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

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, α and β. 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, AαCα, AαCβ, AβCα, and AβCβ, and each core enzyme could in theory give rise to multiple holoenzymes. Differences between Cα and Cβ in expression and subcellular localization during early embryonic development have been reported, which imply that Cα and Cβ have different functions. To address the question of whether these differences might be caused by enzymatic differences between Cα and Cβ, we purified six holoenzymes composed of AαCα or AαCβ core enzyme and B subunits from the B, B′, or B” families. In addition, we purified four holoenzymes composed of AβCα or AβCβ and B′α1 or B“/PR72. The phosphatase activity of each purified form was assayed using myelin basic protein and histone H1 as substrates. We found that Cα and Cβ have identical phosphatase activities when associated with the same A and B subunits. Furthermore, no difference was found between Cα and Cβ in binding A or B subunits. These data suggest that the distinct functions of Cα and Cβ are not based on differences in enzymatic activity or subunit interaction. The implications for the relationship between the structure and function of Cα and Cβ are discussed. 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, α and β. 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, AαCα, AαCβ, AβCα, and AβCβ, and each core enzyme could in theory give rise to multiple holoenzymes. Differences between Cα and Cβ in expression and subcellular localization during early embryonic development have been reported, which imply that Cα and Cβ have different functions. To address the question of whether these differences might be caused by enzymatic differences between Cα and Cβ, we purified six holoenzymes composed of AαCα or AαCβ core enzyme and B subunits from the B, B′, or B” families. In addition, we purified four holoenzymes composed of AβCα or AβCβ and B′α1 or B“/PR72. The phosphatase activity of each purified form was assayed using myelin basic protein and histone H1 as substrates. We found that Cα and Cβ have identical phosphatase activities when associated with the same A and B subunits. Furthermore, no difference was found between Cα and Cβ in binding A or B subunits. These data suggest that the distinct functions of Cα and Cβ are not based on differences in enzymatic activity or subunit interaction. The implications for the relationship between the structure and function of Cα and Cβ are discussed. protein phosphatase 2A hemagglutinin myelin basic protein dithiothreitol Triton X-100 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 (Cα and Cβ), two isoforms of the regulatory/scaffolding A subunit (Aα and Aβ), 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. 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Google Scholar 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 (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Google Scholar). In addition, four core enzymes, AαCα, AβCα, AαCβ, and AβCβ, could exist that lack a regulatory B subunit. Holoenzymes and core enzymes both exist in cells (2Kremmer E. Ohst K. Kiefer J. Brewis N. Walter G. Mol. Cell. Biol. 1997; 17: 1692-1701Google Scholar), 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 Cβ-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 (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Google Scholar, 3Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Google Scholar), 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. 4Myles T. Schmidt K. Evans D.R. Cron P. Hemmings B.A. Biochem. J. 2001; 357: 225-232Google Scholar for further references). Cα and Cβ 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 (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Google Scholar, 5Arino J. Woon C.W. Brantigan D.L. Miller T.B. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4252-4256Google Scholar). Cα and Cβ are both encoded by genes composed of seven exons and six introns (6Khew-Goodall Y. Mayer R.E. Maurer F. Stone S.R. Hemmings B.A. Biochemistry. 1991; 30: 89-97Google Scholar). 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. Cα is expressed at 10-fold higher levels than Cβ because of differences in transcriptional regulation (6Khew-Goodall Y. Mayer R.E. Maurer F. Stone S.R. Hemmings B.A. Biochemistry. 1991; 30: 89-97Google Scholar). 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 (7Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Google Scholar, 8Egloff M.P. Cohen P.T. