©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Dimeric and Catalytic Subunit Forms of Protein Phosphatase 2A from Rat Brain Are Stimulated by C-Ceramide (*)

Brian Law , Sandra Rossie (§)

From the (1) Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Protein phosphatase 2A (PP-2A) is a heterotrimeric enzyme consisting of a catalytic (C) subunit and A and B regulatory subunits. PP-2A is activated by ceramide in vitro suggesting that PP-2A may be a target of this putative second messenger in vivo (Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A.(1993) J. Biol. Chem. 268, 15523-15530). In this study, sensitivity to ceramide was only observed when the B subunit was present, suggesting that the B subunit was required for ceramide activation. Here we show that dimeric PP-2A, produced from trimeric PP-2A by heparin-agarose-induced dissociation of the B subunit and isolated by preparative native electrophoresis, is activated by ceramide. The catalytic subunit of PP-2A, produced from trimeric PP-2A by freezing and thawing in the presence of 0.2 M -mercaptoethanol and isolated by gel filtration, is also activated by ceramide. The trimeric and catalytic subunit forms of PP-2A exhibit a similar dose dependence of activation by ceramide, and are stimulated to a similar extent at ceramide concentrations yielding maximal activation. These findings indicate that neither the A nor the B subunit is required for ceramide stimulation of PP-2A. Together, these results demonstrate that the catalytic subunit contains a ceramide binding site and suggest that efforts to understand the mechanism of activation of PP-2A by ceramide should be focused on this subunit. The discovery that the catalytic subunit contains a ceramide binding site raises the possibility that other members of this serine/threonine phosphatase gene family may contain lipid binding sites and be regulated by ceramide or other lipid second messengers.


INTRODUCTION

The lipid ceramide is released in a variety of cell lines in response to hormones and cytokines such as TNF-(), interleukin-1, -interferon (reviewed in Ref. 1) and nerve growth factor (2) . These agents initiate the sphingomyelin cycle (3) in which sphingomyelinase is activated and hydrolyzes sphingomyelin to produce ceramide and phosphocholine. Ceramide is thought to act as a second messenger because treatment of cells with C-ceramide, a soluble analog of ceramide, mimics the biological effects of agents that elicit ceramide production. Examples include TNF--induced differentiation or apoptosis in HL-60 or U937 cells, respectively (3, 4) , and nerve growth factor-induced growth inhibition and process formation in T9 glioma cells (2) .

The serine/threonine protein phosphatase PP-2A is activated in vitro by ceramide and may be an important target of ceramide acting as a second messenger (1) . Down-regulation of c-myc in HL-60 cells in response to TNF- and C-ceramide, but not phorbol 12-myristate 13-acetate, is blocked by the phosphatase inhibitor okadaic acid (5) . In addition, the ability of ceramide analogs to cause c-myc down-regulation in HL-60 cells (3) and to inhibit yeast growth (6) parallels their ability to activate PP-2A. Understanding the mechanism by which ceramide regulates PP-2A is an important step in determining the role of PP-2A in ceramide signaling, and a step toward determining how ceramide production triggers such cellular responses as differentiation and apoptosis.

Protein phosphatase 2A is one of the major serine/threonine protein phosphatases found in a variety of mammalian tissues (7, 8) . The enzyme is believed to exist in vivo as a heterotrimer consisting of a 36-kDa catalytic subunit (C), a 65-kDa regulatory subunit (A), and one of several B regulatory subunits ranging in molecular mass from 54-74 kDa, or as a heterodimer consisting of the C and A subunits (reviewed in Ref. 7). The catalytic subunit of PP-2A belongs to a gene family which includes the catalytic subunits of phosphatase 1 (PP-1) and calcineurin (PP-2B) (reviewed in Ref. 7). The catalytic subunits of PP-1 and PP-2B are modulated by regulatory subunits and by the direct or indirect action of second messengers (9, 10, 11) .

The mechanisms which control PP-2A activity are not well understood, but recent studies suggest that the catalytic subunit is a target of regulation. The catalytic subunit of PP-2A has been shown to be phosphorylated on tyrosine (13, 14) and carboxymethylated on its C terminus (15, 16, 17, 18) ; its activity is altered by these modifications (13, 17) . In addition, a serine/threonine-specific autophosphorylation-activated protein kinase has been shown to phosphorylate the C and A subunits of PP-2A in vitro; phosphorylation inhibits phosphatase activity by about 80% (34) . The phosphorylation site mediating this effect is not known. The precise functions of the regulatory subunits of PP-2A are not known, but evidence suggests that the isoform of B subunit present in the heterotrimer may control the activity and substrate specificity of the phosphatase (12) .

