The Formation and Activity of PP2A Holoenzymes Do Not Depend on the Isoform of the Catalytic Subunit*

Jin Zhou, Huong T. Pham, and Gernot WalterDagger

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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha  and beta . 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, Aalpha Calpha , Aalpha Cbeta , Abeta Calpha , and Abeta Cbeta , and each core enzyme could in theory give rise to multiple holoenzymes. Differences between Calpha and Cbeta in expression and subcellular localization during early embryonic development have been reported, which imply that Calpha and Cbeta have different functions. To address the question of whether these differences might be caused by enzymatic differences between Calpha and Cbeta , we purified six holoenzymes composed of Aalpha Calpha or Aalpha Cbeta core enzyme and B subunits from the B, B', or B" families. In addition, we purified four holoenzymes composed of Abeta Calpha or Abeta Cbeta and B'alpha 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 Calpha and Cbeta have identical phosphatase activities when associated with the same A and B subunits. Furthermore, no difference was found between Calpha and Cbeta in binding A or B subunits. These data suggest that the distinct functions of Calpha and Cbeta are not based on differences in enzymatic activity or subunit interaction. The implications for the relationship between the structure and function of Calpha and Cbeta are discussed.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Calpha and Cbeta ), two isoforms of the regulatory/scaffolding A subunit (Aalpha and Abeta ), 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, Aalpha Calpha , Abeta Calpha , Aalpha Cbeta , and Abeta Cbeta , 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 Cbeta -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).

Calpha and Cbeta 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). Calpha and Cbeta 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. Calpha is expressed at 10-fold higher levels than Cbeta 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 Calpha and Cbeta are not conserved between PP1, PP2A, and PP2B. Therefore, they are likely not involved in catalysis.

Although very similar in structure, Calpha and Cbeta have different functions. Calpha -/- mice are normal until embryonic day 5.5 but die on embryonic day 6.5, suggesting that Calpha is required for mesoderm formation and gastrulation (9). Furthermore, it has been demonstrated that in normal early embryonic development Calpha is mainly associated with the plasma membrane, whereas Cbeta is localized in the cytoplasm and in nuclei. Interestingly, E-cadherin, which forms a complex with Calpha and beta -catenin and is normally located at the plasma membrane, changes location to the cytoplasm in Calpha -/- mice, suggesting that Calpha but not Cbeta mediates binding of the E-cadherin complex to the plasma membrane (10). It has been suggested that the difference between Calpha and Cbeta could be caused either by a targeting signal in Calpha that directs it to the plasma membrane or that a regulatory B subunit associated with Calpha but not Cbeta is responsible for targeting the Calpha -containing holoenzyme to the plasma membrane where it is involved in Wnt signaling (10).

In the present study, we compared the properties of 10 Calpha - and Cbeta -containing holoenzymes purified from cells transfected with epitope-tagged Calpha or Cbeta , Aalpha or Abeta , and Balpha , B'alpha 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 Calpha and Cbeta 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 Calpha and Cbeta during early embryonic development and in subcellular localization are unlikely because of differences in subunit interaction or enzymatic activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Antibodies-- Rat monoclonal antibody 6G3 recognizing both Aalpha and Abeta 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 Aalpha and Abeta tagged at the C terminus with EE (EEEEYMPME), vectors encoding Balpha , B'alpha 1, and B"/PR72 tagged at the C terminus with KT3 (KPPTPPPEPET), and vectors encoding Calpha and Cbeta 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 Aalpha and Abeta 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-beta -D-galactopyranoside (X-gal) of fixed cells co-transfected with a beta -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 [gamma -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Three-step Purification of Aalpha -Calpha -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 Aalpha (Aalpha EE), KT3-tagged B"/PR72 (B"/PR72KT3), and HA-tagged Calpha (HACalpha ). The goal of the experiment was to purify Aalpha EE-B"/PR72KT3-HACalpha 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 Aalpha EE are eliminated, whereas excess free Aalpha EE as well as core and holoenzymes composed of Aalpha 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 Aalpha EE, B"/PR72KT3 and either HACalpha or the endogenous C subunits (Calpha or Cbeta ) are purified. In the last step, immunoprecipitation with anti-HA, the holoenzyme containing endogenous C is eliminated, and only the desired form, Aalpha EE-B"/PR72KT3-HACalpha , 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 Aalpha or Abeta , C could be Calpha or Cbeta , and B could be B, B', B" or B'''. The exogenous subunits are in color (Aalpha EE, blue; HACalpha , pink; B"KT3, green). EE, KT3, and HA are the peptide tags on the Aalpha , B"/PR72, and Calpha 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 Aalpha EE, B"/PR72KT3, and HACalpha . Superscripts EE and KT3 to the right of Aalpha and B"/PR72 (shown in the figure as B") indicate tagging at the C terminus; superscript HA to the left of Calpha 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 Calpha or Cbeta . Only an exogenously expressed holoenzyme remains: Aalpha EE-B"/PR72KT3-HACalpha .

