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B activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl-terminal phosphorylation of PKC for the ceramide response
Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston, South Carolina 29425, USA
1Correspondence: Department of Biochemistry and Molecular Biology, 173 Ashley Ave., P.O. Box 2550509, Charleston, SC 29425, USA. E-mail: hannun{at}musc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B whereas the lipid mediator ceramide was recently shown to inhibit activation of this transcription factor (1
, 2)
. In this study, the mechanisms by which ceramide interferes with this pathway were examined in Jurkat leukemia and MCF-7 breast cancer cells. Both exogenous and endogenous ceramide inhibited selectively PKC-mediated activation of NF-B by reverting PKC translocation to the membrane. Next, confocal and immunofluorescence studies were performed to evaluate the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)-PKCß2 fusion protein in response to PMA. A mutant PKC in which autophosphorylation sites were mutated to alanine (PKC-DA) was resistant to ceramide. A kinase-inactive mutant (PKC-KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpression of PKC-DA prevented, at least partly, the ability of ceramide to inhibit activation of NF-B. Taken together, these studies show that ceramide has acute effects on translocation of PKC by inducing reverse translocation, and this reversal requires both the kinase activity of PKC and phosphorylation of the autophosphorylation sites.—Signorelli, P., Luberto, C., Hannun, Y. A. Ceramide inhibition of NF-B activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl-terminal phosphorylation of PKC for the ceramide response.
Key Words: diacylglycerol • sphingolipid • glycerolipid • C6-ceramide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 4)
.
DAG, a key lipid mediator, derives from the metabolism of phosphatidylinositols and phosphatidylcholine via the action of phospholipase C and phospholipase D/phosphatidic acid phosphohydrolase, respectively, after stimulation with growth factors and other agonists (5)
. DAG plays various roles as a second messenger, acting mostly as a proliferative, growth-promoting lipid primarily through the activation of members of the protein kinase C (PKC) family (6)
. PKC exists as a family of closely related isoenzymes, grouped into three subfamilies: classical PKCs, cPKCs (composed of PKC
, ß, and 
); the novel PKCs, nPKCs (composed of PKC 
, 
, 
, and 
); and atypical PKCs, aPKCs (composed of PKC
and 
/
) (6
, 7)
. The tumor-promoting phorbol esters exert many, if not most, of their effects by mimicking the action of DAG on PKC, binding the protein with a higher affinity than DAG.
Actions of the sphingolipid ceramide are mostly associated with inhibition of proliferation, induction of differentiation, and induction of cell death and cell senescence (8)
. Ceramide derives from cleavage of complex sphingolipids such as sphingomyelin, through the action of sphingomyelinases, as well as from de novo synthesis (9
, 10)
. Similar to DAG, ceramide has been described to regulate the activity of several cellular proteins (8
, 11)
, including PKC (12
, 13)
.
Although the actions of ceramide and DAG are often antagonistic, little is known about specific molecular targets regulated in an opposing manner by these two lipid-mediated pathways. One key candidate is the nuclear factor kappa B (NF-B), which has been shown to be activated by various members of the PKC family. For example, the aPKCs (,/) have been implicated in activating NF-B in response to tumor necrosis factor (TNF-) and nerve growth factor (14
15
16
17)
whereas conventional PKCs have been reported to be involved in NF-B activation in response to PMA (15
, 18
19
20)
. Some of the novel PKC isoforms have been reported to mediate NF-B activation: PKC in response to LPS (19
, 21)
and PKC in response to stimulation of TCR in T cells (22
, 23)
.
On the other hand, previous studies showed that ceramide prevented activation of NF-B in response to PMA and serum stimulation (1
, 2)
. In a recent study, we showed that bacterial sphingomyelinase (bSMase), which generates ceramide at the plasma membrane, also inhibited activation of NF-B. However, when ceramide was further metabolized to sphingomyelin through the action of sphingomyelin synthase, the resulting DAG (generated from phosphatidylcholine) induced activation of NF-B. These results led us to suggest that ceramide on its own is an inhibitor of NF-B but when it serves as a substrate for SM synthase, thus inducing DAG production, it leads to NF-B activation (2)
. This may explain the initially conflicting reports on the role of ceramide in regulating NF-B (1
, 24
25
26)
.
