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Originally published In Press as doi:10.1074/jbc.M106291200 on August 13, 2001

J. Biol. Chem., Vol. 276, Issue 48, 44848-44855, November 30, 2001
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FAS Activation Induces Dephosphorylation of SR Proteins

DEPENDENCE ON THE DE NOVO GENERATION OF CERAMIDE AND ACTIVATION OF PROTEIN PHOSPHATASE 1*

Charles E. ChalfantDagger , Besim OgretmenDagger , Sehamuddin Galadari§, Bart-Jan Kroesen, Benjamin J. PettusDagger , and Yusuf A. HannunDagger ||

From the Dagger  Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, the § Department of Biochemistry, The United Arab Emirates University, AL AIN, The United Arab Emirates, and the  Department of Pathology and Laboratory Medicine, University Hospital, Groningen, The Netherlands.

Received for publication, July 5, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous D-(e)-C6-ceramide (20 µM) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B1 (100 µM), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with D-(e)-C6-ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B1 and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several lines of evidence have suggested ceramide as an important regulator of various stress responses and growth mechanisms. First, formation of ceramide from the hydrolysis of sphingomyelin or from de novo pathways is observed in response to inducers of stress such as TNFalpha , gamma -interferon, 1alpha ,25-dihydroxyvitamin D3, IL-1, ultraviolet light, heat, chemotherapeutic agents, FAS antigen, and nerve growth factor (1-7). Second, the addition of exogenous ceramide or enhancement of endogenous ceramide levels induces cell differentiation, cell cycle arrest, apoptosis or cell senescence in various cell types (8-10). Third, the action of ceramide relates mechanistically to key regulators of growth such as the retinoblastoma gene product, caspases, Bcl-2, and p53 (11-17). Fourth, studies in yeast have demonstrated an essential role for sphingolipids in many stress responses where sphingolipids function in the adaptation to heat (18, 19). Finally, studies with knock-out mice lacking acid sphingomyelinase or with fumonisin B1, an inhibitor of ceramide synthesis, have disclosed necessary roles for ceramide in several pathways of growth regulation (20, 21).

These emerging roles of ceramide necessitate a mechanistic understanding of ceramide action. This has led to the identification of several candidate ceramide-regulated enzymes, including ceramide-activated protein kinase, protein kinase Czeta , protein kinase Calpha , cathepsin D, phospholipase A2, and ceramide-activated protein phosphatase (CAPP)1 (22-29). CAPP was first identified as a member of the 2A class of serine/threonine phosphatases (PP2A) (25, 29-31). Several in vivo substrates have now been described for PP2A in response to ceramide, including Bcl-2, protein kinase Calpha , c-jun, and AKT/PKB (24, 32-36).

Protein phosphatase 1 (PP1) was reported as a target for natural ceramides in vitro (37). With the demonstration that PP1 is a possible downstream target for endogenous ceramides, known PP1 substrates become potential targets downstream of ceramide. SR proteins, a family of serine/arginine domain containing proteins and known modulators of mRNA splicing, have been demonstrated to be specific substrates for PP1 in nuclear extracts and in permeabilized nuclei (38, 39). In addition, two spliceosome targeting subunits of PP1 have been described, pyrimidine tract-associated splice factor (PSF) and NIPP-1 (40, 41). In this study, we examined the effect of exogenous ceramide and FAS activation on SR proteins in Jurkat acute leukemia T-cells and A549 lung adenocarcinoma cells. We demonstrate that SR proteins are dephosphorylated by endogenous ceramide derived from the de novo sphingolipid pathway and through PP1 activation.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

D-(e)-C6-ceramides were obtained from Matreya Inc. The catalytic subunit of human protein phosphatase 2A (PP2Ac) was purchased from Promega Corporation. The recombinant catalytic subunit of human protein phosphatase 1gamma (PP1gamma c), okadaic acid, caspase inhibitors, and calyculin A were purchased from Calbiochem. Mouse anti-FAS IgM (CH-11) was purchased from Upstate Biotechnology. mAb104 (phospho-SR protein antibody) hybridoma cells were purchased from ATCC. Mouse anti-human ASF/SF2 (SRp30a) was the gracious gift of Dr. Adrian Krainer (Cold Spring Harbor Laboratory, New York, NY). Fumonisin B1 was purchased from Alexis Corporation.