The putative second messenger ceramide activates PP-2A present in cell extracts (19) and purified heterotrimeric PP-2A (20) . Because ceramide did not activate the dimeric or catalytic subunit forms of PP-2A (20) , it was suggested that the B subunit is responsible for ceramide sensitivity. Knowledge of which subunit(s) contain the ceramide binding site is important since the C and A subunits found in different PP-2A holoenzymes are thought to be similar, while there are at least three different B subunit gene families (8) . In the present study we have specifically examined the question of which subunit(s) bear the ceramide binding site. In order to determine whether or not the B subunit and/or A subunit are required for ceramide activation of PP-2A, we have purified trimeric PP-2A from rat brain and used this as the source for the production of the dimeric and catalytic subunit forms of PP-2A. Our findings demonstrate that the catalytic subunit of PP-2A is activated by ceramide and that neither the A nor the B subunit is required for this activation.


EXPERIMENTAL PROCEDURES

Purchased Materials

All purchased materials were obtained from Sigma unless otherwise indicated.

Partial Purification of Trimeric PP-2A

Brains from 8 adult male rats were homogenized in 50 ml of 20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1% -mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µg/ml leupeptin, 1 µM pepstatin A, and 10 µg/ml trypsin inhibitor (buffer A). The homogenate was centrifuged at 100,000 g for 1 h, passed through glass wool and a 0.2-µm filter (Millipore), and loaded onto a Q-Sepharose HP 16/10 column (Pharmacia Biotech Inc.). The column was washed with buffer A until the absorbance at 280 nm reached baseline and was eluted with a 100-ml gradient from 0.0-1.0 M NaCl in buffer A at 1 ml/min. The peak of phosphatase activity observed was pooled and made 55% saturated in (NH)SO, incubated for 30 min at 4 °C, and centrifuged at 6000 g for 30 min. The pellet was dissolved in 1 ml of buffer A containing 0.1 M NaCl and loaded onto a 1.7 96 cm Sephacryl S-300 (Pharmacia) gel filtration column. The column was eluted with buffer A containing 0.1 M NaCl at 0.2 ml/min, and 115 1-ml fractions were collected. The peak of activity was pooled, diluted 2-fold with buffer A, and applied to a 1.7 12 cm aminohexyl-Sepharose column. The column was washed with buffer A until the absorbance at 280 nm had dropped below 0.1 and eluted with a 100-ml linear gradient from 0.0-2.0 M NaCl in buffer A. The peak of activity was pooled and dialyzed extensively against buffer A. The dialysate was applied to a 1.7 12 cm protamine-Sepharose 4B column prepared as described by Tamura et al.(21) . The column was washed and eluted with a 100-ml linear gradient from 0.0-3.0 M NaCl in buffer A. The peak of activity observed was pooled and dialyzed against buffer A. The dialysate was loaded onto a 1.7 22 cm DEAE-Sepharose CL-6B (Pharmacia) column. The column was washed and eluted with a 400-ml linear gradient from 0.0-0.5 M NaCl in buffer A. Two partially resolved peaks of phosphatase activity were observed. The leading edge of the first peak and the trailing edge of the second peak were pooled separately. These two fractions were analyzed by native electrophoresis, phosphatase assays, and immunoblots (described below). These analyses demonstrated that the first pool corresponds to trimeric PP-2A, while the second pool corresponds to dimeric PP-2A. The pool corresponding to trimeric PP-2A was used as starting material in subsequent experiments.

Purified Proteins and Antibodies

The catalytic subunit of cyclic AMP-dependent protein kinase was purified from bovine heart (22) . A mouse monoclonal antibody raised against the catalytic subunit of PP-2A (23) , and rabbit polyclonal antisera specific for the A (24) , B (25) , and B` (26) regulatory subunits of PP-2A were kindly provided by Dr. Marc Mumby, Dept. of Pharmacology, University of Texas Health Science Center, Dallas, TX.