Given these opposing effects of ceramide and DAG on NF-B activation, we investigated in this study the mechanisms by which ceramide interferes with the PKC-mediated activation of NF-B. We demonstrate that, in Jurkat T cell leukemia and in MCF7 adenocarcinoma cells, PMA induced NF-B nuclear translocation through activation of PKC. Exogenous cell-permeable ceramides (C6-ceramide) or endogenous ceramide generated through the action of bSMase inhibited PKC-induced translocation and activation of NF-B but did not inhibit the activation of NF-B by TNF-, demonstrating that this effect is specific to a PKC-mediated pathway. Investigation of this mechanism resulted in tracing the inhibitory action to a reversal of translocation of phosphorylated and active cPKCs. This required both the kinase activity of PKC and the phosphorylation of its carboxyl terminus autophosphorylation sites. A mutant PKCß2 in which the two autophosphorylation sites at the carboxyl terminus were mutated to alanine (PKCß2-DA) was resistant to the action of ceramide upon reverse translocation of PKC, and PKCß2-DA partially prevented the inhibitory action of ceramide on NF-B. These results provide novel insight on the mechanisms of NF-B activation induced by phorbol esters and on the role of ceramide in the regulation of PKC translocation and signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was purchased from PeproTech (Rocky Hill, NJ); C6-ceramide was purchased from Matreya (Pleasant Gap, PA); [-32]ATP was purchased from NEN Life Science Products (Boston, MA). Poly (dI-dC) and poly(dN)6, protein A-Sepharose CL-4B, and the ECL detection system were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). T4 polynucleotide kinase was purchased from Promega (Madison, WI). Anti-phospho-serine 660 and anti-ERK1/2, anti-phospho ERK1/ERK2 antibodies were purchased from New England Biolab (Beverly, MA). Anti-PKC phospho-threonine 500 antibodies were a kind gift from Dr. A. Newton (University of California of San Diego, La Jolla, CA). Primary antibodies against PKC , ß2, , , , , and NF-B (p65) HRP-conjugated, secondary anti-mouse, -goat, and -rabbit IgG were purchased from Santa Cruz. Texas red dye-conjugated anti-mouse antibodies were purchased from Jackson ImmunoResearch Lab. (West Grove, PA). Okadaic acid, wortmannin, Ly 294002, herbimycin A, bisindolylmaleimide I, and G0 6976 were purchased from Calbiochem (San Diego, CA). Phorbol 12-myristate,13-acetate (PMA), sphingomyelinase from Staphylococcus aureus (bSMase), and histone type III:S from calf thymus were purchased from Sigma (St. Louis, MO); 35 mm glass-bottom poly-L-lysine-coated culture dishes were purchased from MatTek Corp. (Ashland, MA).
Cells culture and transfection
Jurkat and MCF7 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C. For experiments, Jurkat cells were seeded at 6 x 105 cells/ml in RPMI supplemented with 2% fetal FBS and starved 12–14 h before treatments; MCF7 cells were seeded at 1 x 106 cells/100 mm dish in RPMI supplemented with 10% FBS, rested for 24 h, then starved in RPMI supplemented with 2% FBS for 12 h before treatments. For transient transfection, 3.5 x 105 MCF7 cells were seeded in 35 mm culture dishes, rested for 24 h in RPMI 10% FCS, and transfected with Superfect reagent (Qiagen, Chatsworth, CA) using 2 µg of plasmids/dish. After 4 h of incubation with the mixture, cells were washed with PBS and fresh RPMI medium with 2% FBS. Cells were grown for 15–20 h before treatments.
Plasmid constructs
pBK-CMV-GFP-PKCß2, pKB-CMV-GFP-PKCß2-T642A-S660A (double mutant: DA), pKB-CMV-GFP-PKCß2-S660E (SE), pKB-CMV-GFP-PKCß2-KR were constructed as described previously (27)
.
Nuclear extracts
The nuclear extraction procedure was modified from Dignam et al. (28)
, with all procedures performed at 4°C. After treatments, the medium was removed; cells (6x106) were washed with PBS, resuspended in 300 µl of lysis buffer (10 mM HEPES, pH7.9, 10 mM KCl, 100 mM EGTA, 100 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride), and incubated for 10 min on ice. Just before centrifugation, 5 µl of 10% Nonidet P40 was added, the cell suspension was pipetted four times, and nuclei were pelleted by centrifugation for 10 min at 1300 g. The supernatant was removed and nuclei were resuspended in 50 µl of extraction buffer (20 mM HEPES pH 7.9, 400 mM NaCl, 25% (v/v) glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). The suspension was mixed gently by shaking for 30 min and centrifuged at 18,000 g for 15 min. The supernatants were flash-frozen and stored at -80°C. Protein concentration was determined using the Bio-Rad assay.
Electrophoretic mobility shift assay (EMSA)
Assays were performed in a final volume of 20 µl using 10 µg of nuclear extract and incubated in HDKE buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 5% (v/v) glycerol, 1 mM EDTA, 5 mM dithiothreitol) containing 1 µg poly (dI-dC), 1 µg poly (dN)6, and 10 µg of bovine serum albumin; 1 µl of radiolabeled oligonucleotide probe (80,000–120,000 cpm) was added to each reaction and incubated at room temperature for 20 min. The reaction was terminated by the addition of 6 µl of 15% Ficoll solution containing indicator dyes (bromphenol blue and xylene cyanol). Equal amounts (20 µl) of reaction mixture were loaded on a 5% nondenatured polyacrylamide gel in 90 mM Tris borate, 2 mM EDTA and run at 200 V. Gels were placed onto Whatman filter paper, dried, and autoradiographed.
Oligonucleotides
A synthetic NF-B consensus oligonucleotide from Promega with the following sequence: 5'-ACTTGAGGGGACTTTCCCAGGC-3' was end-labeled using polynucleotide T4 kinase and [-32]ATP and purified on a microspin G25 column.
