogenesis have not yet been defined. Both biophysical (lipid microdomains) and biochemical (ceramide binding targets) mechanisms have been proposed to address this issue. The present study presents an integrated version of these theories.
Published and preliminary data demonstrate that protein kinase C (PKC) is a direct and selective target for ceramide (2)(3)(4)(5)(6) and that ceramide-activated PKC is necessary for inactivation of Akt-dependent mitogenesis in vascular smooth muscle cells (2). These results have been recapitulated in vivo where cell-permeable ceramide analogues function as novel therapeutics to limit VSM cell growth, in part by abrogating trauma-induced Akt activity in a model of neointimal hyperplasia after stretch injury (1).
PKC is an atypical isoform of the PKC superfamily and is directly or indirectly regulated by several lipids including ceramides and phosphatidylinositol 3,4,5-trisphosphate but not by diacylglycerol and calcium (7). Generally, PKCs reside in the cytosol and translocate to membranes upon stimulation to interact with activating lipid cofactors. PKC may be directly recruited to membranes via interactions with the C1 domain or indirectly recruited to membranes by formation of complexes with PDK1 and scaffolding proteins (7). As lipid micro-and macrodomains have been identified as "signaling hot-spots," we investigated the roles of ceramide-enriched microdomains in the activation of PKC and subsequent inhibition of cellular proliferation via Akt inactivation.
Isolation of Buoyant Fractions by Sucrose Gradient Centrifugation-After treatment, low buoyant fractions were isolated by two different methodologies. The first is based upon the detergent-free method originally described by Song et al. (8). Briefly, A7r5 cells were washed twice with cold phosphate-buffered saline followed by the addition of 500 mM sodium carbonate buffer (pH ϭ 11.0) containing 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM ␤-glycerolphosphate, and a protease inhibitor tablet (Roche Applied Science). Cell lysates were subsequently homogenized using a Dounce glass homogenizer followed by sonication with three 15-s bursts. The lysates were then mixed with equal volumes of 90% sucrose in MBS buffer (25 mM MES, pH 6.5, 150 mM NaCl) and layered at the bottoms of ultracentrifuge tubes. Samples were then overlaid with 4 ml of 35% and 4 ml of 5% sucrose MBScarbonate buffer. The samples were centrifuged at 35,000 rpm for 20 h at 4°C using a Beckman swinging bucket rotor (SW41ti). 1-ml fractions were collected from the top down of each gradient. An additional methodology to obtain buoyant fractions was also employed, based upon Tween 20 solubility, originally described by Ref. 9 with some modifications. Here, after treatment, A7R5 cells were lysed in 2 ml of MBS (25 mM MES, pH 6.5, 150 mM NaCl, 2 mM EDTA) containing 0.5% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO 3 , 1 mM ␤-glycerolphosphate, and a protease inhibitor tablet (Roche Applied Science). The lysate was subsequently homogenized using a Dounce glass homogenizer followed by sonication with three 15 s bursts. Cell lysates were mixed 1:1 (v/v) with 80% sucrose in MBS (final 40% sucrose) and loaded into Beckman ultracentrifuge tubes. The 40% sucrose solution was then overlaid with 4 ml of 30% sucrose in MBS followed by 4 ml of 5% sucrose in MBS to form a 5-30 -40% discontinuous sucrose gradient. Samples were centrifuged at 35,000 rpm for 18 h using a SW41ti rotor. The 12-ml gradient was then fractionated into 1-ml fractions. All gels included a protein lysate control to ensure equivalent visualization between gels. Aliquots of each fraction were immunovisualized by Western blotting and quantified by laser densitometry.
Co-immunoprecipitation-Lysates from A7r5 cells were incubated overnight with pan-14-3-3-, PKC-, or PKC␣-specific antibodies. The next day, GammaBindG Sepharose (GE Healthcare) was added to each sample and incubated for 2 h. Immunocomplexes were then pelleted by brief centrifugation and washed twice with Nonidet P-40 lysis buffer, eluted with sample buffer, and subjected to Western blotting with a pananti-14-3-3 or pPKC antibody.