Electrophoresis

SDS-PAGE was performed according to the method of Laemmli as described by Maizel (27) , using a 3% stacking gel and either a 12% running gel for analysis of the catalytic subunit or a 10% running gel for analysis of the A and B regulatory subunits. Samples for SDS-PAGE were concentrated by acetone precipitation. Samples were added to 5 volumes of (-20 °C) acetone and incubated at -20 °C for 20 min, followed by centrifugation at 4 °C for 10 min at 12,000 g. The acetone was removed, and the pellets were dried with a gentle stream of air and dissolved in 1 SDS sample buffer (1.5% SDS (w/v), 15 mM Tris-HCl, 6 mM EDTA, 10% glycerol (v/v), 0.05% (w/v) bromphenol blue, 0.1% -mercaptoethanol (v/v), pH 6.7). Native PAGE under alkaline conditions was performed using a 5% stacking gel and a 7% running gel (28) .

Immunoblot Analysis

After SDS-PAGE, proteins were electrophoretically transferred to nitrocellulose at 100 V for 30 or 45 min when using 0.75- or 1.5-mm gels, respectively. Transfers were performed in 25 mM Tris-HCl, 192 mM glycine, 20% methanol (v/v), pH 8.3. Blots were incubated for 1 h at room temperature in 50 mM Tris-HCl, pH 8.0, 80 mM NaCl, 2 mM CaCl (buffer C), containing 0.2% Tween 20 and 5% BSA, and then probed with PP-2A subunit-specific antibodies. Blots were then washed five times in buffer C containing 1% Tween 20, 1% BSA, and 0.2% SDS, 5 min per wash. Immunoblots were developed using Zymed alkaline phosphatase/5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium immunostaining kits specific for mouse or rabbit IgG according to the instructions provided. Prestained molecular weight markers (Life Technologies, Inc.) were used to monitor transfer efficiency, and biotinylated molecular weight markers (Bio-Rad) were used for molecular weight estimation.

Analysis of Samples after Native Gel Electrophoresis

Duplicate native gel lanes were cut into 2-mm slices horizontally. For measurement of phosphatase activity, one set of gel slices was extracted by agitation overnight at 4 °C with 200 µl of buffer A containing 20% glycerol. Extracts were assayed for phosphatase activity the following morning. For immunoblot analysis, a set of identical gel slices was extracted with 200 µl of 2 SDS sample buffer. Slices were incubated for 30 min at room temperature, 5 min at 100 °C, and agitated overnight at room temperature. The extracts were frozen until SDS-PAGE was performed.

Preparation of P-Labeled Substrates

P-Labeled casein was prepared using the catalytic subunit of cyclic AMP-dependent protein kinase as described by Sheng and Charbonneau (29) . P-Labeled phosphorylase a was prepared as described by Cohen et al.(30) . The specific activity of the labeled substrates was approximately 25 µCi/µmol and 143 µCi/µmol for casein and phosphorylase, respectively.

Phosphatase Assays

Assays were performed as described (19) with modifications. All dilutions were made into argon-purged 50 mM Tris-HCl, pH 7.6, 1 mM EDTA. Phosphatase assays were performed at 37 °C for 60 min in a 30-µl reaction volume containing 10 µl of phosphatase sample, 10 µl of P-labeled substrate, and 10 µl of 60 µM C-ceramide (Biomol) or an ethanol vehicle control. Casein and phosphorylase a substrates were present at final concentrations of 8.3 µM and 10 µM, respectively. Reactions were terminated by the addition of 100 µl of ice-cold 10% trichloroacetic acid. Twenty µl of 7.5 mg/ml BSA were added, and the samples were incubated for 2 min, then microcentrifuged for 5 min. The P released during the assay was quantitated by liquid scintillation counting of the supernatant. When assaying column fractions, the percentage of the total phosphate released from the substrate was kept under 20% unless otherwise indicated. In ceramide dose dependence experiments, assays were designed such that approximately 10% of the phosphate present in the substrate was released in the presence of C-ceramide, since maximal stimulation of phosphatase activity was observed under these conditions. When phosphorylase a was used as the substrate, 5 mM caffeine was present in the assay mixture. In all cases, phosphate release was linear with respect to time from 10 to 60 min, indicating that enzyme denaturation was not a problem and that substrate concentrations were not limiting under these conditions. C-ceramide was stored under argon as a 20 mM stock solution in absolute ethanol. The final ethanol concentration in the assays was 0.1% and had no effect on phosphatase activity. One unit of phosphatase is defined as the amount of phosphatase activity releasing 1 nmol of phosphate from casein per min under the assay conditions described.


