The Formation and Activity of PP2A Holoenzymes Do Not Depend on
the Isoform of the Catalytic Subunit*
Jin
Zhou,
Huong T.
Pham, and
Gernot
Walter
From the Department of Pathology, University of California at San
Diego, La Jolla, California 92093
Received for publication, October 31, 2002, and in revised form, December 17, 2002
 |
ABSTRACT |
The protein phosphatase 2A holoenzyme is
composed of one catalytic C subunit, one regulatory/scaffolding
A subunit, and one regulatory B subunit. The core enzyme consists of A
and C subunits only. The A and C subunits both exist as two closely
related isoforms,
and
. The B subunits belong to four weakly
related or unrelated families, designated B, B', B", and B
, with
multiple members in each family. The existence of two A and two C
subunit isoforms permits the formation of four core enzymes, A
C
,
A
C
, A
C
, and A
C
, and each core enzyme could in theory
give rise to multiple holoenzymes. Differences between C
and C
in
expression and subcellular localization during early embryonic
development have been reported, which imply that C
and C
have
different functions. To address the question of whether these
differences might be caused by enzymatic differences between C
and
C
, we purified six holoenzymes composed of A
C
or A
C
core
enzyme and B subunits from the B, B', or B" families. In addition, we
purified four holoenzymes composed of A
C
or A
C
and B'
1
or B"/PR72. The phosphatase activity of each purified form was assayed
using myelin basic protein and histone H1 as substrates. We found that
C
and C
have identical phosphatase activities when associated
with the same A and B subunits. Furthermore, no difference was found
between C
and C
in binding A or B subunits. These data suggest
that the distinct functions of C
and C
are not based on
differences in enzymatic activity or subunit interaction. The
implications for the relationship between the structure and function of
C
and C
are discussed.
 |
INTRODUCTION |
Protein phosphatase 2A
(PP2A)1 is the most abundant
serine/threonine phosphatase in mammals and is a major player in many
fundamental processes including differentiation, embryonic development,
and growth control. The great versatility of PP2A is a result of the existence of a large number of subunits: two isoforms of the catalytic C subunit (C
and C
), two isoforms of the regulatory/scaffolding A
subunit (A
and A
), and numerous regulatory B subunits that fall
into four families designated B, B', B", and B
. Each B subunit family consists of several isoforms or splice variants (see Ref. 1 for
review). Together all possible subunit combinations could give rise to
more than 70 different holoenzymes, each composed of one A, one B, and
one C subunit (1). In addition, four core enzymes, A
C
, A
C
,
A
C
, and A
C
, could exist that lack a regulatory B subunit.
Holoenzymes and core enzymes both exist in cells (2), whereas
"free" catalytic C subunits have not been clearly identified. It is
unknown whether all possible forms of PP2A actually exist, and only a
few forms have been purified and characterized. Importantly, not a
single C
-containing core or holoenzyme has been purified and
studied. One reason for this lack in our knowledge is that multiple
forms of PP2A may co-exist in cells that are difficult to separate. In
addition, some forms may represent only a minor fraction of the total
cellular PP2A. Furthermore, PP2A is associated with a large number of
other proteins (1, 3), making the task of separation and purification
even more difficult. One way to solve this problem could be to assemble
different forms of PP2A from purified recombinant subunits. However,
difficulties in expressing soluble C subunits have limited this
approach (see Ref. 4 for further references).
C
and C
both consist of 309 amino acids and are 97% identical in
sequence. They differ by only eight amino acids, seven of which are
localized within a sequence of 30 amino acids at the N terminus (1, 5).
C
and C
are both encoded by genes composed of seven exons and six
introns (6). It has been suggested that exons I and VII encode
sequences involved in regulation, whereas exons II-VI are important
for substrate binding and catalysis. C
is expressed at 10-fold
higher levels than C
because of differences in transcriptional
regulation (6). Interestingly, all catalytically relevant amino acids
in protein phosphatase 1 (PP1), whose three-dimensional structure has
been elucidated, are completely conserved in PP2A and PP2B (7, 8),
suggesting that the three-dimensional structures of PP2A and PP2B are
similar to that of PP1. Importantly, all eight amino acids that are
different between C
and C
are not conserved between PP1, PP2A,
and PP2B. Therefore, they are likely not involved in catalysis.