Cell Culture

Jurkat (acute lymphocytic T-cell leukemia) cells were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with L-glutamine, 10% (v/v) fetal bovine serum (Sigma), 200 units/ml of penicillin G sodium, and 200 µg/ml of streptomycin sulfate. Cells were maintained at densities between 2 × 105 and 1.2 × 106 cells/ml under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C). For treatments with ceramide and anti-FAS monoclonal antibody, Jurkat cells were diluted to 3.75 × 105 cells/ml in RPMI 1640 supplemented with 2% (v/v) fetal bovine serum and incubated for at least 2 h under standard incubator conditions prior to treatment.

A549 lung adenocarcinoma cells were grown in 50% RPMI 1640 (Life Technologies, Inc.) supplemented with L-glutamine/50% low glucose Dulbecco's modified Eagle's medium (Life Technologies) supplemented with L-glutamine, 10% (v/v) fetal bovine serum (Life Technologies, Inc.), 200 units/ml of penicillin G sodium, and 200 µg/ml of streptomycin sulfate. Cells were maintained under 80% confluency and standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C). For treatments with ceramide, A549 cells were plated at 4 × 105 cells/ml in 35-mm dishes 16 h prior to treatment.

Western Blotting

Both Jurkat and A549 cells were directly lysed using Laemmli buffer as described (42). Jurkat and A549 whole cell lysates (20 µg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were electrophoretically transferred to PDVF membranes, blocked with phosphate-buffered saline/0.1% Tween 20 containing 5% nonfat dried milk, washed with phosphate-buffered saline/0.1% Tween 20, and incubated 1.5 h with primary antibody in phosphate-buffered saline/0.1% Tween 20 containing 5% nonfat dried milk. Blots were washed in phosphate-buffered saline/0.1% Tween 20 and incubated 45 min with the secondary antibody in phosphate-buffered saline/0.1% Tween 20 containing 5% nonfat dried milk. Detection was performed using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).

Phosphatase Assays

Reactions were carried out in 1.5 ml of polypropylene microcentrifuge tubes. Buffer A (50 mM Tris-HCl, pH 7.4) and the appropriate amount of ceramide or ethanol vehicle were mixed in a 1.5-microcentrifuge tube. Stock enzyme (PP1gamma c or PP2Ac) was diluted 1:100 in 50 mM Tris-HCl, pH 7.4, and 10 milliunits were added to each tube. Tubes containing PP2Ac or PP1gamma c and Buffer A were pre-incubated for 10 min at 30 °C before addition of purified SR proteins. Reactions were initiated by the addition of 1 µl of purified phosphorylated SR proteins (1 mg/ml) in Buffer A. After 1 h at 30 °C, the assay was terminated by the addition of Laemmli buffer and boiling of the reactions for 10 min. Quantitative dephosphorylation of SR proteins was observed by Western immunoblotting and mAb104 as described above.

Quantification of Ceramide Levels

Pulse Labeling with [3H]Palmitic Acid-- 2 × 106 Jurkat cells were incubated with 1 µCi/ml [3H]palmitic acid (16.7 nM) with simultaneous addition of anti-FAS CH-11 antibody or control mouse IgM. After 7 h, lipids were extracted from the radiolabeled cells using the Bligh-Dyer method as described (43). Ceramide levels were measured following TLC analysis and normalized to total lipid phosphate as described (44, 45).

Diacylglycerol Kinase Assay-- At the indicated time points, lipids were extracted from 2 × 106 cells using the Bligh-Dyer method as described (43). Ceramide levels were measured using the Escherichia coli diacylglycerol kinase assay and normalized to total lipid phosphate as described (44).