Cytosol and membrane fractionation
Cells (6x106) were harvested on ice; all procedures were performed at 4°C. Cells were washed with PBS, resuspended in 250 µl homogenization buffer (20 mM Tris pH 7.4, 10 mM EGTA pH 7.4, 2 mM EDTA pH 7.4, 10 mM sodium orthovanadate, 20 mM sodium fluoride, 1 mM ß-glycerophosphate, 50 nM okadaic acid, 0.02% leupeptin, 0.01% aprotinin, 0.01% trypsin-chymotrypsin inhibitor, 1 mM phenyl methyl sulfonyl), and lysed by sonication three times for 15 s. Nuclei and unbroken cells were pelleted by centrifugation at 1300 g for 10 min and supernatants were centrifuged at 120,000 g for 30 min. Supernatants (cytosolic fraction) were stored at -80°C. Pellets (membrane fractions) were resuspended in 100 µl of homogenization buffer, sonicated once for 20 s, and stored at -80°C. Protein concentrations of the fractions were determined using Bio-Rad assay.
Western blotting
For detection of cytosolic proteins (I-B and ERKs), 6 x 106 cells were lysed on ice in 150 µl of lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 10 mM EGTA pH 7.4, 2 mM EDTA pH 7.4, 10 mM sodium orthovanadate, 20 mM sodium fluoride, 0.01% leupeptin, 0.01% aprotinin, 0.01% trypsin-chymotrypsin inhibitor, 1 mM phenyl methyl sulfonyl, 1 mM dithiothreitol, 0.5% Nonidet P-40). The lysates were centrifuged at 500 g for 10 min (to discard unbroken cells) and the protein concentration of the supernatants was determined by Bio-Rad assay. For PKC translocation studies, cytosolic and membrane fractions were separated as described above and protein concentration was determined in both fractions. Equal amounts of protein (50 µg for I-B, 5 µg for ERKs and phospho-ERKs, 15 µg for novel PKCs, 5 µg for classical PKCs) from cell extracts were diluted in Laemmli buffer and separated on 7.5 or 10% polyacrylamide gel by SDS-PAGE as described (29)
. Proteins were electrophoretically transferred to a nitrocellulose membrane (30)
. The nitrocellulose membranes were blocked in 5% dried milk-PBS containing 0.1% Tween 20, then incubated 1–3 h with primary antibodies diluted in 5% dried milk-PBS 0.1% Tween 20 (anti-PKCs antibodies were diluted 1:500; anti-phospho PKC, anti-ERKs and anti-phospho-ERKs were diluted 1:1000). Membranes were washed in PBS containing 0.1% Tween 20 and incubated with secondary antibodies diluted 1:3000 in 5% dried milk-PBS 0.1% Tween 20 for 1 h. Membranes were developed using the ECL detection system.
Immunoprecipitation
Proteins (200 µg) from cell extracts were resuspended in a total volume (150–200 µl) of homogenization buffer containing 0.25% Nonidet P40 and incubated with the appropriate antibody in a final dilution of 1:200 for 1 h while shaking. Protein A-Sepharose (5 mg) was added to each sample and incubation was continued for another 30–60 min. Beads were washed three times with homogenization buffer containing 0.5% Nonidet P40.
In vitro kinase assay
The protein A-Sepharose beads conjugated with PKC were washed twice with kinase buffer (50 mM Tris pH 7.4, 10 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM magnesium chloride, 1 mM phenylmethylsulfonyl fluoride, 0.01% leupeptin, aprotinin, trypsin-chymotrypsin inhibitor) and resuspended in a total volume of assay buffer (20 mM Tris pH 7.4, 10 mM magnesium chloride, 0.4 mg/ml histone type III:SS, 200 µM calcium chloride, 80% phosphatidylserine/20% diacylglycerol/3% Triton X-100, 50 µM unlabeled ATP). Reaction was started by addition of 2.5 µCi of [-32] ATP/sample carried out for 20 min in water bath at 30°C and terminated by addition of 2x Laemmli buffer. Samples were boiled and loaded in a 10% polyacrylamide gel and separated by SDS-PAGE. Gels were dried at 80°C for 90 min and radioactive-labeled phosphorylated histone was visualized by autoradiography.
Confocal microscopy and immunocytochemistry
Cells were seeded at 3.5 x 105 cells/dish on glass-bottom, poly-L-lysine-coated 35 mm dishes transiently transfected as mentioned above. Once treated, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, then washed with PBS. Cells expressing GFP fluorescent PKCßII or its mutants were observed at confocal microscope, Olympus IX 70, Perkin-Elmer Ultraview software, spinning disk; using Olympus 60 X, 1.4 NA, oil immersion lens and fluorescence signals were collected after single line excitation at 488 nm (green). For immunocytochemical analysis, cells were fixed with 4% paraformaldehyde in PBS for 30 min. All procedures were performed at room temperature. Once fixed, cells were permeabilized in 4% paraformaldehyde, 0.2% Triton X-100 in PBS for 30 min at room temperature. After washing with PBS, cells were blocked with 10% fetal calf serum (FCS) in PBS for 30 min. Incubation with mouse monoclonal antibody anti-NF-B diluted in 5% FCS in PBS was carried out for 1 h. Cells were washed three times with PBS and incubated with secondary anti-mouse antibody Texas red-conjugated diluted 1:200 in 5% FCS in the same saline. Cells were washed with PBS and fluorescence was observed at confocal microscope with excitation at 488 nm (green) or 543 nm (red).