Although very similar in structure, C
and C
have different
functions. C
/
mice are normal until embryonic day
5.5 but die on embryonic day 6.5, suggesting that C
is required for
mesoderm formation and gastrulation (9). Furthermore, it has been
demonstrated that in normal early embryonic development C
is mainly
associated with the plasma membrane, whereas C
is localized in the
cytoplasm and in nuclei. Interestingly, E-cadherin, which forms a
complex with C
and
-catenin and is normally located at the plasma
membrane, changes location to the cytoplasm in C
/
mice, suggesting that C
but not C
mediates binding of the
E-cadherin complex to the plasma membrane (10). It has been suggested
that the difference between C
and C
could be caused either by a
targeting signal in C
that directs it to the plasma membrane or that
a regulatory B subunit associated with C
but not C
is responsible for targeting the C
-containing holoenzyme to the plasma membrane where it is involved in Wnt signaling (10).
In the present study, we compared the properties of 10 C
- and
C
-containing holoenzymes purified from cells transfected with epitope-tagged C
or C
, A
or A
, and B
, B'
1, or
B"/PR72. Purification was carried out by consecutive
immunoprecipitations using anti-peptide antibodies. This approach has
been successfully applied in the past to highly purify a complex of
polyoma virus middle T antigen with two cellular proteins (11) that
were subsequently identified as the A and C subunits of PP2A (12, 13).
We demonstrated that C
and C
have the same affinity for A and B
subunits. In addition, they have identical phosphatase activities when
associated with the same type of A and B subunits. Therefore, the
marked functional differences between C
and C
during early
embryonic development and in subcellular localization are unlikely
because of differences in subunit interaction or enzymatic activity.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
Rat monoclonal antibody 6G3 recognizing
both A
and A
and mouse monoclonal anti-peptide antibodies anti-EE
and anti-KT3 were described previously (2, 14, 15). IgG fractions of
the above antibodies were isolated by standard procedures using protein G-Sepharose CL-6B beads (GammaBind Plus from Amersham
Biosciences). Mouse monoclonal antibody anti-hemagglutinin
(anti-HA) was purchased (Roche Molecular Biochemicals). For
immunoprecipitations, anti-EE and anti-KT3 antibodies were covalently
coupled to the protein G-Sepharose (GammaBind Plus-Sepharose, Amersham
Biosciences) using dimethylpimelimidate as described (16).
Plasmids--
Vectors encoding A
and A
tagged at the C
terminus with EE (EEEEYMPME), vectors encoding B
, B'
1, and
B"/PR72 tagged at the C terminus with KT3 (KPPTPPPEPET), and vectors
encoding C
and C
tagged at the N terminus with HA (YPYDVPDYA)
were described previously (17).
Cell Culture and Transfection--
The human embryonic kidney
cell line 293 was obtained from the American Type Culture Collection
and grown in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum. For transfection, 1.5 × 106 293 cells
were plated per 10-cm dish, grown for 24 h, and transfected with a
total of 3.9 µg of plasmid DNA using 30 µl of LipofectAMINE and 20 µl of PLUS reagent following Invitrogen's instructions. Transfection
conditions were optimized 1) for high and similar expression levels of
EE-tagged A
and A
as determined by Western blotting with anti-EE
antibodies, 2) for high expression of co-transfected tagged B or C
subunits, and 3) for high transfection efficiency as determined by
staining with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
of fixed cells co-transfected with a
-galactosidase vector (18).