Tandem Mass Spectrometry-- Lipids were extracted from 2 × 106 cells using the Bligh-Dyer method, and ceramide levels were measured using normal phase high performance lipid chromatography coupled to atmospheric pressure chemical ionization-mass spectrometry (43, 46). Ceramide levels were normalized to total lipid phosphate.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ceramide Treatment of Jurkat Cells Leads to Dephosphorylation of SRp70, SRp55, SRp40, and SRp30-- We have reported that PP1 is a ceramide-activated protein phosphatase in vitro (37). Therefore, SR proteins, specific PP1 substrates, were examined for modulation in response to exogenous ceramide treatment in Jurkat cells (38). Using a monoclonal antibody, mAb104, that recognizes only phosphorylated SR proteins, it was demonstrated that treatment of Jurkat cells with 20 µM D-(e)-C6-ceramide produced a time-dependent decrease in the immunoreactivity of all detectable SR protein species, SRp70, SRp55, SRp40, and SRp30 (Fig. 1A). To demonstrate that the effect is phosphorylation and not proteolysis, we used a non-phosphoepitope antibody specific for the 30-kDa SR protein species, ASF/SF2 (SRp30a). The total immunoreactive SRp30 did not decrease when using this antibody, but an apparent shift in molecular weight was observed (Fig. 1A). The effect of D-(e)-C6-ceramide on SR proteins was dose-dependent, and a minimal dose of 5 µM was necessary to produce significant dephosphorylation in SR proteins with a complete loss of mAb104 detectability using a maximal dose of 20 µM after 6 h (Fig. 1B). Treatment of Jurkat cells with the biologically inactive D-(e)-dihydro-C6-ceramide (20 µM) had no effect on SR proteins after 6 h (Fig. 1C). These results, therefore, demonstrate a specific effect of D-(e)-C6-ceramide on dephosphorylation of SR proteins.


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  		Fig. 1.   Effects of exogenous ceramide on the phosphorylation state of SR proteins. A, time course of the dephosphorylation of SR proteins by ceramide. Jurkat cells were treated for various times with 20 µM D-(e)-C6-ceramide, and total protein lysates were produced. Total protein lysates (20 µg) were subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for either phosphorylated SR proteins (mAb104) or total SRp30a. Data are representative of five separate determinations reproduced on four separate occasions. B, dose response of the dephosphorylation of SR proteins by ceramide. Jurkat cells were treated for 6 h with various concentrations of D-(e)-C6-ceramide, and total protein lysates were produced. Total protein lysates (20 µg) were subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of four separate determinations reproduced on two separate occasions. C, specificity of the dephosphorylation of SR proteins by ceramide. Jurkat cells were treated for 6 h with either 20 µM D-(e)-C6-ceramide or 20 µM D-(e)-dihydro-C6-ceramide. Total protein lysates were produced, and 20 µg of the lysate was subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of three separate determinations reproduced on two separate occasions.

Ceramide is known to activate caspases and induce cleavage of PARP and other substrates (45-48). Therefore, to determine the relationship of the effects on the dephosphorylation of SR proteins to the effects on caspases, Jurkat cells were pretreated with either 25 µM Z-VAD-fmk (a pan-caspase inhibitor), 50 µM ac-YVAD-cmk (caspase 1 inhibitor), or 25 µM ac-DEVD-fmk (a caspase 3 inhibitor). None of the inhibitors had an effect on the shift in molecular weight of each SR protein species or on the loss of detectability by mAb104 in response to 20 µM C6-ceramide treatment after 6 h (Fig. 2A) but did have distinct effects on inhibiting PARP proteolysis (ac-ZVAD-fmk = 100% inhibition, ac-YVAD-cmk = 50% inhibition, and ac-DEVD-cmk = 75% inhibition) (Fig. 2A). These results show that ceramide exerts two effects, activation of caspases and dephosphorylation of SR proteins, and that the latter is not dependent on the former.