Reproducibility of results
Results shown are representative of three to five independent experiments with similar results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B in a PKC-dependent pathwayB induced by serum stimulation or phorbol esters in Wi-38 human fibroblasts (2)
. To investigate the mechanism of inhibition, Jurkat cells were treated for 30 min with PMA (10 nM), either with or without pretreatment with bSMase (200 mU/ml for 10 min), and the activation of NF-B was evaluated by EMSA (Fig. 1
A). The results show that PMA activated NF-B, and bSMase had no effect of its own on activation of NF-B, consistent with previous studies that ceramide does not activate NF-B in this cell line (1)
. bSMase inhibited significantly the ability of PMA to activate NF-B (Fig. 1A
). Another well-known inducer of nuclear translocation of NF-B is TNF-; some (31)
, but not all (1
, 2
, 15
, 18
, 20
, 32)
, studies point to common mediators activated by PMA and TNF- in the pathways leading to activation of NF-B. Therefore, we evaluated the effects of bSMase on TNF--induced NF-B translocation (3 nM for 30 min). Pretreatment with bSMase did not inhibit activation of NF-B induced by TNF- (Fig. 1A
), suggesting that TNF- and PMA activate distinct pathways leading to the activation of NF-B.
|
The activation of NF-B was also evaluated by examining the levels of I-B, which binds to NF-B and prevents its nuclear translocation. Degradation of I-B is required for activation of NF-B by TNF and probably by PMA (33)
. Treatment with PMA resulted in the loss of I-B, as evaluated by Western blot analysis. Pretreatment with bSMase prevented the effects of PMA (Fig. 1B
). Again, bSMase alone had no effect on the basal levels of I-B (Fig. 1B
). These results provide clear evidence for the ability of bSMase to inhibit activation of NF-B by PMA.
Since PMA is a well-known activator of PKC and TNF- is an inconsistent (and indirect) activator of PKC, we determined whether PKC is involved in the selective response to bSMase. Jurkat cells were treated with PMA with or without pretreatment with the PKC inhibitors bisindolylmaleimide (BIS), a broad spectrum inhibitor of PKC, and G06979 (G0), a selective inhibitor of the classical PKC isoforms at low concentrations (34)
. Both G0 and BIS inhibited PMA-induced activation of NF-B at low concentrations (0.2–1 µM), but not the TNF-induced activation (Fig. 2
). These results suggest that PMA activates NF-B in a PKC-dependent manner; they also suggest it is most likely a member of the classical PKCs, less likely a novel PKC since these require higher concentrations of G0 or BIS for inhibition, and unlikely to be atypical PKCs since these are not activated directly by PMA.
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Effects of bSMase on PKC function in cells
Since PKC was selectively involved in PMA-, but not TNF-induced, activation of NF-B and bSMase selectively inhibited the PMA response, we evaluated the effects of bSMase on additional cellular substrates of PKC. Jurkat cells were treated with PMA with or without pretreatment with bSMase, and phosphorylation of ERK1 and ERK2 was evaluated by Western blot analysis using antibodies specific for the phosphorylated forms. PMA markedly induced phosphorylation of ERK1 and 2, and bSMase pretreatment did not reduce these effects significantly (Fig. 3
). bSMase had little effect on phosphorylation of the MARCKS protein in response to PMA (data not shown). Thus, bSMase appears to have selective effects on targets of PKC, blocking preferentially the pathway leading to NF-B activation.
|
In previous studies, it was shown that long-term treatment with C6-ceramide caused dephosphorylation and inactivation of PKC as a consequence of activation of protein phosphatases (35
, 36)
. Since those effects required treatment of cells with C6-ceramide for several hours, we wondered whether the more acute effects of bSMase (and C6-ceramide) on I-B and NF-B share the same mechanism of inactivation of PKC. Therefore, the phosphorylation at the activation loop and at the carboxyl terminus of PKC extracted from total lysates of treated cells was evaluated by Western blot analysis, using antibodies directed against the activation site threonine p500 of ß2 (p497 of ) and against serine 660 of ß2 (657 for ). Treatment with bSMase or PMA did not change significantly the phosphorylated pool of the kinase (Fig. 4
a, b). The activity of the kinase was then evaluated by assaying histone phosphorylation using enzyme immunoprecipitated from treated cell extracts. PKC activity was not affected by PMA treatment; pretreatment with bSMase had little effect as well (Fig. 4c
). These results show that the acute action (within 30 min) of bSMase on PKC is not related to the long-term effect of ceramide on inactivation of the kinase activity of PKC.
|
The cellular activation of PKC is intimately related to its translocation and binding to the membrane. Therefore, the translocation of PKC isoenzymes from the cytosolic to the membrane fractions was evaluated in Jurkat cells after treatment with PMA with or without pretreatment with bSMase (Fig. 5
A). Since PMA activates classical and novel (DAG-responsive) isoforms of PKC, the translocation of PKC , ß, , , , and was followed by Western blot analysis. All isoforms of PKC translocated to the membrane fraction after treatment with PMA, albeit to various extents. Pretreatment with bSMase did not show much effect on the translocation of the novel isoforms, but the classical forms showed a decrease of the membrane-bound and an increase in the cytosolic fraction when bSMase was administered to cells before PMA (Fig. 5A
). Therefore, bSMase exerted a specific effect on translocation of classical PKCs.