48 h after transfection, the cells were harvested either with
SDS-PAGE sample buffer (2% SDS, 100 mM 1,4-dithiothreitol (DTT), 60 mM Tris-HCl, pH 6.8, 10% glycerol, and 0.01%
bromphenol blue) for Western blotting or with TX-100 buffer (0.5%
TX-100, 50 mM Tris-HCl pH 7.5, 150 mM NaCl)
containing 3 mM MgCl2, 1 mM DTT,
and 50 µM leupeptin for immunoprecipitation.
Labeling Transfected 293 Cells with
[35S]Methionine--
1.5 × 106 293 cells were plated per 10-cm dish and transfected as described above.
36 h after transfection, cells were washed twice with prewarmed
phosphate-buffered saline followed by one washing with prewarmed
methionine-free Dulbecco's modified Eagle's medium (Invitrogen). The
cells were then incubated for 12 h in 2 ml of labeling medium
consisting of 10% fetal bovine serum, 15% regular Dulbecco's
modified Eagle's medium, 75% methionine-free Dulbecco's modified
Eagle's medium, 20 mM HEPES, 584 mg/ml glutamine, 110 mg/ml pyruvate, and 300 µCi/ml [35S]methionine
(specific activity, >1000 Ci/mmol, Amersham Biosciences).
Purification of PP2A Holoenzymes--
At 48 h
post-transfection, the 293 cells (labeled or unlabeled with
[35S]methionine) were washed twice with cold
phosphate-buffered saline, placed on ice, and extracted for 10 min with
450 µl per 10-cm dish of cold TX-100 buffer containing 3 mM MgCl2, 1 mM DTT, and 50 µM leupeptin. The extracts were centrifuged at
12,000 × g at 4 °C for 5 min, and the supernatants
were used for immunoprecipitation.
For the first immunoprecipitation, 800 µl of cell extract was
incubated with 60 µl of protein G-Sepharose coupled with anti-EE (17 µg of IgG/µl). After rotating for 1 h at room temperature, the
immune complexes were washed three times with TX-100 buffer and
released in 200 µl of release buffer (2% TX-100, 10 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT)
containing 300 µg/ml EE peptide. After incubation at room temperature
for 30 min while rotating, the protein G-Sepharose was spun down, and
the supernatant was subjected to a second round of precipitation and
release using 20 µl of anti-KT3 protein G-Sepharose containing 15 µg of IgG/µl and 100 µl of release buffer containing 300 µg/ml
KT3 peptide. The protein complex in 100 µl of release buffer was then
subjected to the third precipitation with 10 µl of anti-HA (100 µg/ml). After 1 h of incubation, 5 µl of protein G-Sepharose
was added and the mixture was incubated for another hour. After a
washing with TX-100 buffer, the Sepharose with bound holoenzymes was
divided; half of it was used directly for the phosphatase assays, and
the other half was boiled in SDS-PAGE sample buffer for Western
analysis and quantitation. To monitor purification and subunit
interaction, aliquots from each step were analyzed on 12% SDS-PAGE
followed by Western blotting as described (17).
Preparation of 33P-Labeled
Substrates--
Myelin basic protein (MBP) was phosphorylated with
cAMP-dependent protein kinase following the manufacturer's
instructions (New England Biolabs). To phosphorylate histone H1, 1 mg
of histone H1 (Roche Molecular Biochemicals) was incubated at 30 °C
overnight in 1 ml of reaction buffer (New England Biolabs) containing
300 units of p34cdc2-cyclin B (New England Biolabs), 0.5 mM ATP, and 250 µCi/µmol [
-33P]ATP
(specific activity, >2500 Ci/mmol, Amersham Biosciences). The reaction
was terminated by the addition of trichloroacetic acid to a final
concentration of 25% and kept on ice for 30 min. The mixture was
centrifuged at 12,000 × g at 4 °C for 10 min. The
pellet containing phosphorylated histone H1 was washed twice with 20%
trichloroacetic acid, dissolved in 200 µl of solubilization buffer
(50 mM imidazole, pH 7.4, 1 mM DTT, 5 mM EGTA, 0.01% Brij35), and dialyzed overnight against 1 liter of dialysis buffer (25 mM imidazole, pH 7.4, 1 mM DTT, 5 mM EGTA, 0.01% Brij35) to remove unbound ATP. The stoichiometry of phosphorylation for MBP was 1.4 and
0.6 mol/mol for histone H1. The concentrations of the substrates were
adjusted to 50 µM for MBP and 25 µM for
histone H1 with respect to the incorporated phosphate.