That this approach yields highly purified enzyme is shown in Fig. 2. 293 cells transfected with Aalpha EE-, B"/PR72KT3-, and HACalpha -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 Aalpha EE, which is detectable (lane 1) when compared with untransfected control lysate (lane 2). B"/PR72KT3 and HACalpha were undetectable. Precipitation with anti-EE and release with EE peptide achieved considerable purification so that B"/PR72KT3 and HACalpha can now be seen (lanes 3 and 4). Compared with B"/PR72KT3 and HACalpha , Aalpha EE was in a large molar excess because of the presence of excess "free" monomeric Aalpha 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 Aalpha EE-B"/PR72KT3-HACalpha 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 HACalpha (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 Aalpha EE-B"/PR72KT3-HACalpha holoenzyme. The radioactive bands for Aalpha EE, B"/PR72KT3, and HACalpha 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 Aalpha EE after the first purification step is caused by its high overexpression. The relatively higher intensity of Aalpha in the final product (lane 8) results from its higher methionine content as compared with Calpha 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 Aalpha EE-B"/PR72KT3-HACalpha from transfected 293 cells by sequential immunoprecipitation. 293 cells were transfected with plasmids encoding Aalpha EE, B"/PR72KT3, and HACalpha , 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 Aalpha EE, B"/PR72KT3, and HACalpha . Numbers at the left indicate molecular mass in kDa. Lanes 1-9, 16-h exposure; lanes 10-15, 7-day exposure.

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 Calpha and Cbeta in Holoenzyme Formation-- As mentioned earlier, the Calpha and Cbeta 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 Calpha and Cbeta bind equally well to Aalpha (17). They also bind equally well to Abeta , although binding to Abeta is 8-fold weaker than to Aalpha (17). Whether Calpha - and Cbeta -containing core enzymes differ in B subunit binding has not been studied. To address this question, we expressed Aalpha with Calpha or Cbeta and Balpha , B'alpha 1, or B"/PR72 in 293 cells and carried out three-step purification of holoenzymes. As described above, Aalpha was EE-tagged, Calpha and Cbeta were HA-tagged, and the B subunits were tagged with KT3. Each step was monitored by Western blotting with 6G3 recognizing tagged and untagged Aalpha and Abeta , anti-KT3 recognizing tagged Balpha , B'alpha 1, and B"/PR72, and anti-HA recognizing tagged Calpha and Cbeta . 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 Calpha and Cbeta . As shown in Fig. 3a, similar amounts of Calpha or Cbeta were associated with Aalpha and Balpha , B'alpha 1, or B"/PR72 at all steps of purification. For example, in the second immunoprecipitation with anti-KT3, similar amounts of Calpha and Cbeta were co-immunoprecipitated with Balpha KT3 (lanes 15 and 16), B'alpha 1KT3 (lanes 17 and 18), and B"/PR72KT3 (lanes 19 and 20). In the third immunoprecipitation with anti-HA, similar amounts of Balpha , B'alpha 1, or B"/PR72 were co-precipitated with Calpha and Cbeta (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'alpha 1 (lanes 3 and 4) and Balpha (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.   Aalpha Calpha and Aalpha Cbeta bind equally well to Balpha , B'alpha 1, and B"/PR72 (a); Abeta Calpha and Abeta Cbeta bind equally well to B'alpha 1 and B"/PR72 (b). In panel a, 293 cells were co-transfected in parallel with expression vectors for Aalpha EE; Balpha KT3, B'alpha 1KT3, or B"/PR72KT3; and HACalpha or HACbeta . In panel b, 293 cells were co-transfected with vectors for Abeta EE; B'alpha 1KT3 or B"/PR72KT3; and HACalpha or HACbeta . Holoenzymes consisting of Aalpha Calpha or Aalpha Cbeta and Abeta Calpha or Abeta Cbeta 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 Aalpha and Abeta 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 Aalpha EE (lanes 1-21), 16 h for Aalpha EE (lanes 22-28), and 5 min for the Bs and Cs. In panel b, the exposure times are 30 s for Abeta EE (lanes 1-10), 20 min for Abeta 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.

