Fig. 7. PP2A methylation functions in the assembly of PP2A ABC holoenzymes. The PP2A methyltransferase (PPMT) and methylesterase (PPME) bind to unmethylated and methylated AC dimers, respectively, in place of the regulatory B subunits. Methylation dramatically enhances the binding of B and B′ regulatory subunits to facilitate the assembly of PP2A holoenzyme trimers.

Fig. 4. Methylated PP2A trimers are not demethylated by PPME. Demethylation assays were performed as described previously (Lee et al., 1996). (A) Aliquots of 25 nM [³H]methyl-labeled AC dimers (open circles) or [³H]methyl-labeled AB′_εC trimers (filled circles) were incubated with 10 nM PPME for the indicated times. (B) Aliquots of 1.0 nM [³H]methyl-labeled AC dimers were incubated in buffer alone (filled circles), in the presence of 1.0 nM PPME (open circles) or in the presence of 1.0 nM PPME plus 2.5 nM B′_ε (filled triangles), 5.0 nM B′_ε (open triangles), 10 nM B′_ε (filled squares) or 20 nM B′_ε (open squares). (C) [³H]methyl-labeled AC dimer, 10 nM in buffer alone (filled circles), was incubated in the presence of 5.0 nM PPME (open circles) or in the presence of 5.0 nM PPME plus 250 nM B_α (open squares).

Fig. 3. Methylated forms of PP2A. (A) A monoclonal antibody that specifically recognizes methylated PP2A. The indicated forms of PP2A were subjected to western blot analysis using the non-specific monoclonal antibody 6A3 and the methylation-specific antibody 4D9. (B) Separation of [³H]methyl-labeled PP2A in bovine brain extract by hydrophobic interaction chromatography. Fractions were analyzed by the methanol diffusion assay to detect [³H]methyl-labeled C subunits of PP2A (open circles) and by immunoblotting with non-specific anti-C monoclonal antibody 6A3, methylation-specific anti-C monoclonal antibody 4D9 and anti-B′ polyclonal antibody. (C) Purification of PP2A from peaks I and II. Coomassie Blue-stained SDS–polyacrylamide gels of the purified PP2A components obtained from each peak are indicated. Subunit identities were confirmed by western blotting with polyclonal antibodies as well as by microsequence analysis.

Fig. 6. Effects of methylation on PP2A phosphatase activity depend on methylation-induced changes in the association of B subunits with AC dimers. (A) Unmethylated AC dimers (120 nM) in the presence (filled circles) or absence (open circles) of equimolar B_α were incubated with 12 nM PPMT plus 10 µM [³H]AdoMet (5.5 Ci/mmol). Aliquots were removed at the indicated times and assayed for incorporation of [³H]methyl esters. The results are presented as the percentage of total possible methyl ester incorporation, assuming a maximum of 1 mol of methyl esters per mol PP2A. (B) AC alone, AC plus one equivalent of B_α or AC plus two equivalents of B_α was incubated for 20 min at 32°C. In a parallel experiment, AC alone or AC plus B_α were incubated in the presence of PPMT plus AdoMet for 20 min at 32°C. Aliquots of each protein mixture were then assayed for pNPP phosphatase activity. The data presented, expressed as change in A₄₁₀/min/nmol PP2A, are the averages of triplicate determinations (standard deviation <10%).

Fig. 1. Resolution of different forms of PP2A from bovine brain. (A) Hydrophobic interaction chromatography. Bovine brain extracts were subjected to hydrophobic interaction chromatography and 20 ml fractions were analyzed for PP2A [³H]methyl-accepting (open circles) and pNPP phosphatase (filled circles) activities as described in Materials and methods. Aliquots of indicated column fractions were analyzed by immunoblotting with anti-A, -B′, -B_α and -C antibodies. The positions of subunits A, B_α, B′ and C are shown on the right. (B) Purification of PP2A dimers. The major peak of methyl-accepting activity (fractions 44–56) was pooled, dialyzed and subjected to DEAE–Toyopearl column chromatography. Fractions with methyl-accepting activity were pooled (13 mg protein), concentrated and chromatographed on a Superose 6 HR column. Peak fractions were pooled, concentrated, desalted and subjected to Mono Q HR column chromatography. A Coomassie Blue-stained SDS–polyacrylamide gel of the purified PP2A dimer obtained from this procedure is indicated. Subunit identities were confirmed by western blotting with polyclonal antibodies as well as by microsequence analysis.

Fig. 5. Methylation increases the affinity of AC dimers for B_α. Size exclusion HPLC and western blot analysis of complex formation between B_α and methylated (A) or unmethylated (B) AC dimers. AC dimers (120 µM) were incubated for 30 min at 32°C in the presence (A) or absence (B) of 6.25 µM PPMT plus 50 µM AdoMet. An equal volume of 120 µM B_α was then added and the resulting mixtures were separated by size exclusion chromatography using a Superose 12 HR column. Samples to be analyzed were prepared in 200 µl of column buffer (50 mM MOPS pH 7.2, 0.20 M NaCl, 1.0 mM EDTA, 1.0 mM DTT, 5% glycerol) containing the carrier proteins β-amylase, alcohol dehydrogenase and ovalbumin (100 µg each). The samples were chromatographed at a flow rate of 0.40 ml/min and the indicated 0.40 ml fractions were collected and analyzed by western blotting using a mixture of anti-A, -B_α and -C antibodies. Control experiments in which AC dimer and B_α were incubated with either AdoMet or PPMT alone (not shown) gave the same results as those obtained with unmethylated AC. The elution profiles of free B_α, AC dimer, AB_αC trimer and molecular weight standards are indicated.

Fig. 2. Methyltransferase binding to PP2A AC dimers. Protein samples were prepared in 200 µl of column buffer (50 mM MOPS pH 7.2, 0.2 M NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol) containing the carrier proteins β-amylase, alcohol dehydrogenase and ovalbumin (100 µg each). The samples were chromatographed at a flow rate of 0.40 ml/min on a Superose 12 column and fractions were collected every 30 s. Methyltransferase activity was measured by adding [³H]AdoMet plus excess AC to aliquots of each fraction (filled circles); AC–PPMT complexes were detected by measuring [³H]methyl-labeled AC after incubation with [³H]AdoMet (open circles). (A) A mixture of purified AC (100 pmol, 10 µg) and PPMT (10 pmol, 0.4 µg) was incubated for 5 min at 24°C. PPMT activity and AC methyl-accepting activity co-eluted at an apparent molecular weight corresponding to AC–PPMT trimers (∼150 kDa). (B) After incubation for 15 min in the presence of unlabeled 50 µM AdoMet, the AC–PPMT complex dissociated into free PPMT plus methylated AC. The latter is not subject to further methylation by [³H]AdoMet and therefore has no methyl-accepting activity. Unmethylated AC remained associated with PPMT. (C) Addition of 1.0 µM okadaic acid caused the dissociation of PPMT from AC. No AC–PPMT complex was detected in this experiment.