RESULTS

The partially purified trimeric PP-2A used for these studies contains a mixture of two isoforms, ACB` and ACB. Immunoblots of the partially purified trimeric PP-2A used in the present study exhibited one major band when probed with antibodies specific for either the C, the A, or the B subunits (Fig. 1). The B` subunit appeared as a doublet as observed previously by Ruediger et al.(31) .


Figure 1: Specificity of antibodies directed toward PP-2A. SDS-PAGE followed by immunoblot analysis was performed on trimeric PP-2A (1.9 10 units) as described under ``Experimental Procedures,'' with antibodies specific for either the catalytic subunit (C), the A (A), or one of the B (B, B`) subunits of PP-2A. For each immunoblot, trimeric PP-2A and molecular mass standards were run on adjacent lanes of the gel. The biotinylated standards shown are rabbit muscle phosphorylase b (97.4 kDa), BSA (66.2 kDa), hen egg white ovalbumin (45 kDa), and bovine carbonic anhydrase B (31 kDa).



Trimeric PP-2A was subjected to preparative native electrophoresis, then gel slice extracts were prepared and analyzed for phosphatase activity and for the presence of the C, A, and B subunits. Phosphatase activity was observed primarily in extracts of slices 5 and 6 (Fig. 2A), and, as expected, these extracts were activated by 20 µM C-ceramide. Immunoblot analyses (Fig. 2B) show that all three PP-2A subunits were contained primarily in slices 5 and 6. Heparin treatment of trimeric PP-2A is known to cause dissociation of the B subunit (32) . Pretreatment of trimeric PP-2A with heparin-agarose before native electrophoresis resulted in phosphatase activity which eluted primarily in slice 11 rather than slices 5 and 6 (Fig. 3A), but was still sensitive to C-ceramide (Fig. 3, A and C). Immunoblot analyses (Fig. 3B) showed that slice 11 contained the C and A subunits, but neither of the B subunits. Together, these data demonstrate that dimeric PP-2A can be activated by C-ceramide and that the activation does not require the presence of the B subunit. The loss of staining for the B` subunit indicates that the free subunit does not comigrate with the dimeric or trimeric forms of PP-2A. The staining for the B subunit in slices 6 and 7 represents undissociated trimeric PP-2A. It has been observed (25) that heterotrimeric PP-2A containing the B` subunit is more susceptible to heparin-induced B subunit dissociation than trimeric enzyme containing the B subunit.


Figure 2: Ceramide stimulation of trimeric PP-2A. Partially purified trimeric PP-2A (0.17 unit) was subjected to native PAGE. Gel slices were processed as described under ``Experimental Procedures,'' and extracts from the gel slices were assayed for casein phosphatase activity (see ``Experimental Procedures'') in the presence (--) or absence (--) of 20 µM C-ceramide (panel A). Identical slices were analyzed by SDS-PAGE and immunoblotting (panel B) with antibodies specific for either the catalytic (C), the A (A), or one of the B regulatory subunits (B, B`). The experiment was repeated four times using two different preparations of trimeric PP-2A, with similar results.




Figure 3: Dimeric PP-2A is stimulated by C-ceramide. Native gel slices were prepared and processed as described in Fig. 2 except that the trimeric PP-2A (0.17 units) used was treated overnight with heparin-agarose as described under ``Experimental Procedures.'' Slice extracts were analyzed for casein phosphatase activity (see ``Experimental Procedures'') in the presence (--) or absence (--) of 20 µM C-ceramide (panel A). Immunoblots (panel B) of extracts from identical slices were performed with antibodies specific for either the catalytic (C), the A (A), or one of the B regulatory subunits (B, B`). Extracts from slice 11 were reassayed for casein phosphatase activity (panel C) in quadruplicate in the presence or absence of 20 µM C-ceramide. The experiment was repeated four times with two different preparations of PP-2A, with similar results. Samples in Figs. 2 and 3 were run on the same native gel in each replicate.