We also co-expressed Calpha or Cbeta with Abeta and B'alpha 1 or B"/PR72 and purified the B'alpha 1- and B"/PR72-containing holoenzymes. As shown in Fig. 3b, both the Abeta Calpha and Abeta Cbeta core enzymes bound equally well to B'alpha 1 or B"/PR72. The Balpha subunit was not included in this experiment because, as shown previously (17), it does not bind to Abeta -containing core enzyme. The conclusion from these experiments is that the core enzymes Aalpha Calpha and Aalpha Cbeta on the one hand and Abeta Calpha and Abeta Cbeta on the other hand are indistinguishable in their ability to bind B subunits.

Comparison of Calpha and Cbeta in Holoenzyme Activity-- We tested whether Aalpha Calpha and Aalpha Cbeta differ in phosphatase activity when associated with Balpha , B'alpha 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 Aalpha Balpha Calpha and Aalpha Balpha Cbeta have the same phosphatase activities with MBP (panel a) or histone H1 (panel b) as substrates. Similarly, Aalpha B'alpha 1Calpha and Aalpha B'alpha 1Cbeta have the same activities toward MBP (panel a) or histone H1 (panel b). Furthermore, Aalpha B"/PR72Calpha and Aalpha B"/PR72Cbeta also have indistinguishable activities toward both substrates. The results with histone H1 and MBP show that the sole determining factors for activity are Balpha , B'alpha 1, and B"/PR72, whereby with histone H1 the Balpha -containing holoenzyme had the highest activity followed by the B"/PR72- and B'alpha 1-containing holoenzymes (panel b).


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Fig. 4.   Specific phosphatase activity of six PP2A holoenzymes. Holoenzymes consisting of Aalpha and Balpha , B'alpha 1, or B"/PR72, and Calpha or Cbeta 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).

We also compared Abeta B'alpha 1Calpha with Abeta B'alpha 1Cbeta and Abeta B"/PR72Calpha with Abeta B"/PR72Cbeta and found no differences in activity between the respective Calpha - and Cbeta -containing forms. However, Abeta -containing holoenzymes are unstable because of the low affinity of Abeta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The goal of our study was to understand how the marked functional differences between Calpha and Cbeta (9, 10) could be explained in view of the small differences in their sequences. We considered three possibilities. First, Calpha and Cbeta 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 Calpha and Cbeta 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, Calpha and Cbeta might interact differently with A or B subunits. For example, if the N termini of Calpha and Cbeta are involved in A subunit binding, Calpha and Cbeta 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 Calpha and Cbeta 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 Calpha and Cbeta 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 Calpha and Cbeta have identical phosphatase activities when associated with the same A and the same type of B subunit. We also showed that Calpha and Cbeta 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, Calpha and Cbeta are identical in their catalytic domains (7, 8). We cannot exclude that the activities of Calpha and Cbeta might differ had we used other substrates. It is also possible that other regulatory subunits besides Balpha , B'alpha 1, and B"/PR72 exist that bind differentially to Calpha and Cbeta . Nonetheless, we favor the third possibility, i.e. that the 30 N-terminal amino acids direct Calpha - and Cbeta -containing holoenzymes to different subcellular locations by interacting with distinct target proteins. For example, Calpha may tether Calpha -containing holoenzymes to the plasma membrane in early embryonic development (10). On the other hand, Cbeta 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 Aalpha and Calpha , 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 Aalpha and Calpha is not surprising, because most cells and tissues, including rabbit skeletal muscle, express at least 10-fold more Aalpha and Calpha than Abeta and Cbeta (6, 22). Therefore, no direct and quantitative comparison of Aalpha Calpha with Abeta Calpha or of Aalpha Cbeta with Abeta Cbeta in binding B subunits was made. In addition, as we showed recently, Abeta is significantly weaker in binding B and C subunits than Aalpha (17).

Ample evidence exists that the Calpha subunit directly associates with proteins other than A and B subunits (see Refs. 1 and 3 for review). Calpha binds to axin and might be involved in Wnt signaling by regulating the level of beta -catenin (23). Calpha also binds to B cell receptor-associated protein alpha 4 (24). Interestingly, alpha 4 does not associate with the core enzyme, suggesting that alpha 4 competes with the A subunit for binding to Calpha . Other examples of Calpha 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 Calpha . 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 Cbeta remain to be identified.

    ACKNOWLEDGEMENTS

We thank Ralf Ruediger for helpful discussions and reading the manuscript. We thank Marc Mumby for Balpha , B'alpha 1, and Calpha 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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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