Translocation of PKC-A7r5 cells were plated in 100-mm dishes and grown overnight followed by a 24-h serum starvation. Cells were subsequently treated as described in the text and washed twice with cold phosphate-buffered saline followed by the addition of detergent-free lysis buffer (50 mM HEPES, 150 mM NaCl, 5 mM NaF, 1 mM ␤-glycerolphosphate, protease inhibitor mixture (Roche Applied Science)). Cell lysates were homogenized by Dounce homogenization and then centrifuged at 100,000 ϫ g for 1 h. The supernatant (cytosolic fraction) was collected. The pellet was resuspended in detergent-free lysis buffer and recentrifuged to diminish any cytosolic contamination. The supernatant was removed, and the pellet was resuspended in Nonidet P-40 detergent lysis buffer and sonicated briefly. After incubation on ice for 30 min, the sample was centrifuged at 14,000 rpm for 10 min. The supernatant was collected (membranous fraction) and analyzed by Western blotting.
Western Blot Analysis-Western blots were done as described previously (2) with some modifications. Briefly, after selected treatment, cells were washed once with cold phosphate-buffered saline followed by the addition of cold lysis buffer (1% Nonidet P-40, 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na 4 P 2 O 7 , 1 mM ␤-glycerolphosphate, 1 mM Na 3 VO 4 , protease inhibitor mixture, and 1 mM phenylmethylsulfonyl fluoride in double distilled H 2 O, pH 7.5) on ice. Cells were lysed for 15 min on ice, and cell lysate was harvested and centrifuged at 14,000 ϫ g for 10 min at 4°C. Cell lysate or sucrose gradient fractions were loaded in 4 -12% precasted SDS-polyacrylamide gel electrophoresis gradient gels, and the resolved proteins were transferred to Hybond C nitrocellulose membranes (GE Healthcare). The membranes were blocked in 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h and then incubated with the appropriate primary antibody overnight at 4°C. After incubation, the membranes were washed with Tris-buffered saline (3 ϫ 10 min). The membranes were then incubated with the appropriate secondary antibody for 3 h at room temperature. After three more washes with Tris-buffered saline containing 0.1% Tween 20, the protein bands were detected by the enhanced chemiluminescence method and quantitated by laser densitometry.
Expression Constructs-Human Akt3 was cloned from human coronary aortic smooth muscle cell cDNA by conventional methodologies. This Akt3 was inserted into pcDNA3.1 (Invitrogen) with a FLAG epitope tag added by PCR to the C terminus. Site-directed mutagenesis was performed to convert serine 34 to glutamic acid (S34E) or alanine (S34A). All constructs were sequence verified by the Molecular Genetics Core Facility of the Section of Research Resources, Penn State College of Medicine. The myristoylated-PKC construct was graciously provided by Dr. Alex Toker (10).
Transfections-A7r5 cells were transiently transfected by either an electroporation kit from Amaxa Biosystems (Cologne, Germany) or FuGENE 6 from Roche Applied Science. For FuGENE 6, A7r5 cells were seeded to 50 -60% confluency and transfected with a 3:1 ratio of FuGENE 6 to DNA.
Proliferation Assay-DNA synthesis was analyzed essentially as described previously (2). Briefly, transfected cells were plated in a 24-well plate and grown overnight prior to 24 h of serum starvation. Following serum starvation, cells were treated with C 6 -ceramide for 1 h followed by treatment Ϯ platelet-derived growth factor (PDGF) for an additional 16 h. Cellular proliferation was assayed with the addition of 0.5 mCi/ml [ 3 H]thymidine for the final 4 h of treatment. Cells were washed once with cold 10% trichloroacetic acid and then incubated with cold trichloroacetic acid for 20 min. Cells were solubilized with 0.4 N NaOH for 30 min, and [ 3 H]thymidine incorporation into acid-insoluble DNA was assessed with a scintillation counter.
Statistical Analysis-One-way analysis of variance with Bonferroni multiple comparison post test and t tests analyses were performed using GraphPad Prism 4.0 software. A statistically significant difference was reported if p Ͻ 0.05. Data are reported at the mean Ϯ S.E. from at least n ϭ 3 separate experiments.