Phosphatase Assay and Specific Activity
Calculation--
Phosphatase activity was determined by measuring the
release of radioactive phosphate. After the final immunoprecipitation, one half of the PP2A holoenzymes bound to the protein G-Sepharose was
suspended in 40 µl of assay buffer (New England Biolabs) and then
mixed with 10 µl of MBP to a final concentration of 10 µM with respect to the incorporated phosphate. The
phosphatase reaction was carried out at 30 °C for 10 min followed by
precipitation of the unreleased phosphate with 10% cold
trichloroacetic acid. The released phosphate was counted in a
scintillation counter. The specific activity of PP2A (nmol/min/mg) was
calculated according to the NEB manual. Quantitation of the amount of
holoenzyme used in the phosphatase reaction was done by Western
blotting of the remaining half of the purified holoenzymes with 6G3 and
comparing the A subunit after the final purification to a serial
dilution of pure A. The amount of the purified holoenzyme was
calculated according to the molecular mass ratio of A/ABC. The
phosphatase assays with histone H1 were carried out similarly, except
that the final concentration of histone H1 substrate was 2.5 µM with respect to the incorporated phosphate, and
unreleased phosphate was precipitated by trichloroacetic acid at a
final concentration of 25%.
 |
RESULTS |
Three-step Purification of A
-C
-B"/PR72 Holoenzyme
Using Anti-peptide Antibodies--
Fig.
1 illustrates schematically how the
consecutive use of two monoclonal anti-peptide antibodies, anti-EE and
anti-KT3, combined with anti-HA monoclonal antibodies yields a defined
form of PP2A holoenzyme. As a source for the enzyme we used an extract from 293 cells transfected with vectors encoding EE-tagged A
(A
EE), KT3-tagged B"/PR72 (B"/PR72KT3), and
HA-tagged C
(HAC
). The goal of the
experiment was to purify
A
EE-B"/PR72KT3-HAC
holoenzyme
free of any other form of PP2A. As shown in Fig. 1, the cell lysate
contains a mixture of core and holoenzymes composed of endogenous
(white) and exogenous (color) subunits. In the
first immunoprecipitation with anti-EE and the subsequent solubilization of the immune complexes with EE peptide, all forms of
enzyme not containing A
EE are eliminated, whereas excess
free A
EE as well as core and holoenzymes composed of
A
EE combined with untagged endogenous or tagged
exogenous B or C are retained. In the second step, immunoprecipitation
with anti-KT3 and release with KT3 peptide, holoenzymes composed of
A
EE, B"/PR72KT3 and either
HAC
or the endogenous C subunits (C
or C
) are
purified. In the last step, immunoprecipitation with anti-HA, the
holoenzyme containing endogenous C is eliminated, and only the desired
form, A
EE-B"/PR72KT3-HAC
,
remains.

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Fig. 1.
Scheme of holoenzyme purification by
sequential immunoprecipitations with anti-peptide antibodies.
Models of PP2A core and holoenzymes are drawn according to Ruediger
et al. (28, 29). The endogenous subunits are in
white; A could be A or A , C
could be C or C , and B could be B, B', B" or B . The
exogenous subunits are in color
(A EE, blue;
HAC , pink;
B"KT3, green). EE, KT3, and HA are
the peptide tags on the A , B"/PR72, and C subunits,
respectively. The holoenzyme to be purified is framed by a
rectangle. The lysate panel shows possible forms
of core and holoenzymes in 293 cells transfected with
A EE, B"/PR72KT3, and HAC .