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  		Fig. 2.   Effects of inhibitors of caspases and protein phosphatases on ceramide-induced dephosphorylation of SR proteins. A, effects of caspase inhibitors. Jurkat cells were pretreated 2 h with either 25 µM ac-ZVAD-fmk, 50 µM ac-YVAD-cmk, or 25 µM ac-DEVD-cmk followed by a 6-h treatment with 20 µM D-(e)-C6-ceramide. Total protein lysates were produced, and 20 µg of the lysate was subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104) and PARP. Data are representative of three separate determinations reproduced on two separate occasions. B, effects of inhibitors of serine/threonine protein phosphatases. Jurkat cells were pretreated 2 h with either 10 nM okadaic acid or 5 nM calyculin A followed by a 6-h treatment with 20 µM D-(e)-C6-ceramide. Total protein lysates were produced, and 20 µg of the lysate was subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of three separate determinations reproduced on three separate occasions.

Calyculin A, but Not Okadaic Acid, Inhibits Ceramide-induced Dephosphorylation of SR Proteins-- To determine whether SR protein dephosphorylation is mediated by ceramide-activated protein phosphatases in vivo, specifically PP1, we pretreated cells with the serine/threonine phosphatase inhibitors, okadaic acid (in vitro IC50: PP2A IC50 = 0.1 nM, PP1 IC50 = 10 nM), and calyculin A (in vitro IC50: PP2A IC50 = 1 nM, PP1 IC50 = 2 nM) followed by a 6-h treatment with 20 µM D-(e)-C6-ceramide. Calyculin A inhibited ceramide effects on SR protein dephosphorylation at >=  5 nM (Fig. 2B). The inhibitor alone increased the phosphorylation of each detectable SR protein species (Fig. 2B). On the other hand, okadaic acid at a concentration that only inhibits PP2A in T-cell leukemias (10 nM) did not have any effect on SR protein dephosphorylation in response to exogenous ceramide treatment nor was any increase in basal SR protein phosphorylation observed (Fig. 2B) (34). We have used various concentrations of okadaic acid up to 600 nM without any effects on the basal phosphorylation of SR proteins or ceramide-induced dephosphorylation of SR proteins (data not shown). Therefore, these results suggest that PP1 is the most likely phosphatase involved in mediating the effects of ceramide on the dephosphorylation of SR proteins.

FAS Activation Induces the Dephosphorylation of SR Proteins in a Time-dependent Manner-- Ceramide is generated via two main pathways, hydrolysis of sphingomyelin by sphingomyelinases and by de novo production (49). To date, activation of protein phosphatase 1 by endogenous ceramide in cells has not been demonstrated. With the establishment of PP1 activation in cells by short chain ceramides, we examined FAS activation as a model for agonist-induced generation of ceramide to determine the effects of endogenous ceramide on PP1 activation. Treatment of Jurkat cells with the anti-FAS IgM, CH-11, induced dephosphorylation of SR proteins in a time-dependent manner with decreased dephosphorylation of SR proteins occurring as early as 3 h (Fig. 3A). Furthermore, calyculin A (Fig. 3B) and not okadaic acid (Fig. 3C) blocked this effect. Importantly, FAS did not induce proteolysis or loss of SRp30 as shown by Western immunoblotting of the same samples (Fig. 3A). Therefore, these data suggest that activation of PP1 leading to the dephosphorylation of SR proteins occurs in response to FAS activation.


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  		Fig. 3.   Effects of FAS activation on the phosphorylation state of SR proteins. A, effects of FAS antibody. Jurkat cells were treated for various times with 150 ng/ml of CH-11 or control IgM, and total protein lysates were produced. Total protein lysates (20 µg) were subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for either phosphorylated SR proteins (mAb104) or total SRp30a. Data are representative of four separate determinations reproduced on four separate occasions. B and C, effects of inhibitors of serine/threonine protein phosphatases. Jurkat cells were pretreated for 2 h with either 5 nM calyculin A (B) or 10 nM okadaic acid (C) followed by a 7-h treatment with 150 ng/ml of CH-11 or control IgM. Total protein lysates were produced, and 20 µg were subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104).

