From the
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
The lipid ceramide is released in a variety of cell lines in
response to hormones and cytokines such as
TNF-
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-
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.
The partially purified trimeric PP-2A used for these studies
contains a mixture of two isoforms, ACB` and ACB
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
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
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-
Different forms of PP-2A were incubated
with either
-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.
(
)
, 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) .
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.
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.
. 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.
-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.
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.
,
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
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.
, tumor
necrosis factor-
; PP, protein phosphatase; PAGE, polyacrylamide
gel electrophoresis; BSA, bovine serum albumin; IgG, immunoglobulin G.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.