We next examined whether the catalytic subunit of PP-2A can be activated by C-ceramide. The catalytic subunit of PP-2A was produced by freezing and thawing trimeric PP-2A in the presence of 0.2 M -mercaptoethanol (21) , then separated from undissociated PP-2A by gel filtration. Phosphatase assays of the gel filtration fractions revealed two peaks of phosphatase activity. The first peak eluted near the void volume, while the second peak eluted as a 34-kDa species (Fig. 4A), consistent with its identity as the C subunit of PP-2A. Both peaks of phosphatase activity were stimulated by 20 µM C-ceramide. Since the fractions corresponding to the trailing edge of the second peak are also ceramide-sensitive (Fig. 4B), contamination of the second peak by the first is an unlikely explanation for the ceramide sensitivity of the 34-kDa species. Immunoblots (Fig. 4C) showed that the C, A, B, and B` subunits comigrated with the first peak, indicating that it consists of undissociated trimeric or dimeric PP-2A. The second peak stained positively only for the catalytic subunit.


Figure 4: The catalytic subunit of PP-2A is activated by ceramide. Partially purified trimeric PP-2A (0.58 unit) was made 0.2 M in -mercaptoethanol, placed at -20 °C for 1 h, thawed, and immediately loaded onto a 1.7 47 cm Sephadex G-75 gel filtration column. The column was eluted at 0.3 ml/min until 75 1-ml fractions had been collected. The elution volumes of the molecular mass standards: a, BSA (66 kDa); b, carbonic anhydrase (29 kDa); and c, cytochrome c (12.4 kDa) are shown in panel A. Casein phosphatase assays of 0.5 µl (panel A) or 2.0 µl (panel B) of the resulting fractions were performed in the presence (--) or absence (--) of 20 µM C-ceramide. The arrow in panel B denotes 20% phosphate release. Samples corresponding to every other fraction from 22-46 were concentrated by acetone precipitation (see ``Experimental Procedures''), subjected to SDS-PAGE, and analyzed by immunoblot (panel C) using antibodies specific for either the catalytic (C), the A (A), or one of the B regulatory subunits (B, B`) of PP-2A. The experiment was repeated three times with similar results.



The free A subunit is expected to elute near the void volume owing to its size (7) and elongated shape (33) and should be resolved from the C subunit. Based on protein standards, the free B subunit should elute in fractions 30-32 and should also be separated from the C subunit. Immunoblots for B and B` verify that little if any B subunit is present in fractions containing the free C subunit. Furthermore, if the ceramide activation was mediated by either the A or the B subunits, the first peak should be more highly stimulated by ceramide than the second peak. This is not observed (Fig. 4, A and B). Finally, in separate experiments utilizing a longer (1.7 96 cm) G-75 column, the two peaks of phosphatase activity could be completely resolved, and the peak of phosphatase activity eluting as a 34-kDa species was still activated to the same extent by C-ceramide (data not shown). Together, these results show that the catalytic subunit of PP-2A can be activated by C-ceramide in the absence of the A and B subunits.

To determine whether the catalytic subunit and trimeric forms of PP-2A have a similar sensitivity to ceramide, ceramide dose-response curves were performed for both enzyme forms using casein as substrate (Fig. 5). The sensitivity of the two enzyme forms to C-ceramide is similar. The extent of activation of the catalytic subunit by ceramide varied from experiment to experiment, but was equal to or greater than that observed with trimeric PP-2A and sometimes approached 6-fold activation (Fig. 5). The reason for the increased activation of the catalytic subunit relative to the trimer is not known. In order to determine whether the ceramide activation of different forms of PP-2A is substrate-specific, PP-2A subunit isoforms were assayed in the presence or absence of 20 µM ceramide using either casein or phosphorylase a as substrates (). These experiments demonstrated that ceramide activates the phosphatase activity of the catalytic subunit whether casein or phosphorylase a is used as substrate, suggesting that ceramide activation of the catalytic subunit is not substrate-specific. In summary, the catalytic subunit and trimeric forms of PP-2A do not differ dramatically with respect to their sensitivity to or extent of activation by C-ceramide, demonstrating that the A and B subunits are not required for ceramide activation of the enzyme.