Exogenous C 6 -ceramide Localizes to Low Buoyant, Caveolinenriched
Fractions-It has been demonstrated by others that ceramide accumulates in lipid microdomains as a consequence of augmented sphingomyelinase activity or de novo synthesis (11). We now provide evidence showing that microdomains are enriched with exogenously delivered ceramide in A7r5 vascular smooth muscle cells. When A7r5 are treated with fluorescently labeled NBD-C 6 -ceramide, the majority of exogenous C 6 -ceramide was detected in the low buoyant density fractions (fractions 4 and 5) isolated by a sodium carbonate discontinuous sucrose gradient. C 6 -ceramide rapidly accumulated within the low buoyant fractions within 30 min and persisted for at least 4 h after administration (Fig. 1A). Similar results were obtained with a 3 H-radiolabeled C 6 -ceramide (data not shown). We next assessed the distribution of caveolin-1 (Fig. 1B) and 14-3-3 (Fig. 1C) within a discontinuous sucrose gradient. Caveolin-1 is a common raft-associated protein that is a marker protein for caveolae and also serves as a scaffolding protein (12), whereas 14-3-3 is another important scaffold protein (13). Western blot analysis of individual sucrose fractions confirmed an enrichment of caveolin-1 in fractions 4 and 5 ( Fig.  1B), suggesting that we had successfully isolated caveolinrich/low buoyant microdomains. In contrast, 14-3-3 expression is localized to fractions 9 -12 ( Fig. 1C). C 6 -ceramide (10 M, 1 h) treatment of A7r5 cells did not appreciably change the distribution or expression of either caveolin-1 or 14-3-3. These data indicate that exogenous C 6 -ceramide localizes to the low buoyant fractions that are consistent with caveolin-enriched microdomains.
Ceramide Dissociates PKC from 14-3-3 Scaffold Proteins-14-3-3 proteins have been demonstrated to regulate signaling proteins, including PKC isotypes (14). Although ceramide did not change the distribution or expression of 14-3-3, it did specifically reduce the association of PKC with 14-3-3 (Fig. 2). Specifically, immunoprecipitated PKC and PKC␣ were immunoblotted for 14-3-3 (pan), a scaffolding protein that was not associated with these microdomains (Fig. 1C). Ceramide reduced the interaction of PKC, but not PKC␣, with 14-3-3, (Fig. 2A). To confirm that ceramide reduced an interaction between 14-3-3 and an activated PKC, we utilized another  Ceramide selectively dissociates PKC from 14-3-3. A, treatment with 10 M C 6 -ceramide reduces the association between the scaffolding protein, 14-3-3, and PKC, but not PKC␣, as determined by co-immunoprecipitation (IP) experiments. In these experiments, either PKC or PKC␣ was immunoprecipitated from A7r5 cells and blotted for 14-3-3. IB, immunoblotted. B, treatment with 10 M C 6 -ceramide reduces association between 14-3-3 and phosphorylated PKC. In these experiments, 14-3-3 was immunoprecipitated and blotted for phospho-PKC. This is a representative blot of n ϭ 3 separate experiments, in which ceramide delivered either in a Me 2 SO/ bovine serum albumin (DMSO/BSA) or in liposomal formulation reduced interactions by 38 Ϯ 2 and 30 Ϯ 3%, respectively. co-immunoprecipitation strategy in which we now immunoprecipitated with pan-14-3-3 antibody and reprobed with a phospho-PKC antibody (Fig. 2B). C 6 -ceramide treatment reduced the interaction between 14-3-3 and the activated kinase. These experiments were confirmed when the C 6 -ceramide was delivered in either a Me 2 SO/bovine serum albumin formulation or a liposomal formulation. The fact that C 6 -ceramide treatment did not change the distribution of 14-3-3 (Fig.  1C) suggests that 14-3-3 and phospho-PKC disassociate upon C 6 -ceramide treatment. Thus, it is hypothesized that ceramide might specifically recruit activated PKC to microdomains. This may occur, in part, by inducing dissociation of PKC from scaffold proteins, including 14-3-3, that are not associated with structured membrane domains or reside loosely or transiently associated with the membrane. Ceramide-induced disassociation of PKC from 14-3-3 may serve as a mechanism to ensure optimal activation of PKC.