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Effects of bSMase on phosphorylation and activity of subcellular distributed PKC
In a previous study, we showed that phosphorylation of PKC promoted its reverse translocation in response to activation of the angiotensin receptor (27
, 37)
. Therefore, we evaluated the phosphorylation state of the protein in unstimulated and PMA-stimulated conditions in either the cytosolic or membrane fraction. Jurkat cells were treated with PMA with or without pretreatment with bSMase, and Western blotting was performed with antibodies directed against the phosphorylated forms of PKC: anti-threonine p497–500 and anti-serine p657–660. PMA induced significant translocation of phosphorylated classical PKCs and bSMase reduced significantly membrane association of phosphorylated PKC (Fig. 5B
).
Since phosphorylation of PKC at threonine 500 is required for activity and p660 is involved in stability and membrane binding of the enzyme, we next assessed the effects of bSMase on activity of translocated PKC. PKC was immunoprecipitated from cytosolic and membrane fractions of Jurkat cells treated with PMA. Treatment with PMA resulted in decreased total PKC activity in the soluble fraction and an increase in activity in the membrane fraction (Fig. 5C
). On the other hand, pretreatment with bSMase reversed the PMA effects with most of the activity found in the cytosolic fraction. Thus, the modulation by bSMase of translocation of the activity of PKC paralleled the modulation of translocation of the phosphorylated forms.
According to the above data, the effects of bSMase could be due to the loss of membrane SM and/or the generation of ceramide. Treatment with bSMase in this cell line resulted in an acute elevation in the levels of endogenous ceramide; this did not affect cell viability (data not shown), consistent with previous results that many cell lines do not undergo cell death after generation of ceramide on the outer leaflet of the plasma membrane (38)
.
To determine whether exogenously added C6-ceramide was able to induce the same effects on the subcellular distribution of phospho-PKC observed with bSMase, Jurkat cells were treated with PMA (10 nM) for 30 min either with or without pretreatment with C6-ceramide 10 and 20 µM for 60 min (Fig. 6
). As with bSMase, cotreatment with exogenous ceramide caused a decrease of membrane-bound phosphorylated PKC after PMA stimulation and a consequent increase of the cytosolic pool. Moreover, these results support the ability of endogenous ceramide, formed at the plasma membrane, to exert regulatory effects by inhibiting a PMA-mediated event.
|
Ceramide reverts the translocation of PKC
To further understand the mechanism by which ceramide affects the subcellular localization of PKC, the effects of ceramide administered either before or after PMA-induced translocation were compared. Jurkat cells were stimulated with PMA (10 nM) for 5, 10, or 20 min, and the membrane translocation of PKC was evaluated. The results showed that ceramide (30 µM) was effective in preventing translocation whether added before (30 min) or after PMA (Fig. 6B
). Therefore, ceramide does not prevent PKC translocation to the membrane; rather, it induces detachment from the membrane, thus reverting PKC to the cytosol.
Evaluation of the effects of bSMase and C6-ceramide on translocation of PKC in vivo by confocal microscopy
To better understand ceramide effects on reversal of translocation of PKC, MCF7 cells were transiently transfected with a GFP-tagged PKCß2 and translocation events were followed by confocal microscopy. PMA stimulation of MCF7 cells (25 nM) markedly induced translocation of GFP-PKCß2 to the membrane, which started to be detectable in 
5–10 min, became clearly visible at 30 min, and persisted for longer than 6 h. However, pretreatment with bSMase (200 µm/ml) or C6-ceramide (30 µM) for 45 min before PMA addition caused a diffuse cytosolic location of GFP-PKCß2 (Fig. 7
A). The effects of C6-ceramide were dose dependent, being totally effective at 30 µM, partially effective at 15 µM, and ineffective at lower concentrations (Fig. 7B
, panels 1–10). Similar to the results in Jurkat cells, ceramide also induced reversal of translocation of PKC when added after PMA (Fig. 7B
, panel 11); dihydro-C6-ceramide was ineffective (Fig. 7B
, panels 12, 13).
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Requirement for kinase activity for ceramide-induced reverse translocation
The membrane association of PKC has been shown to be regulated by kinase activity of the enzyme and by autophosphorylation (27
, 39)
. This suggested the possibility that bSMase may require kinase activity in order to affect the interaction of PKC with membranes. Therefore, Jurkat cells were treated with PMA with or without pretreatment with the PKC kinase inhibitor bisindolylmaleimide (BIS), and cytosolic and membrane fractions were analyzed for phosphorylated PKC by Western blotting (Fig. 8
A). Inactivation of the enzyme significantly impaired the ability of bSMase to reverse the translocation of PKC. Similarly, using confocal microscopy when BIS was administered to cells together with C6-ceramide, the effects of ceramide on the reversal of translocation of PKC were abrogated and GFP-PKCß2 remained bound to the membrane (Fig. 8B, WT
, column 5). To confirm these results and exclude the possibility that BIS acts on targets other than PKC, we overexpressed in MCF7 cells the kinase-deficient mutant GFP-PKCß2-KR in which a conserved lysine was substituted with an arginine at the ATP binding site (K371R). This mutation causes the complete loss of ability to autophosphorylate and phosphorylate other substrates (37
, 40)
. In MCF7 cells overexpressing GFP-PKCß2-KR, PMA induced membrane translocation of the mutated protein, but pretreatment with C6-ceramide did not affect the association of PKC with the membrane (Fig. 8C
). These experiments show clearly that ceramide acts only on the active kinase. Moreover, the results with the BIS inhibitor and the kinase inactive mutant confirm and support the thesis that ceramide does not interfere with initial membrane translocation of PKC but induces reverse translocation of membrane-associated PKC.