Superscripts EE and KT3 to the right of A and B"/PR72
(shown in the figure as B") indicate tagging at the C
terminus; superscript HA to the left of C indicates
tagging at the N terminus. After the first immunoprecipitation
(IP) with anti-EE and release with EE peptide, endogenous A
subunit-containing core and holoenzymes are eliminated. A second
immunoprecipitation with anti-KT3 and release with KT3 peptide
eliminates all enzymes that do not contain B"/PR72KT3. The
third precipitation with anti-HA eliminates holoenzymes with endogenous
C or C . Only an exogenously expressed holoenzyme remains:
A EE-B"/PR72KT3-HAC .
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|
That this approach yields highly purified enzyme is shown in Fig.
2. 293 cells transfected with
A
EE-, B"/PR72KT3-, and
HAC
-encoding vectors were labeled with
[35S]methionine from 36-48 h post-transfection.
Cytoplasmic extract was prepared as described under "Experimental
Procedures." The extract contained numerous
[35S]methionine-labeled proteins including
A
EE, which is detectable (lane 1) when
compared with untransfected control lysate (lane 2).
B"/PR72KT3 and HAC
were undetectable.
Precipitation with anti-EE and release with EE peptide achieved
considerable purification so that B"/PR72KT3 and
HAC
can now be seen (lanes 3 and
4). Compared with B"/PR72KT3 and
HAC
, A
EE was in a large molar excess
because of the presence of excess "free" monomeric
A
EE. That the holoenzyme in the EE-released material
still contained a large number of cellular proteins is apparent after a
longer exposure time (compare lanes 10 and 4).
The second precipitation with anti-KT3 (lanes 6 and
12) and the release with KT3 peptide (lanes 7 and
13) considerably improved the purity of the
A
EE-B"/PR72KT3-HAC
holoenzyme. After immunoprecipitation with anti-HA, the product was
only slightly purer than after the previous step (compare lanes
7 and 13 with lanes 8 and 14).
One difference was the loss of two bands just below HAC
(compare lanes 13 and 14) that might represent
untagged forms of endogenous C subunit expected to be removed at this
step. Note that B"/PR72 is heterogeneous, suggesting that it might be
modified by phosphorylation. Despite the high degree of purity of the
final product, some proteins are still present at submolar quantities. Because they were not found in the untransfected control (lane 15), they might represent proteins specifically associated with the A
EE-B"/PR72KT3-HAC
holoenzyme. The radioactive bands for A
EE,
B"/PR72KT3, and HAC
were quantitated by
using a Molecular Dynamics Storm Gel and Blot Imaging System and
ImageQuant software. Adjustments were made for the number of methionine
residues in each subunit, the amount of cell extract used, and the
amount of protein loaded for each step of purification. The results of
quantitation show that in the course of purification, the molar ratio
of the A, B, and C subunits approached 1:1:1 (data not shown). The
large excess of A
EE after the first purification step is
caused by its high overexpression. The relatively higher intensity of
A
in the final product (lane 8) results from its higher
methionine content as compared with C
and B"/PR72. The yield of
holoenzyme after the third immunoprecipitation was ~3% compared with
the first immunoprecipitation (100%) (data not shown). Losses occurred
both during the immunoprecipitations and releases, but the yield could
be improved by optimizing each step. Under the present conditions, one
10-cm dish with 1.5 × 106 293 cells (~200 µg of
total protein and 600 ng PP2A) yielded 10-20 ng of purified holoenzyme
after the third precipitation.

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Fig. 2.
Purification of holoenzyme
A EE-B"/PR72KT3-HAC
from transfected 293 cells by sequential
immunoprecipitation. 293 cells were transfected with plasmids
encoding A EE, B"/PR72KT3, and
HAC , and labeled with [35S]methionine as
described under "Experimental Procedures." Five aliquots of
transfected cell extract were used to purify the holoenzyme to various
degrees as outlined in Fig. 1: 1) first immunoprecipitation
(IP) with anti-EE, 2) EE release, 3) second
immunoprecipitation with anti-KT3, 4) KT3 release, and 5) third
immunoprecipitation with anti-HA. Equivalent amounts from each step of
purification were analyzed. As a control, a mock purification was
carried out in parallel using lysate from pcDNA3-transfected cells
(lanes 2, 5, 9, 11, and
15). Transf Lys indicates lysate from cells
transfected with A EE, B"/PR72KT3, and
HAC . Numbers at the left indicate molecular
mass in kDa. Lanes 1-9, 16-h exposure; lanes
10-15, 7-day exposure.