Dephosphorylation of SR Proteins by FAS Activation Correlates with Increased Levels of Endogenous Ceramide-- To establish whether dephosphorylation of SR proteins in response to FAS activation occurred in conjunction or following increases in endogenous ceramide following CH-11 treatment, a time course of ceramide generation in response to acute CH-11 treatment was examined. Endogenous ceramide levels began to increase as early as 2 h post-CH-11 treatment (Fig. 4). Thus, activation of PP1 and subsequent dephosphorylation of SR proteins following FAS activation occurs after a significant accumulation of endogenous ceramides.


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  		Fig. 4.   Effects of FAS activation on the levels of endogenous ceramide. Jurkat cells were treated for various times with 150 ng/ml of CH-11 or control IgM, and total lipids were extracted using the Bligh-Dyer method. The extracted lipids were based hydrolyzed and the levels of ceramide determined by diacylglycerol kinase assay. The results are presented as picomoles of total ceramide normalized to nanomoles of total phosphate. Data are representative of four separate determinations on four separate occasions.

Ceramide Generated by the de Novo Sphingolipid Pathway Is Responsible for Activation of PP1/Dephosphorylation of SR Proteins in Response to FAS Activations-- It has been previously reported that ceramide levels are increased in response to FAS activation, but the reports differ as to the ceramide pathway involved (50-55). In this study, pretreatment of Jurkat cells with fumonisin B1 (an inhibitor of de novo ceramide generation) inhibited 80% (10.7-fold increase to a 2.14-fold increase) of the total mass increase of ceramide (primarily C16:0 and C24:1 ceramides) in response to 7 h of CH-11 exposure (Fig. 5A). To examine the de novo synthesis of ceramide directly, we pulse labeled Jurkat cells with [3H]palmitic acid. Pretreatment with fumonisin B1 blocked 83.8% of the increase in [3H]ceramide following 7 h of CH-11 exposure. Similar to the ceramide mass data, basal ceramide levels were reduced by 67% (Fig. 5B). Thus, the main pathway of increasing ceramide levels in response to FAS activation is via the de novo sphingolipid pathway.


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  		Fig. 5.   Effect of fumonisin B1 on the induction of endogenous ceramide by FAS activation. A, effects of fumonisin B1 on the levels of endogenous ceramide. Jurkat cells were pretreated for 2 h with 100 µM fumonisin B1 followed by a 7-h treatment with either 150 ng/ml CH-11 or control IgM. Total lipids were extracted using the Bligh-Dyer method, and the extracted lipids were subjected to normal phase high performance lipid chromatography coupled to atmospheric pressure chemical ionization-mass spectrometry. The results are presented as arbitrary mass units of total ceramide normalized to nanomoles of total phosphate. The data presented are representative of two separate determinations on one occasion, but the results were confirmed by two separate determinations on one occasion by diacylglycerol kinase assay. B, effects of fumonisin B1 on ceramide measured by pulse labeling. Jurkat cells were pretreated 2 h with 100 µM fumonisin B1 followed by a 7-h cotreatment with either 150 ng/ml CH-11 or control IgM and 16.7 nM [3H]palmitate. Total lipids were extracted using the Bligh-Dyer method, and the extracted lipids were base-hydrolyzed and subjected to thin layer chromatography. The results are presented as cpm of labeled ceramide normalized to nanomoles of total phosphate. Data are representative of four separate determinations reproduced on two separate occasions.

To establish that the de novo ceramide pathway was responsible for activating PP1 leading to the dephosphorylation of SR proteins in response to FAS activation, Jurkat cells were again pretreated with fumonisin B1. Pretreatment with fumonisin B1 (100 µM) blocked the dephosphorylation of SR proteins in response to FAS activation (Fig. 6, A and B). Since fumonisin B1 not only inhibits ceramide synthesis but could also divert sphingolipid synthesis toward free sphingoid bases (e.g. dihydrosphingosine) and phosphorylated bases (e.g. dihydrosphingosine phosphate), the role of the de novo sphingolipid pathway in mediating the effect of FAS activation on SR proteins was examined by pretreating Jurkat cells with 50 nM myriocin, a specific inhibitor of serine palmitoyltransferase (the first enzyme in sphingolipid biosynthesis). Myriocin pretreatment completely blocked the dephosphorylation of SR proteins in response to FAS activation (Fig. 6C). Thus, the dephosphorylation of SR proteins and activation of PP1 in response to FAS are dependent on the generation of endogenous ceramide via the de novo sphingolipid pathway.