|
Role of phosphorylated residues on PKC in ceramide-regulated translocation
We then investigated the role of phosphorylation and autophosphorylation of PKC by transiently transfecting cells with GFP-PKCß2 mutated in the carboxyl-terminal phosphorylated residues. Replacement of serine 660 and threonine 641 in PKCß2 with alanine (negating the possibility of either auto- or trans-phosphorylation of the protein on these sites) generated the double-point mutant PKCß2-DA. On the other hand, replacement of the same residues with glutamic acid (PKCß2-SE) mimicked the charge of the phosphate. In MCF7 cells, PMA markedly induced translocation to the membrane of GFP-PKCß2-DA and GFP-PKCß2-SE (Fig. 8B
, column 3). Treatment with C6-ceramide before stimulation with PMA reverted the translocation of the glutamate mutant GFP-PKCß2-SE, but was unable to induce detachment from membrane of the nonphosphorylatable GFP-PKCß2-DA (Fig. 8B
). These results suggest that phosphorylation at the carboxyl terminus of the protein is essential for ceramide to be able to revert protein translocation.
The requirement for both kinase activity and the autophosphorylation sites suggests (as the easiest scenario) a single requirement for kinase activity to induce autophosphorylation. However, this scenario is not consistent with the inability of PMA to induce significant changes in phosphorylation of PKC (Fig. 4a, b
). To determine whether phosphorylation was the only requirement for the induction of reverse translocation, we next evaluated the role of kinase activity in autophosphorylation site mutants. MCF7 cells were transfected with mutated GFP-PKCß2-DA or GFP-PKCß2-SE, treated with BIS (1 µM) for 30 min, then with ceramide (30 µM) for an additional 45 min, and finally with PMA (25 nM) for another 30 min. After kinase inhibition, both mutated forms translocated to the membrane and remained bound to it, with ceramide having no effect upon reverting their translocation (Fig. 8B
, column 5). Since the SE mutation should mimic the phosphorylation at the autophosphorylation sites in the carboxyl terminus, these results suggest that kinase activity is not required simply to induce phosphorylation at the carboxyl terminus sites.
PKCß2-DA overcomes ceramide inhibition of NF-B activation in MCF7 cells
Since the DA mutant was resistant to the effects of ceramide on translocation, we wondered whether this mutant could overcome the effects of ceramide on NF-B activation. GFP-PKCß2-WT, -DA, -SE overexpressing MCF7 cells were treated with ceramide and PMA; after stimulation, cells were fixed and stained with anti-NF-B antibodies. The nuclear/cytosolic distribution of NF-B was analyzed by confocal microscopy. In cells overexpressing either WT or mutated forms, PMA treatment induced PKC translocation to the membrane and NF-B translocation to the nucleus. Pretreatment with C6-ceramide reverted membrane translocation of WT- and SE-PKCs (Fig. 9
A). C6-ceramide also prevented significantly translocation of NF-B to the nucleus (seen as red nuclear staining in Fig. 9A
). Pretreatment with C6-ceramide did not affect PMA-induced GFP-DA-PKC translocation to the membrane; more important, C6-ceramide did not prevent NF-B translocation to the nucleus (Fig. 9A
). As shown in Fig. 9A
, the lack of effect of ceramide on blocking NF-B nuclear translocation was seen only in cells overexpressing GFP-PKCß2-DA (green cells) and not in adjacent cells not expressing the DA mutant. Thus, cells not overexpressing the DA mutant were still sensitive to ceramide inhibition of PMA-induced NF-B activation. These results suggest that the persistence of an active enzyme on the membrane is required for the signaling pathway that leads to NF-B translocation.
|
To further support this observation, we evaluated NF-B translocation by EMSA. Although in normal and wild-type-PKC transfected cells C6-ceramide reduced NF-B activation, such an effect was significantly attenuated in the GFP-PKCß2-DA transfected cells (Fig. 9B
). Given the transfection efficiency of around 40%, 60% of cells do not express the DA-PKC mutant and therefore are expected to show the effects of ceramide on wild-type PKC and NF-B.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B by phorbol esters. As models we chose two human cancer cell lines, T cell leukemia (Jurkat) and breast adenocarcinoma (MCF7), which provided concordant and complementary results. These results showed that exogenous short-chain ceramide and endogenous ceramide, formed at the plasma membrane by the action of bSMase, both resulted in selective inhibition of activation of NF-B by phorbol esters but not by TNF. This was traced to an action of ceramide on the membrane association of classical PKCs. The ability of ceramide to induce this reverse translocation depended on PKC activity as well as its carboxyl-terminal phosphorylation. These results point to several important mechanisms in the cross-talk of the DAG/PKC pathway with that of the ceramide-regulated one.