Figure 5: Dose dependence of activation of the trimeric and catalytic subunit forms of PP-2A by C-ceramide. Trimeric PP-2A (--) or the catalytic subunit of PP-2A (--) were diluted to yield approximately equal activity in the absence of ceramide, then assayed for casein phosphatase activity in the presence of increasing concentrations of C-ceramide. The catalytic subunit used was prepared as in Fig. 4 except that a longer (1.7 96 cm) Sephadex G-75 column was used to completely resolve the activity of the catalytic subunit from the activity corresponding to the higher molecular mass forms of PP-2A. Results are expressed as the -fold increase in activity relative to control. The values are from a single representative experiment and are expressed as the mean ± S.D. of triplicate assays. Abberant data points were discarded using the Q-test with 90% confidence limits. The experiment was repeated using three different preparations of catalytic subunit with similar results.




DISCUSSION

In this report we have shown that the dimeric and catalytic subunit forms of PP-2A are stimulated by ceramide. The catalytic subunit is similar to trimeric PP-2A in its sensitivity to ceramide and can be stimulated to a similar or greater maximal extent than trimeric PP-2A. These data indicate that the catalytic subunit is the target for ceramide activation, and that additional subunits are not required for ceramide sensitivity.

The response of heterotrimeric PP-2A to ceramide in the present study was similar to that reported by Dobrowsky et al.(20) in that: stimulation was independent of the substrate used; a similar sensitivity to and maximal stimulation by ceramide were observed and; the magnitude of stimulation observed was dependent on the amount of phosphatase present in the assay. In contrast, we found that dimeric PP-2A and the catalytic subunit alone can be stimulated by ceramide, whereas Dobrowsky and colleagues (20) were unable to detect stimulation of either dimeric or catalytic subunit forms of PP-2A. Differences between our results and theirs (20) may be due to differences in experimental details between the two studies or may be attributed to differences in post-translational modifications of the phosphatase preparations used.

In the present study, dimeric PP-2A was produced by heparin-agarose treatment of partially purified trimeric PP-2A, while the dimeric PP-2A used in the previous study (20) was prepared either by purification of dimeric PP-2A from crude extracts or treatment of purified trimeric PP-2A with trypsin or soluble heparin. The B subunit may protect the ceramide sensitivity of the catalytic subunit during purification even though it is not directly required for ceramide stimulation. If this is the case, isolation of dimeric PP-2A from crude extracts may result in the loss of a putative ceramide-sensitive element during purification, whereas in our studies partial purification of trimeric PP-2A prior to release of the B subunit may have preserved this element. Indeed, we also observed that dimeric PP-2A which was purified from crude extracts could not be stimulated by ceramide (data not shown). Likewise, generation of the catalytic subunit of PP-2A from crude extracts by ethanol precipitation, as was done in the study of Dobrowsky and colleagues (20) , may expose a ceramide-sensitive element that is lost during further purification steps. Again, this ceramide-sensitive element could be preserved by purifying trimeric enzyme, then dissociating the catalytic subunit, as was done in the present study. Trypsinization, an alternative strategy used by Dobrowsky et al.(20) to release the B subunit, can also cause cleavage of the C terminus of the catalytic subunit (32) . Time course experiments of trypsin treatment of trimeric PP-2A show that the C terminus is cleaved shortly after the B subunit is degraded (32) , consistent with the possibility that the B subunit protects the C subunit from cleavage by making it less accessible to trypsin. If the ceramide sensitivity of the catalytic subunit resides in its C terminus, cleavage of the C terminus, which has no observable effect on phosphatase activity (32) , could potentially alter ceramide binding.

Finally, although heparin was used to cause B subunit dissociation in both studies, in our study, heparin-agarose was used to treat PP-2A, then removed by sedimentation before activity was assayed; Dobrowsky and colleagues (20) treated PP-2A with soluble heparin. It is possible that heparin remaining in the assay mixture bound ceramide or competed with ceramide for a binding site on the phosphatase.

Alternatively, loss of a post-translational modification during purification may explain the differential ceramide sensitivity of the different phosphatase preparations. The catalytic subunit is methylated on the C terminus (15, 16, 17, 18) and phosphorylated on Tyr both in vitro(13) and in vivo(14) . Carboxymethylation was shown to increase the phosphorylase phosphatase activity of the enzyme (17) . Thiophosphorylation of Tyr was shown to inhibit phosphatase activity dramatically (13) . In addition, an autophosphorylation-activated serine/threonine kinase was shown to phosphorylate PP-2A on its C and A subunits and inhibit its phosphatase activity (35) . The phosphorylation site affecting activity, however, was not identified.