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  		Fig. 6.   Effects of fumonisin B1 and myriocin on the dephosphorylation of SR proteins in response to FAS activation. A, effects of FAS antibody on the phosphorylation state of SR proteins. Jurkat cells were treated for various times with 150 ng/ml of CH-11 or control IgM, and total protein lysates were produced. Total protein lysates (20 µg) were subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of four separate determinations reproduced on three separate occasions. B, effects of fumonisin B1 on the dephosphorylation of SR proteins in response to FAS antibody. Jurkat cells were pretreated 2 h with 100 µM fumonisin B1 followed by treatment for various times with either 150 ng/ml CH-11 or control IgM. Total protein lysates (20 µg) were subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of three separate determinations reproduced on three separate occasions. C, effects of myriocin and fumonisin B1 on the dephosphorylation of SR proteins in response to FAS antibody. Jurkat cells were pretreated 2 h with either 50 nM myriocin or 100 µM fumonisin B1 followed by treatment for 7 h with either 150 ng/ml CH-11 or control IgM. Total protein lysates (20 µg) were subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of three separate determinations reproduced on two separate occasions.

Ceramide Generated by the de Novo Sphingolipid Pathway Is Responsible for Activation of PP1/Dephosphorylation of SR Proteins in Response To D-(e)-C6-Ceramide-- To further extend the role of ceramide generated by de novo sphingolipid pathway in activating PP1 and inducing the dephosphorylation of SR proteins, we examined another cell model of de novo sphingolipid synthesis, A549 lung adenocarcinoma cells treated with D-(e)-C6-ceramide. In this model, endogenous ceramide is increased by 4-fold in a fumonisin B1-inhibitable manner2 (56). Treatment of A549 cells with 20 µM D-(e)-C6-ceramide (IC50 = 37 µM) for 16 h induced significant dephosphorylation of all detectable SR protein species (Fig. 7A). Pretreatment of A549 cells with 100 µM fumonisin B1 completely blocked this effect (Fig. 7A).


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  		Fig. 7.   Effects of fumonisin B1 and overexpression of glucosylceramide synthase on the dephosphorylation of SR proteins in response to exogenous ceramide. A, effects of fumonisin B1. A549 cells were pretreated 2 h with 100 µM fumonisin B1 followed by treatment for 16 h with 20 µM D-(e)-C6-ceramide. Total protein lysates were produced, and 20 µg of lysate was subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of three separate determinations reproduced on two separate occasions. B, effects of the overexpression of GCS. A549 cells overexpressing GCS or vector only were treated for 16 h with 20 µM D-(e)-C6-ceramide. Total protein lysates were produced, and 20 µg of lysate was subjected to 10% SDS-PAGE analysis, transferred to PDVF, and immunoblotted for phosphorylated SR proteins (mAb104). Data are representative of three separate determinations reproduced on two separate occasions.

The A549 cell model also allowed further determination of the role of endogenous ceramide using overexpression of glucosylceramide synthase (GCS). It was previously shown that overexpression of GCS inhibits increases in de novo ceramide in response to extracellular agonists (56-59). SR proteins were significantly dephosphorylated in vector control cells treated with D-(e)-C6-ceramide (20 µM). In contrast, overexpression of GCS completely abrogated the effect of ceramide on the phosphorylation status of SR proteins (Fig. 7B). Therefore, activation of the de novo sphingolipid pathway and activation of PP1 to induce the dephosphorylation of SR proteins are intrinsically linked. Taken together, these results show that endogenous ceramide derived from the de novo pathway also activates PP1 leading to dephosphorylation of SR proteins in the A549 cells.