Different studies have suggested either common or distinct pathways for PMA- and TNF-induced activation of NF-B (31
, 41)
. It was reported that PMA activates NF-B through direct activation of classical and/or novel PKC isoforms (15
, 18
19
20)
whereas TNF- has been shown to stimulate atypical PKC isoform activity (13
, 14
, 42)
and, via these PKCs, to activate the IKKs up stream of I-B (15
, 16)
. Our results dissociate the mechanisms of activation of NF-B by PMA from those in response to TNF. Activation by PMA required classical PKCs and was inhibited by PKC inhibitors and ceramide. TNF, on the other hand, did not require PKC (at least the classical or novel isoforms) and its action was not inhibited by ceramide. These results clearly show that TNF- and phorbol esters signal to NF-B via distinct pathways.
These initial results prompted us to investigate the mechanisms by which ceramide inhibits NF-B, and thus to implicate the action of ceramide on translocation of classical PKCs. We found that NF-B activation in response to PMA was prevented by blocking the activity of classical isoforms of PKC with low concentrations (200 nM) of G0676. These results were further confirmed and extended by studies with ceramide. Ceramide inhibited selectively the translocation of classical PKCs, which correlated with the inhibition of NF-B. Moreover, ceramide had modest effects on phosphorylation of ERKs (and no effect on phosphorylation of the MARCKS; data not shown) in response to PMA, suggesting they may be substrates for the novel PKCs. Finally, a ceramide-resistant mutant of PKC (the DA double mutant) overcame ceramide inhibition of NF-B. Together, these results support a selective role of classical isoforms of PKC in the activation of NF-B in response to PMA and in the inhibition of this response by ceramide.
A major focus of this study was the determination of mechanisms by which ceramide inhibited the ability of PKC to function in cells. We considered different possibilities based on previously published results. It has been shown that ceramide has no effect on PKC activity in vitro (43)
, although recently it has also been shown that ceramide binds in vitro to PKC and , induces activation of alpha and decreases dephosphorylation of . It has been reported in vivo that C16-ceramide increased PKC translocation (44)
. In other in vivo studies, ceramide was demonstrated to activate PKC (45)
, induce translocation from the membrane to the cytosol of PKC and in HL-60, U937, HBP-ALL cells (46)
, and inhibit the activity of PKC (35
, 36)
. Two studies reported the inhibition of PKC activity by bSMase and short-chain ceramides (35
, 47)
. In one of these studies, the authors showed that sphingomyelinase reduced the activity of PKC without affecting its translocation after induction with phorbol esters but not with growth factors (47)
. In another line of investigation, it was shown that prolonged action (several hours) of ceramide leads to activation of protein phosphatases (35
, 48)
, which can target and inactivate PKC without major effects on membrane translocation (35)
. This was traced mechanistically to the ability of ceramide-activated phosphatases to induce dephosphorylation of PKC at the critical p500 site (phosphorylation in the activation loop, required for enzyme activity; see ref 49
). We first evaluated the in vitro activity of PKC in cytosolic and membrane fraction in order to see whether the translocated or the reverted pool of PKC was inactivated by bSMase treatment. These results showed that ceramide did not inactivate PKC or induce dephosphorylation of the p500 site within the time frame of our study. Thus, the acute effects of ceramide were due to a mechanism distinct from the long-term effects on inactivation of PKC.
Our results led to the observation of early and acute effects of ceramide in inhibiting the membrane association of PKC, which was most noticeable with the active phosphorylated PKC. Moreover, our results suggest that the action of ceramide was more likely on reverse translocation rather than inhibiting initial translocation. In Jurkat cells, the administration of C6-ceramide at different times after PMA stimulation was still able to reduce membrane-bound PKC and to induce the return of the protein back to the cytosol. Similarly, exogenous ceramide administrated after GFP-PKC translocation induced by PMA caused detachment of PKC from the membrane.
Thus, the results from this study suggest that bSMase and C6-ceramide induce/facilitate the detachment of PKC from the membrane and disclose two specific requirements for the reversal of PKC translocation in response to ceramide. First, there was a clear requirement for kinase activity. This was supported by the use of kinase inhibitors of PKC that act as competitive inhibitors with the Mg2+-ATP substrate and by the expression of the kinase-deficient mutant. The second requirement was for phosphorylation at the carboxyl-terminal phosphorylation sites. These sites are known autophosphorylation sites (39)
that regulate the association of the enzyme with the plasma membrane (27
, 37)
. Thus, the mutant PKC with a double alanine substitution (DA, nonphosphorylatable residues) was unable to respond to ceramide. The SE mutant (substitution of threonine 643 and serine 660 with glutamic acid), which mimics a constitutively phosphorylated enzyme, behaved more or less as a wild-type PKC, responding to ceramide treatment with detachment from the membrane. Thus, ceramide-induced reverse translocation of PKC requires the kinase activity of PKC and phosphorylation of the autophosphorylation sites.