Although the A and B subunits are not required for ceramide activation of PP-2A, these regulatory subunits may modulate the responses of PP-2A to ceramide in vivo. Modulation may occur through a direct mechanism such as controlling the specificity of the enzyme for different forms of ceramide (20) , or through an indirect mechanism such as controlling the subcellular localization of PP-2A and therefore its exposure to ceramide, or controlling the substrate specificity of the enzyme and hence which substrates are dephosphorylated in response to increased ceramide levels.

Because the free catalytic subunit of PP-2A can be activated by ceramide, attempts to determine the basis of ceramide stimulation should be focused on this subunit. PP-2A is a member of a rapidly growing gene family of serine/threonine protein phosphatases. It is possible, based upon the degree of sequence identity shared between these phosphatases, that the catalytic subunits of other phosphatases in this family also have lipid binding sites. Regulation of PP-2A by ceramide binding in coordination with multiple covalent modifications and regulatory subunit interactions suggests that the phosphatase activity of PP-2A is under complex control.

Since PP-2A is one of the major serine/threonine phosphatases in many tissues, understanding the mechanisms by which PP-2A is regulated is important in determining how the state of phosphorylation of a variety of proteins is controlled. In particular, the putative regulation of PP-2A by ceramide may implicate PP-2A as a downstream effector of hormones and cytokines which include TNF-, interleukin-1, -interferon, and nerve growth factor and raises the possibility that the cell biological processes triggered by these agents such as apoptosis, differentiation, growth inhibition, and changes in gene expression are mediated in part by PP-2A. It will be important to identify physiological substrates of PP-2A whose state of phosphorylation is controlled by ceramide levels. The identification of these substrates and their biological functions will verify that PP-2A is a target of ceramide and explain the role of PP-2A in bringing about the physiological responses to this second messenger.

  
Table: Activation of various forms of PP-2A by 20 µM ceramide

Different forms of PP-2A were incubated with either P-labeled casein or phosphorylase for 60 min at 37 °C in the presence of 20 µM C-ceramide or an ethanol vehicle control. Data are presented as percent activity of the ceramide-treated samples relative to the corresponding vehicle controls. The values presented are means ± S.D. of the number of experiments indicated. Values from individual experiments are the means of triplicate or quadruplicate determinations.



FOOTNOTES

*
This work was supported by Grant NS31221 from the National Institutes of Health (to S. R.). This is Journal Paper Number 14629 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: 1153 Dept. of Biochemistry, Purdue University, West Lafayette, IN 47907-1153. Tel.: 317-494-3112; Fax: 317-494-7897.

The abbreviations used are: TNF-, tumor necrosis factor-; PP, protein phosphatase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; IgG, immunoglobulin G.