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|
Compared with more conventional methods of protein purification, the
procedure described in the present study has several advantages. It is
quick and gentle, requires very small amounts of starting material, and
can be carried out in parallel to purify at least six forms of holoenzymes.
Comparison of C
and C
in Holoenzyme Formation--
As
mentioned earlier, the C
and C
polypeptides differ only by eight
amino acids, seven of which are located within the N-terminal 30 amino
acids (1). We asked whether the reported biological differences between
the two isoforms might result from differences in their phosphatase
activity or from differences in their ability to interact with the
other subunits. We previously demonstrated that C
and C
bind
equally well to A
(17). They also bind equally well to A
,
although binding to A
is 8-fold weaker than to A
(17). Whether
C
- and C
-containing core enzymes differ in B subunit binding has
not been studied. To address this question, we expressed A
with C
or C
and B
, B'
1, or B"/PR72 in 293 cells and carried out
three-step purification of holoenzymes. As described above, A
was
EE-tagged, C
and C
were HA-tagged, and the B subunits were tagged
with KT3. Each step was monitored by Western blotting with 6G3
recognizing tagged and untagged A
and A
, anti-KT3 recognizing
tagged B
, B'
1, and B"/PR72, and anti-HA recognizing tagged C
and C
. As a control, the purification was carried out with extracts
from cells transfected with empty vector pcDNA3
(Invitrogen). The lysates were adjusted to contain equal amounts of
C
and C
. As shown in Fig.
3a, similar amounts of C
or
C
were associated with A
and B
, B'
1, or B"/PR72 at all steps of purification. For example, in the second
immunoprecipitation with anti-KT3, similar amounts of C
and C
were co-immunoprecipitated with B
KT3 (lanes
15 and 16), B'
1KT3 (lanes 17 and 18), and B"/PR72KT3 (lanes 19 and
20). In the third immunoprecipitation with anti-HA, similar
amounts of B
, B'
1, or B"/PR72 were co-precipitated with C
and
C
(compare lane 22 versus 23,
24 versus 25, 26 versus 27). Note that in cell lysates, B"/PR72
was the most highly expressed B subunit (lanes 5 and
6), followed by B'
1 (lanes 3 and 4)
and B
(lanes 1 and 2). Accordingly, the yields
of B"/PR72-containing holoenzymes after the second and third
immunoprecipitation were the highest (lanes 19,
20, 26, and 27). The control lysate
(lane 7) showed an A subunit signal coming from endogenous A
subunit recognized by the 6G3 antibody. As expected, this signal
disappeared after precipitation with anti-EE antibodies (lane
14). To obtain sufficient signal strength, various fractions of
the totals were loaded for Western analysis: 1/80 of the lysate,
1/20 of the first, 1/10 of the second, and 1/2 of
the third immunoprecipitation. The exposure times were adjusted to
visualize weak signals (see the legend to Fig. 3).

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Fig. 3.
A C and
A C bind equally well
to B , B' 1, and
B"/PR72 (a);
A C and
A C bind equally well
to B' 1 and B"/PR72 (b).