SR Proteins Are Substrates for PP1 in Vitro-- Protein phosphatase 1 has been demonstrated to affect the nuclear distribution of SR proteins in permeabilized nuclei and specifically dephosphorylate SR proteins in nuclear extracts (38, 39). To demonstrate that SR proteins are specific substrates for PP1, the effects of the two identified CAPPs, PP1c and PP2Ac, on SR proteins in vitro were examined. For these experiments, recombinant PP1c and purified PP2Ac along with SR proteins purified from Jurkat cells were used. PP1c efficiently dephosphorylated all SR protein species in vitro, and ceramide significantly increased this effect (Fig. 8). In contrast, the closely related serine/threonine phosphatase, PP2Ac, was unable to dephosphorylate any SR protein species in the presence or absence of ceramide in vitro (Fig. 8). Therefore, SR proteins are specific substrates for PP1, and ceramide directly activates PP1c leading to increased dephosphorylation of SR proteins in vitro.


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  		Fig. 8.   Effects of serine/threonine protein phosphatases on SR proteins in vitro. PP1c and PP2Ac were assayed with purified SR proteins in the presence and absence of 10 µM D-(e)-C6-ceramide. The assays were carried out at 30 °C for 1 h in 50 mM Tris-HCl, pH 7.4. Data are representative of three separate determinations reproduced on two separate occasions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, Bcl-2, c-Jun, and protein kinase Calpha (PKCalpha ) were demonstrated to be dephosphorylated in response to ceramide in cells, and this effect was mediated by PP2A, a CAPP (32, 34, 35). In this study, we demonstrate a novel in vitro and in vivo pathway involving PP1 as a CAPP with SR proteins as substrates. We further demonstrate that ceramide generated through activation of the de novo sphingolipid pathway activates PP1 in response to FAS. These findings are important for several reasons. First, protein phosphatase 1 is now shown to be a ceramide-activated protein phosphatase in cells, and specific roles for different CAPP species are better defined. Second, for the first time, a specific pathway/pool of ceramide, generated in the de novo pathway, is shown to activate protein phosphatase 1 in cells. Third, SR proteins, an important family of RNA splicing factors, have been demonstrated as novel targets for certain apoptotic agonists.

Treatment with either exogenous ceramide or FAS antibody led to dephosphorylation of SR proteins. This differs from a previous report from Utz et al. who reported that FAS activation increased the phosphorylation of SR proteins in Jurkat cells (60). This observation may be a result of the Nonidet P-40 lysis system employed. This cell lysis method does not solubilize the nuclei in Jurkat cells. Using a direct lysis method, as done in this study, the phosphorylation status of total SR proteins is directly determined. We clearly demonstrate that SR proteins are dephosphorylated in response to ceramide and FAS activation. This effect on SR proteins was not due to proteolytic degradation as caspase inhibitors had no effect on ceramide-induced dephosphorylation of SR proteins. The non-phosphoepitope antibody to the SR proteins, ASF/SF2 (SRp30a), demonstrated only changes in molecular weight and no loss in immunoreactivity in contrast to the specific phospho-SR protein antibody, mAb104. Furthermore, calyculin A, an inhibitor of both PP1 and PP2A, completely blocked ceramide- and FAS-induced SR protein dephosphorylation, while okadaic acid at concentrations that only affect PP2A in cells had no effect. Therefore, both FAS and ceramide induce phosphatase-dependent and caspase-independent dephosphorylation of SR proteins.

Moreover, the results with the phosphatase inhibitors along with the observation that only PP1c and not PP2Ac is able to dephosphorylate SR proteins in vitro in response to ceramide clearly define PP1 as the CAPP-regulating SR protein dephosphorylation. These findings are consistent with several previous studies. First, two spliceosomal-targeting subunits for PP1, PSF, and NIPP-1 were reported (40, 41). Second, Lamond and co-workers reported that PP1c and not PP2Ac dephosphorylate SR proteins in nuclear extracts (38). Third, Misteli and Spector demonstrated that exposure of permeabilized nuclei to PP1 will change the nuclear distribution of SR proteins (39).