As this work progressed, the results unexpectedly began to parallel a model we had investigated on the reverse translocation of PKC in response to receptor stimulation (27
, 37)
. The action of angiotensin II on its receptor results in activation of phospholipase C, formation of DAG, and acute translocation of PKC to the membrane. We found that this translocation persisted for 40–120 s, after which PKC ß2 reverted to the cytosol. We found this required kinase activity of PKC as well as phosphorylation of the carboxyl-terminal autophosphorylation sites. At that time, we concluded that the simplest explanation is that the kinase activity of PKC was required for autophosphorylation (27)
. The results from this study show a similar mechanism in the action of ceramide on reverse translocation of PKC; however, they favor a more complex mechanism where the kinase activity and the autophosphorylation sites provide for two distinct (independent) requirements for ceramide-induced PKC reverse translocation. That is, the kinase activity is not only involved in autophosphorylation. This is supported by 1) the ability of kinase inhibitors to inhibit the ceramide effect, without much change in PKC phosphorylation at the carboxyl sites. If the kinase activity was required simply for autophosphorylation, then stimulation with PMA should have increased this phosphorylation and PKC inhibitors would have inhibited it,2 and 2) translocation of the SE mutant of PKC, which should mimic the phosphorylation of the autophosphorylation sites, was still sensitive to the action of the kinase inhibitors. These results therefore suggest that kinase activity is required for the phosphorylation of a distinct substrate (or, less likely, PKC itself at a previously undisclosed site) that may be involved in regulating the membrane association of PKC. Current studies are aimed at exploring some candidate substrates.
All these observations suggest several hypothetical models for ceramide action. The dual requirement for kinase activity and autophosphorylation suggests a need for phosphorylation of a PKC-interacting protein. One possible scenario is that tight or persistent interaction of PKC with the membrane requires an interaction with a protein partner. Once PKC phosphorylates this partner and is itself phosphorylated at the carboxyl terminus (even constitutively), it becomes a target for ceramide-induced reversal of translocation. Ceramide may achieve this effect by interfering with interaction of PKC with the membrane and/or its putative protein partner(s). This could be a consequence of a physical interaction or may be an indirect effect involving ceramide-regulated targets. Clearly, other more complex scenarios can be envisioned.
Another direct implication of these observations is whether endogenous ceramide plays a physiological role in regulating the reverse translocation of PKC. It is conceivable that certain ceramide-inducing agonists would elevate ceramide at the plasma membrane, thus interfering with PKC translocation.
Other implications emanate from these results. First, coupled with previous published data, they clearly show that the site of action of ceramide in the cell regulates selective functions. Several stimuli activate sphingomyelinases and/or de novo synthesis of ceramide, leading to increases in endogenous ceramide, which has been shown to induce cell death (8)
. Short-chain ceramides are effective in inducing such death pathways, including specific biochemical actions on several targets in vivo such as caspases (50)
, PKC (35)
, Rb (51)
, bad (52)
, bcl2 (48)
, and cyclin-dependent kinases (53)
, COX-2 (54)
, and a few direct targets in vitro: CAPPs (48
, 51
52
53
, 55
, 56)
, CAPK/KSR (57)
, CDK2 (53)
, and PKC (12)
. bSMase, on the other hand, which generates ceramide at the plasma membrane, is unable to reproduce many of these effects (38)
. In this present study, both bSMase and C6-ceramide inhibited activation of NF-B and PKC translocation to the plasma membrane, yet only C6-ceramide was capable of activating caspases and inducing cell death in these same cells (50
, 58)
. In one study, it was shown that bSMase activated caspases and induced cell death when expressed endogenously, but not at the cell surface in a related leukemia model (38)
. Thus, ceramide formed at the plasma membrane induces only a subset of the total ceramide responses, supporting important roles for the topology of ceramide metabolism and function.
Second, these results show that the effects of ceramide on NF-B do not appear to be related to the proapoptotic effects of ceramide even though NF-B is considered a major anti-apoptotic factor and its inhibition should result in cell death. bSMase, which inhibited activation of NF-B in response to PKC activation and serum, did not cause cell death (data not shown). This suggests that inhibition of NF-B is not sufficient for induction of apoptosis in response to ceramide. It is possible that NF-B can exert its anti-apoptotic functions as long as it can respond to other activators (not inhibited by ceramide).
Another implication concerns the role of PKC in activation of NF-B physiologically. DAG-activated PKCs clearly do not have a role in TNF--induced activation (see above). However, bSMase was shown to inhibit serum-induced activation of NF-B (2)
. Serum stimulation is known to activate the DAG/PKC pathway and phospholipase D (59)
. Thus, it is tempting to speculate that PKC plays a key role in serum-induced activation of NF-B. This is currently under investigation in our laboratory.
In conclusion, this study demonstrates an important and specific cross-talk between the two neutral lipids DAG and ceramide. Acting on the plasma membrane, ceramide selectively inhibits translocation of PKC. This does not appear to be a generalized or nonspecific effect, but is mechanistically determined and requires the kinase activity of PKC and the carboxyl autophosphorylation sites. Future studies should explore this unique mechanism in regulating PKC translocation and mediating ceramide actions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 23, 2001.
Revision received June 20, 2001.
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