In panel a, 293 cells were co-transfected in parallel with
expression vectors for A EE; B KT3,
B' 1KT3, or B"/PR72KT3; and
HAC or HAC . In panel b, 293 cells were co-transfected with vectors for A EE;
B' 1KT3 or B"/PR72KT3; and HAC
or HAC . Holoenzymes consisting of A C or A C
and A C or A C and one of the B subunits were purified as
outlined in Fig. 1. Aliquots from cell lysates (1/80), the first
precipitation and release (1/20), the second precipitation and
release (1/10), and the third precipitation (1/2) were
analyzed by Western blotting with 6G3 recognizing endogenous and tagged
A and A subunits, anti-KT3 recognizing tagged B subunits, and
anti-HA recognizing tagged C subunits. As a control, a mock
purification was carried out in parallel using lysate from cells
transfected with empty vector pcDNA3. In panel a, the
exposure times are 20 min for A EE (lanes
1-21), 16 h for A EE (lanes 22-28),
and 5 min for the Bs and Cs. In panel b, the exposure times
are 30 s for A EE (lanes 1-10), 20 min
for A EE (lanes 11-20), 5 min for the Bs and
Cs (lanes 1-5), and 30 min for the Bs and Cs (lanes
6-20). IP, immunoprecipitation.
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We also co-expressed C
or C
with A
and B'
1 or B"/PR72 and
purified the B'
1- and B"/PR72-containing holoenzymes. As shown in
Fig. 3b, both the A
C
and A
C
core enzymes bound
equally well to B'
1 or B"/PR72. The B
subunit was not included in
this experiment because, as shown previously (17), it does not bind to
A
-containing core enzyme. The conclusion from these experiments is
that the core enzymes A
C
and A
C
on the one hand and
A
C
and A
C
on the other hand are indistinguishable in their
ability to bind B subunits.
Comparison of C
and C
in Holoenzyme
Activity--
We tested whether A
C
and A
C
differ in
phosphatase activity when associated with B
, B'
1, or B"/PR72
subunits. Phosphatase assays were carried out with two different
substrates, MBP phosphorylated by cAMP-dependent protein
kinase and histone H1 phosphorylated by p34cdc2-cyclin B. These
substrates have been used in the past to differentiate between the
activities of different forms of PP2A (19). As shown in Fig.
4, the purified holoenzymes A
B
C
and A
B
C
have the same phosphatase activities with MBP
(panel a) or histone H1 (panel b) as substrates.
Similarly, A
B'
1C
and A
B'
1C
have the same activities
toward MBP (panel a) or histone H1 (panel b).
Furthermore, A
B"/PR72C
and A
B"/PR72C
also have
indistinguishable activities toward both substrates. The results with
histone H1 and MBP show that the sole determining factors for activity
are B
, B'
1, and B"/PR72, whereby with histone H1 the
B
-containing holoenzyme had the highest activity followed by the
B"/PR72- and B'
1-containing holoenzymes (panel b).

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Fig. 4.
Specific phosphatase activity of
six PP2A holoenzymes. Holoenzymes consisting of A and B ,
B' 1, or B"/PR72, and C or C were purified as outlined in Fig.
1. Phosphatase activity was determined using 10 µM
33P-labeled MBP (panel a) and 2.5 µM 33P-labeled histone H1 (panel
b) as described under "Experimental Procedures." In each
assay, the amount of released phosphates was less than 20% of the
total incorporated phosphates used. Each experiment was repeated at
least twice. Shown are two representative experiments (Exp 1 and Exp 2).
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We also compared A
B'
1C
with A
B'
1C
and A
B"/PR72C
with A
B"/PR72C
and found no differences in activity between the respective C
- and C
-containing forms. However, A
-containing holoenzymes are unstable because of the low affinity of A
for C and
B subunits (17), and they partially dissociated in the course of
purification. Therefore, the products after the third immunoprecipitation probably contained free C subunit and core enzyme,
and the phosphatase activities obtained were not representative of the
pure holoenzymes (data not shown).
 |
DISCUSSION |
The goal of our study was to understand how the marked functional
differences between C
and C
(9, 10) could be explained in view of
the small differences in their sequences. We considered three
possibilities. First, C
and C
might have different catalytic activities, although their catalytic domains are nearly identical. It
is conceivable that the sequence of 30 N-terminal amino acids, which
contains seven of the eight differences between C
and C
and is
located outside the catalytic domain (7, 8), regulates the
catalytic activity in a similar fashion as the C terminus of
pp60c-src regulates
pp60c-srckinase activity (see Ref. 20 for review).