REFERENCES
  1. Hannun, Y. A.(1994) J. Biol. Chem. 269, 3125-3128 [Free Full Text]
  2. Dobrowsky, R. T., Werner, M. H., Castellino, A. M., Chao, M. V., and Hannun, Y. A.(1994) Science 265, 1596-1599 [Medline] [Order article via Infotrieve]
  3. Kim, M.-Y., Linardic, C., Obeid, L., and Hannun, Y.(1991) J. Biol. Chem. 266, 484-489 [Abstract/Free Full Text]
  4. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A.(1993) Science 259, 1769-1771 [Medline] [Order article via Infotrieve]
  5. Wolff, R. A., Dobrowsky, R. T., Bielawska, A., Obeid, L. M., and Hannun, Y. A.(1994) J. Biol. Chem. 269, 19605-19609 [Abstract/Free Full Text]
  6. Fishbein, J. D., Dobrowsky, R. T., Bielawska, A., Garrett, S., and Hannun, Y. A.(1993) J. Biol. Chem. 268, 9255-9261 [Abstract/Free Full Text]
  7. Cohen, P.(1989) Annu. Rev. Biochem. 58, 453-508 [CrossRef][Medline] [Order article via Infotrieve]
  8. Mumby, M. C., and Walter, G.(1993) Physiol. Rev. 73, 673-699 [Abstract/Free Full Text]
  9. Dent, P., Campbell, D. G., Caudwell, F. B., and Cohen, P.(1990) FEBS Lett. 259, 281-285 [CrossRef][Medline] [Order article via Infotrieve]
  10. Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P.(1982) FEBS Lett. 137, 80-84 [CrossRef][Medline] [Order article via Infotrieve]
  11. Klee, C. B., Draetta, G. F., and Hubbard, M. J.(1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 149-200 [Medline] [Order article via Infotrieve]
  12. Cegielska, A., Shaffer, S., Derua, R., Goris, J., and Virshup, D. M. (1994) Mol. Cell. Biol. 14, 4616-4623 [Abstract]
  13. Chen, J., Martin, B. L., and Brautigan, D. L.(1992) Science 257, 1261-1264 [Medline] [Order article via Infotrieve]
  14. Chen, J., Parsons, S., and Brautigan, D. L.(1994) J. Biol. Chem. 269, 7957-7962 [Abstract/Free Full Text]
  15. Xie, H., and Clarke, S.(1993) J. Biol. Chem. 268, 13364-13371 [Abstract/Free Full Text]
  16. Lee, J., and Stock, J.(1993) J. Biol. Chem. 268, 19192-19195 [Abstract/Free Full Text]
  17. Favre, B., Zolnierowicz, S., Turowski, P., and Hemmings, B. A.(1994) J. Biol. Chem. 269, 16311-16317 [Abstract/Free Full Text]
  18. Xie, H., and Clarke, S.(1994) J. Biol. Chem. 269, 1981-1984 [Abstract/Free Full Text]
  19. Dobrowsky, R. T., and Hannun, Y. A.(1992) J. Biol. Chem. 267, 5048-5051 [Abstract/Free Full Text]
  20. Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530 [Abstract/Free Full Text]
  21. Tamura, S., Kikuchi, H., Kikuchi, K., Hiraga, A., and Tsuiki, S.(1980) Eur. J. Biochem. 104, 347-355 [Abstract]
  22. Kaczmarek, L. K., Jennings, K. R., Strumwasser, F., Nairn, A. C., Walter, U., Wilson, F. D., and Greengard, P.(1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7487-7491 [Abstract]
  23. Mumby, M. C., Green, D. D., and Russell, K. L.(1985) J. Biol. Chem. 260, 13763-13770 [Abstract/Free Full Text]
  24. Walter, G., Ruediger, R., Slaughter, C., and Mumby, M.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2521-2525 [Abstract]
  25. Kamibayashi, C., Estes, R., Lickteig, R. L., Yang, S.-I., Craft, C., and Mumby, M. C.(1994) J. Biol. Chem. 269, 20139-20148 [Abstract/Free Full Text]
  26. Mumby, M. C., Russell, K. L., Garrard, L. J., and Green, D. D.(1987) J. Biol. Chem. 262, 6257-6265 [Abstract/Free Full Text]
  27. Maizel, J. V.(1971) Methods Virol. 51, 179-224
  28. Bollag, D. M., and Edelstein, S. J.(1991) Protein Methods, pp. 145-160, Wiley-Liss, Inc., New York
  29. Sheng, Z., and Charbonneau, H.(1993) J. Biol. Chem. 268, 4728-4733 [Abstract/Free Full Text]
  30. Cohen, P., Alemany, S., Hemmings, B. A., Resink, T. J., Stralfors, P., and Tung, H. Y. L.(1988) Methods Enzymol. 159, 390-408 [Medline] [Order article via Infotrieve]
  31. Ruediger, R., VanWartHood, J. E., Mumby, M., and Walter, G.(1991) Mol. Cell. Biol. 11, 4282-4285 [Medline] [Order article via Infotrieve]
  32. Kamibayashi, C., Estes, R., Slaughter, C., and Mumby, M. C.(1991) J. Biol. Chem. 266, 13251-13260 [Abstract/Free Full Text]
  33. Chen, S.-C., Kramer, G., and Hardesty, B.(1989) J. Biol. Chem. 264, 7267-7275 [Abstract/Free Full Text]
  34. Guo, H., Reddy, S. A. G., and Damuni, Z.(1993) J. Biol. Chem. 268, 11193-11198 [Abstract/Free Full Text]

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