Endogenous ceramide levels have been observed to increase in response to FAS by several laboratories (50-55). In this study, most of the endogenous ceramide produced in response to FAS activation resulted from activation of de novo ceramide synthesis. This conclusion is based on the observation that fumonisin B1, an inhibitor of the generation of de novo ceramide, decreased endogenous ceramide in response to FAS activation by 80%. Of interest to note, fumonisin B1 pretreatment did not block all of the endogenous ceramide generated in response to FAS. This may indicate specificity of the de novo sphingolipid pathway for PP1 activation or that the magnitude of the ceramide response plays a role in PP1 activation. We hypothesize that the remaining 20% increase in response to FAS activation may be generated by either neutral or acid sphingomyelinases, both of which have been suggested to mediate ceramide increases in response to FAS activation. Further studies to answer both questions are currently being investigated.

The effect of FAS activation on the phosphorylation state of SR proteins was not observed until 3 h posttreatment with anti-FAS IgM. Thus, dephosphorylation of SR proteins did not begin until there was a significant increase in endogenous ceramide levels. Using several methods, it was clearly demonstrated that the activation by PP1 is dependent on this increase in endogenous ceramide. First, two specific inhibitors of the de novo sphingolipid pathway, fumonisin B1 and myriocin, completely blocked FAS-induced dephosphorylation of SR proteins in Jurkat cells. Second, in a different de novo ceramide model, A549 cells treated with exogenous ceramide, fumonisin B1 also completely inhibited the dephosphorylation of SR proteins. Furthermore, overexpression of glucosylceramide synthase completely abrogated the ability of exogenous ceramide to induce SR protein dephosphorylation in A549 cells. Thus, in this study, the dephosphorylation of SR proteins in response to FAS and exogenous ceramide was dependent on endogenous ceramide generation.

Another implication of this study pertains to the regulation of the phosphorylation of SR proteins in response to apoptotic stimuli. Thus the question is, what are the consequences of the dephosphorylation of SR proteins in the apoptotic process? SR proteins are known to modulate alternative mRNA processing, and dephosphorylation of certain SR protein species has been shown to regulate alternative splicing (38, 61-68). Many apoptotic regulators such as Bax, Bcl-x, caspase 9, and caspase 2 have been shown to be alternatively spliced with the splice variants having opposite/dominant-negative activities (69-81). By dephosphorylating SR proteins, posttranscriptional processing of various genes can be affected, and therefore any regulation of SR proteins can affect the gene expression of key apoptotic regulators via changes in alternative splicing inducing or inhibiting apoptosis. Increased phosphorylation of SR proteins has been shown to occur in response to various mitogens; thus, dephosphorylation of SR proteins in response to apoptotic agonists may enhance the execution of the apoptotic response (62, 65, 82, 83).

In this paper, it was demonstrated that SR proteins are dephosphorylated by FAS activation and exogenous ceramide treatment. Mechanistically, this effect is dependent on endogenous ceramide generation from de novo ceramide synthesis. Furthermore, PP1 is implicated as a ceramide-activated protein phosphatase in cells and as the protein phosphatase species regulating SR protein dephosphorylation. Therefore, this study defines a novel and specific module of in vivo activation of PP1 by de novo ceramide leading to subsequent dephosphorylation of SR proteins (Fig. 9).


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  		Fig. 9.   Schematic of de novo ceramide, activation of PP1, and dephosphorylation of SR proteins.


    ACKNOWLEDGEMENTS

We thank Drs. Stefan Stamm, Adrian Krainer, Rebecca Taub, and Denise Cooper for providing antibodies to the SR protein family.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA87584 (to Y. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Rm. 501, Basic Science Bldg., Medical Univ. of South Carolina, 173 Ashley Ave., P.O. Box 250509, Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322; E-mail: hannun@musc.edu.

Published, JBC Papers in Press, August 13, 2001, DOI 10.1074/jbc.M106291200

2 B. Ogretmen, Y. A. Hannun, and L. M. Obeid, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: CAPP, ceramide-activated protein phosphatase; PP2A, protein phosphatase 2A; PP1, protein phosphatase 1; PP2Ac, catalytic subunit of protein phosphatase 2A; PP1gamma c, catalytic subunit of protein phosphatase 1gamma ; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; GCS, glucosylceramide synthase; PARP, poly(ADP-ribose)polymerase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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