Ceramide Enhances PKC Activation within Caveolin-rich Domains-Confirming earlier published studies (2), we now show that exogenous ceramide, as well as agents that produce endogenous ceramide (interleukin-1␤ and bacterial sphingomyelinase), increase membrane-bound PKC phosphorylation at Thr 410 (Fig. 3A). In contrast, PDGF does not increase mem-brane-associated PKC phosphorylation in A7r5 cells. Western blots were reprobed and normalized for total PKC protein. It should be noted that ceramide, interleukin-1␤, and bacterial sphingomyelinase elevated only membranous PKC phosphorylation and did not induce "global" PKC translocation from the cytosol to the membrane. It also should be noted that these studies using Thr 410 as a surrogate marker for activated PKC support our previous work reporting ceramide activation of PKC in intact and in cell-free reconstitution assays using either immunoprecipitated or recombinant PKC (2).
To evaluate the possibility of intramembranous movement and activation of PKC, we utilized discontinuous sucrose gradients and again assessed the phosphorylation state of PKC at Thr 410 . As shown in Fig. 3B, ceramide increased the phosphorylation of PKC in the low buoyant fractions 4 and 5. In fact, there was approximately a 7-fold increase (Fig. 3B) in ceramide-induced PKC phosphorylation in fractions 4 and 5 when compared with the control levels (2.2 versus 14.7%). Consistent with these findings, bacterial sphingomyelinase ( Fig. 3C) also induced a shift of phosphorylated PKC to the low buoyant fractions. These results suggest that the ceramide localized PKC pool is preferentially phosphorylated/activated.
In the above discontinuous sucrose gradient experiments, A7r5 VSM cells were fractionated with a sodium carbonate method, which better preserves membrane structure and only maintains strongly associated protein-membrane interactions. To further support intramembranous movement of activated PKC to ceramide-enriched caveolin-containing domains, we employed a second detergent-inclusive (0.5% Tween) methodology to isolate lipid microdomains. Using this method of sucrose gradient fractionation, there was a preferential localization of caveolin-1 in fraction 5. In serum-starved A7r5 cells, activated PKC (pThr 410 PKC) was predominantly localized in fractions 9 -12. However, upon treatment with ceramide or sphingomyelinase, there was an increase in the amount of phospho-PKC found in fractions 5-8, with the greatest increase occurring in fraction 5 (Fig. 3D). Treatment with ceramide and bacterial sphingomyelinase led to an appreciable increase in the amount of phospho-PKC in fractions 5-8 when compared with vehicle-treated cells. Sucrose gradient fractions 5-8 of the lysates of vehicle-treated cells contained only 0.2% of the total phospho-PKC, whereas fractions 5-8 of lysates from cera- mide-and sphingomyelinase-treated cells contained 1.4 and 1.9%, respectively, of the total phospho-PKC. Confirming results with the detergent-free procedure, ceramide and sphingomyelinase treatment led to a 7-and 9-fold increase, respectively, in the percentage of total phospho-PKC found in the lower buoyant fractions when compared with that of vehicletreated cells.
To further explore PKC activation within ceramide-enriched microdomains, A7r5 cells were transfected with a myristoylated PKC construct. Discontinuous sucrose gradient analysis revealed that a small percentage of myristoylated PKC localized to the caveolin domain (Fig. 3, E and F). More importantly, exogenous ceramide, which localized within fraction 4 and 5, increased the percentage of myristoylated PKC within the caveolin domain. With the detergent-free sucrose gradient fractionation, ceramide treatment led to more than a 50% increase (4.9 versus 7.6%) in the percentage of myristoylated PKC detected in fractions 4 and 5 when compared with vehicle-treated cells (Fig. 3E). Similarly, in detergent-inclusive sucrose gradient fractionation, the percentage of myristoylated PKC in the raft-containing fractions increased over 2-fold, from 3.3 to 8%, after treatment with ceramide (Fig. 3F). These data further support the hypothesis that the recruitment of PKC to the structured ceramide-enriched microdomain may be necessary for PKC activation and not just membrane localization.