Second, C
and C
might interact differently with A or B subunits.
For example, if the N termini of C
and C
are involved in A
subunit binding, C
and C
might differ in core enzyme formation.
This in turn might affect the assembly of holoenzymes, which depends on
the prior generation of core enzymes. If C
and C
bind equally
well to the A subunits, they might differ in B binding provided that the N-terminal 30 amino acids play a role in B binding. Third, if C
and C
associate with A and B subunits in an identical fashion, they
might differ in their ability to bind other regulatory proteins.
In the present report, we demonstrate that C
and C
have identical
phosphatase activities when associated with the same A and the same
type of B subunit. We also showed that C
and C
are identical in
their ability to associate with A and B subunits. These results render
the first two possibilities mentioned above unlikely. They are
consistent with the previous observation that, except for one amino
acid, C
and C
are identical in their catalytic domains (7, 8). We
cannot exclude that the activities of C
and C
might differ had we
used other substrates. It is also possible that other regulatory
subunits besides B
, B'
1, and B"/PR72 exist that bind
differentially to C
and C
. Nonetheless, we favor the third
possibility, i.e. that the 30 N-terminal amino acids direct
C
- and C
-containing holoenzymes to different subcellular locations by interacting with distinct target proteins. For example, C
may tether C
-containing holoenzymes to the plasma membrane in
early embryonic development (10). On the other hand, C
may bind to a
cytoplasmic or nuclear protein consistent with earlier findings
(10).
In a previous study, two holoenzymes containing either B or B"/PR72
were isolated from rabbit skeletal muscle. Because both enzymes
contained predominantly A
and C
, it was concluded that the
isoforms of A or C do not determine which B subunit associates with the
core enzyme (21). However, the fact that both enzymes contained
predominantly A
and C
is not surprising, because most cells and
tissues, including rabbit skeletal muscle, express at least 10-fold
more A
and C
than A
and C
(6, 22). Therefore, no direct and
quantitative comparison of A
C
with A
C
or of A
C
with
A
C
in binding B subunits was made. In addition, as we showed
recently, A
is significantly weaker in binding B and C subunits than
A
(17).
Ample evidence exists that the C
subunit directly associates with
proteins other than A and B subunits (see Refs. 1 and 3 for review).
C
binds to axin and might be involved in Wnt signaling by regulating
the level of
-catenin (23). C
also binds to B cell
receptor-associated protein
4 (24). Interestingly,
4 does not
associate with the core enzyme, suggesting that
4 competes with the
A subunit for binding to C
. Other examples of C
subunit binding
partners are the class C L-type calcium channel (25), the homeobox
protein HOX11 (26), and the translation termination factor eRF1 (27).
The latter case is of particular interest because it was demonstrated
that the eRF1 binding domain is located within the N-terminal 50 amino
acids of C
. In all other cases, the binding domains have not been
identified. It will be of interest to find out whether they are located
within the N-terminal 30 amino acids. In addition, specific binding
partners for C
remain to be identified.
 |
ACKNOWLEDGEMENTS |
We thank Ralf Ruediger for helpful
discussions and reading the manuscript. We thank Marc Mumby for B
,
B'
1, and C
subunit cDNAs, and Brian Hemmings for B"/PR72 cDNA.
 |
FOOTNOTES |
*
This work was supported by the Tobacco-related Disease
Research Program Grant 8RT-0037 and by Public Health Service Grant CA-36111.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 Pathology
0612, University of California at San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0612. Tel.: 858-534-1894; Fax: 858-534-8942; E-mail:
gwalter@ucsd.edu.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M211181200
 |
ABBREVIATIONS |
The abbreviations used are:
PP2A, protein
phosphatase 2A;
HA, hemagglutinin;
MBP, myelin basic protein;
DTT, dithiothreitol;
TX-100, Triton X-100.
 |
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