Lipid Rafts Are Necessary for PKC and Akt Activation-Lipid rafts are specialized microdomains that are enriched in specific lipids including cholesterol and sphingolipids. Disruption of the lipid rafts is possible through the addition of methyl-␤-cyclodextrin (M␤CD), which depletes the cell of cholesterol (15). We have previously demonstrated that ceramide-activated PKC inhibits PDGF-stimulated Akt phosphorylation (2). To test whether discrete ceramide-enriched structured microdomains are necessary for PKC-dependent Akt inactivation, we assessed PKC Thr 410 phosphorylation (Fig. 4A) as well as Akt Ser 473 phosphorylation (Fig. 4B) after pretreatment with 1% M␤CD. C 6 -ceramide modestly stimulated PKC Thr 410 phosphorylation (Fig. 4A), which correlated with complete inhibition in PDGF-induced phosphorylation of AKT Ser 473 (Fig. 4B). Ceramide-induced phosphorylation of PKC was significantly reduced in the presence of M␤CD (Fig. 4A). Treatment with 1% M␤CD also inhibited the PDGF-induced increase in Akt phosphorylation. In the presence of M␤CD, ceramide did not further reduce Akt phosphorylation levels (Fig. 4B). Upon cholesterol repletion, ceramide was again able to reduce PDGF-induced AKT activity. It should also be noted that raft disruption also reduced PDGF-induced Akt signaling but not growth factor-induced ERK signaling, which is mediated independent of cholesterol depletion (data not shown). These data support the postulate that ceramide-enriched structured microdomains are necessary for ceramide signaling.

Akt3 Ser 34 Is Necessary and Sufficient for Ceramide Inhibition of PDGF-stimulated Akt as well as PDGF-induced
Mitogenesis-In contrast to phosphorylations at Thr 308 and Ser 473 , which indicate activation of Akt, post-translational modifications of Akt indicative of inactivation have also been noted. Ceramide-induced activation of PKC can lead to inac-tivation of Akt1 through phosphorylation of Thr 34 within the pleckstrin homology domain of Akt1. This phosphorylation in turn inhibits its binding to phosphatidylinositol 3 lipids, a necessary cofactor for Akt activation (16). Here, we proposed that such a mechanism holds true for Akt3, the most abundant homolog found in vascular smooth muscle cells (17), which has an analogous Ser 34 in the pleckstrin homology domain. As inac- . Lipid rafts are necessary for PKC and Akt activation. A, A7r5 rat aortic smooth muscle cells were treated with 1% methyl-␤-cyclodextrin for 1 h followed by 10 M C 6 -ceramide (C6) for 1 h. Cell lysates were then immunoblotted for phospho-PKC and PKC. Methyl-␤-cyclodextrin disruption of structured microdomains decreases ceramide phosphorylation of PKC Thr 410 . V, vehicle. B, A7r5 cells were treated with 1% methyl-␤-cyclodextrin for 1 h followed by 10 M C 6 -ceramide (cer) for 1 h. In selected experiments, 0.3 mM cholesterol complexed with M␤CD was added for an additional 30 min prior to ceramide treatment but after cholesterol depletion. 10 ng/ml PDGF or vehicle was then added for 5 min, and cell lysates were immunoblotted for phospho-Akt. Methyl-␤-cyclodextrin disruption of structured microdomains decreases growth factor phosphorylation of Akt Ser 473 . Representative blots of n ϭ 3 separate experiments are shown. cnt, control.
tivated Akt leads to VSM growth arrest, DNA synthesis levels were determined by thymidine incorporation. It is well established that Akt plays a role as a cell survival kinase and has been implicated in cell proliferation in different cell types, including vascular smooth muscle cells. To demonstrate that Ser 34 phosphorylation is sufficient for ceramide-induced growth arrest, we transiently transfected A7r5 cells with either a wild type or a mutant Akt construct. The mutant Akt construct contains a glutamate at amino acid 34 to mimic phosphorylation at this site, which has the potential to constitutively inactivate Akt. In these experiments, equal loading and transfection efficiency for Akt mutants was confirmed using a FLAG-tagged antibody (data not shown). In A7r5 cells transfected with a wild type Akt3, PDGF increased DNA synthesis, which was significantly decreased by ceramide (Fig. 5A). The statistically significant decrease in mitogenesis with exogenous ceramide supports our earlier in vivo work documenting that C 6 -ceramide released from balloon catheters reduces stretch-induced rabbit carotid hyperplasia, as well as Akt in vivo (1). However, when the A7r5 cells were transfected with the mutant S34E Akt3 construct, PDGF no longer increased DNA synthesis levels above basal levels. Ceramide did not further reduce the DNA synthesis levels. To further delineate this mechanism, we demonstrate that Ser 34 phosphorylation is also sufficient for ceramide-induced inactivation of Akt. Using the same transfection strategy with the wild type construct, we again confirmed our initial observations (2) that PDGF activated Akt and ceramide reduced this PDGF-induced activation of Akt (Fig. 5B). However, upon transfection of the mutant Akt S34E construct, PDGF no longer increased Akt activation and ceramide no longer further reduced the phosphorylation levels of Akt. To definitively address the question of whether phosphorylation of Ser 34 is necessary for inactivation of Akt by ceramide, we utilized a Ser to Ala mutation at position 34 to potentially inhibit phosphorylation and subsequent inactivation of AKT (Fig. 5C). In con-trast to wild type overexpression and directly opposite to the S34E mutant construct, A7r5 cells overexpressing the S34A mutant still respond to PDGF, but the ability of ceramide to inactivate Akt, as assessed by Ser 473 phosphorylation, is now impaired. Taken together, these data suggest that ceramide-dependent PKC phosphorylation of Akt3 at Ser 34 is necessary for ceramide-induced vascular smooth muscle cell growth arrest. In addition, these observations support previous data that PDGF antagonizes the growth-arresting actions of ceramide by either inducing ceramide metabolism into pro-mitogenic sphingosine-1-phosphate or activating conventional PKC-dependent signaling cascades (18 -20).

DISCUSSION
The mechanisms by which ceramide exerts its biological functions, including apoptosis and growth arrest, have been under active investigation. Both biophysical (lipid microdomains) and biochemical (ceramide binding targets) mechanisms have been proposed to address this issue. The present study presents an integrated version of these theories.
We, and others, have previously demonstrated evidence that short chain ceramide analogs as well as physiological ceramide contributes to the activation of PKC in vitro and in vivo (biochemical mechanism) (2)(3)(4). This in turn leads to inhibition of cell cycle progression through a PKC-dependent, phosphatidylinositol 3-kinase-independent inactivation of the pro-survival kinase, Akt, in vascular smooth muscle cells (2). We report now that ceramide results in an increased localization and phosphorylation of PKC within lipid microdomains to inactivate Akt, demonstrating both a biophysical and a biochemical mechanism for ceramide-induced growth arrest.
Although the inhibition of Akt by ceramide has been demonstrated by multiple laboratories, including our own, multiple mechanisms have been described. These include mechanisms by which ceramide activates PKC (2-6), PP2A (21), and/or FIGURE 5. Akt3 Ser 34 is necessary and sufficient for ceramide inhibition of PDGF-stimulated Akt as well as PDGF-induced mitogenesis. A, A7r5 cells were transiently transfected with wild type (WT) Akt or the mutant (Mut) Akt-S34E, treated with 10 M C 6 -ceramide and/or 10 ng/ml PDGF, and subjected to a DNA synthesis proliferation assay. The S34E Akt mutation mimics the ability of C 6 -ceramide to inhibit PDGF-stimulated DNA synthesis. Representative blots of n ϭ 3 separate experiments, *, p Ͻ 0.05. B, A7r5 rat aortic smooth muscle cells were transiently transfected with wild type Akt or the mutant Akt-S34E and treated with 10 ng/ml PDGF and/or 10 M C 6 -ceramide. Immunoblotting for phospho-Akt-Ser 473 demonstrates that the S34E Akt mutation mimics the ability of C 6 -ceramide (C6-Cer) to inhibit the activation of Akt stimulated by platelet-derived growth factor. Veh, vehicle. C, A7r5 cells were transiently transfected with wild type Akt or the mutant Akt-S34A and treated with 10 ng/ml PDGF and/or 10 M C 6 -ceramide. Immunoblotting for phospho-Akt-Ser 473 demonstrates that the S34A Akt mutation inhibits C 6 -ceramide-induced inactivation of PDGF-stimulated Akt. B and C, representative blot of n ϭ 3-4 separate experiments.
PTEN (phosphatase and tensin homologue deleted on chromosome ten) (22). We now suggest that a commonality of these multiple mechanisms may be through the localization of ceramide within lipid microdomains. Recently, the Summers laboratory reported that ceramide could inhibit Akt activation in an okadaic acid-(PP2A) sensitive manner and/or via an inhibition of translocation (23). It has been demonstrated that caveolin-1, a resident protein of caveolae microdomains, can directly inhibit PP2A, thus preventing its ability to dephosphorylate/ inactivate Akt (24). Furthermore, ceramide has also been demonstrated to recruit PTEN into these microdomains, which can inhibit Akt activation by decreasing phosphoinositide-3-phosphates (22). Here, we have demonstrated that ceramide results in increased phosphorylated PKC within lipid microdomains. We have utilized both detergent-free and detergent-inclusive sucrose gradient strategies to isolate both endogenous phospho-PKC as well as overexpressed myristoylated PKC. Moreover, only the detergent-free system utilizes sodium carbonate at pH ϭ 11, whereas the detergent-inclusive system used Tween 20 at pH 6.5. Thus, we confirmed the same phenomenon using two distinct isolation methodologies for two versions of an activated and/or post-translationally modified membrane protein.
We provide evidence that a major affect of ceramide in vascular smooth muscle cells is to localize a bioactive, phosphorylated form of PKC within low buoyant caveolin-enriched lipid microdomains. This occurs by the recruitment and disassociation of phospho-PKC from 14-3-3, a non-microdomain yet membrane-associated scaffolding protein, to place phospho-PKC in close proximity to downstream targets such as Akt to induce growth arrest. We also report that ceramide-activated PKC-induced growth arrest is mediated by the phosphorylation of Akt3 at serine 34. This further supports the work previously published by the Hundal laboratory, which demonstrated that ceramide induced PKC phosphorylation of Akt1 at Thr 34 and subsequent inhibition of activation (16). When the analogous site of Akt3 was mutated to a glutamic acid to mimic phosphorylation, PDGF-induced proliferation was decreased, even in the absence of ceramide. Furthermore, in PDGF-stimulated cells overexpressing this mutant, phosphorylation of the activating site Ser 473 was greatly diminished. Thus, ceramideinduced Akt inactivation is a necessary component of VSM growth arrest. Although we demonstrate a role for PKC and Akt in the growth-arresting properties of ceramide, it is unlikely to be this simplistic. We cannot rule out other contributing proteins such as PAR-4, which interacts with PKC to form a pro-apoptotic complex in response to ceramide (4,5).
This recruitment of activated PKC within microdomains is further validated by a recent study demonstrating that upon Fas activation, PKC accumulates within lipid microdomains (25). Although this study did not examine the role of ceramides in this recruitment, other studies have demonstrated that Fas/ FasL interactions lead to acid sphingomyelinase-generated ceramide, which is capable of orchestrating raft reorganization into large cell-surface macrodomains, where the proteins of the death-inducing signaling complex of Fas can oligomerize (26,27). These studies also suggest that ceramide may regulate binding interactions between PKC and upstream elements in the Akt cascade, including phosphoinositide-dependent kinase (PDK), which interact with and activate PKC by phosphorylating Thr 410 (10,28).
Taken together, understanding the mechanism by which PKC is activated within ceramide-enriched microdomains will further define the clinical relevance of ceramide as an antiproliferative therapeutic for inflammatory diseases. Understanding the biophysics and biochemistry of these lipid-protein interactions could eventually lead to the design of selective lipido-or peptidomimetic inhibitors of these interactions.