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Google Scholar), 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 Cα and Cβ are not conserved between PP1, PP2A, and PP2B. Therefore, they are likely not involved in catalysis. Although very similar in structure, Cα and Cβ have different functions. Cα−/− mice are normal until embryonic day 5.5 but die on embryonic day 6.5, suggesting that Cα is required for mesoderm formation and gastrulation (9Götz J. Probst A. Ehler E. Hemmings B. Kues W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12370-12375Google Scholar). Furthermore, it has been demonstrated that in normal early embryonic development Cα is mainly associated with the plasma membrane, whereas Cβ is localized in the cytoplasm and in nuclei. Interestingly, E-cadherin, which forms a complex with Cα and β-catenin and is normally located at the plasma membrane, changes location to the cytoplasm in Cα−/−mice, suggesting that Cα but not Cβ mediates binding of the E-cadherin complex to the plasma membrane (10Götz J. Probst A. Mistl C. Nitsch R.M. Ehler E. Mech. Dev. 2000; 93: 83-93Google Scholar). It has been suggested that the difference between Cα and Cβ could be caused either by a targeting signal in Cα that directs it to the plasma membrane or that a regulatory B subunit associated with Cα but not Cβ is responsible for targeting the Cα-containing holoenzyme to the plasma membrane where it is involved in Wnt signaling (10Götz J. Probst A. Mistl C. Nitsch R.M. Ehler E. Mech. Dev. 2000; 93: 83-93Google Scholar). In the present study, we compared the properties of 10 Cα- and Cβ-containing holoenzymes purified from cells transfected with epitope-tagged Cα or Cβ, Aα or Aβ, and Bα, B′α1, 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 (11Grussenmeyer T. Carbone-Wiley A. Scheidtmann K.H. Walter G. J. Virol. 1987; 61: 3902-3909Google Scholar) that were subsequently identified as the A and C subunits of PP2A (12Pallas D.C. Shahrik L.K. Martin B.L. Jaspers S. Miller T.B. Brautigan D.L. Roberts T.M. Cell. 1990; 60: 167-176Google Scholar, 13Walter G. Ruediger R. Slaughter C. Mumby M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2521-2525Google Scholar). We demonstrated that Cα and Cβ 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 Cα and Cβ during early embryonic development and in subcellular localization are unlikely because of differences in subunit interaction or enzymatic activity. Rat monoclonal antibody 6G3 recognizing both Aα and Aβ and mouse monoclonal anti-peptide antibodies anti-EE and anti-KT3 were described previously (2Kremmer E. Ohst K. Kiefer J. Brewis N. Walter G. Mol. Cell. Biol. 1997; 17: 1692-1701Google Scholar, 14Grussenmeyer T. Scheidtmann K.H. Hutchinson M.A. Eckhart W. Walter G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7952-7954Google Scholar, 15MacArthur H. Walter G. J. Virol. 1984; 52: 483-491Google Scholar). IgG fractions of the above antibodies were isolated by standard procedures using protein G-Sepharose CL-6B beads (GammaBind Plus from AmershamBiosciences). 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, AmershamBiosciences) using dimethylpimelimidate as described (16Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988: 310Google Scholar). Vectors encoding Aα and Aβ tagged at the C terminus with EE (EEEEYMPME), vectors encoding Bα, B′α1, and B“/PR72 tagged at the C terminus with KT3 (KPPTPPPEPET), and vectors encoding Cα and Cβ tagged at the N terminus with HA (YPYDVPDYA) were described previously (17Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Google Scholar). 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 Aα and Aβ 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-β-d-galactopyranoside (X-gal) of fixed cells co-transfected with a β-galactosidase vector (18Ruediger R. Brewis N. Ohst K. Walter G. Virology. 1997; 238: 432-443Google Scholar). 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, mm mm 1 mm and 50 for 1.5 × 106 293 cells were plated per 10-cm and transfected as described h after transfection, cells were with by one with Dulbecco's modified Eagle's medium The cells were for h in of medium of 10% fetal bovine Dulbecco's modified Eagle's Dulbecco's modified Eagle's 20 mm and 48 h the 293 cells or with were with on and for 10 with μl per 10-cm of TX-100 buffer mm 1 mm and 50 The were at × at for and the were for For the μl of cell was with 60 μl of protein G-Sepharose coupled with anti-EE μg of for 1 h at the were with TX-100 buffer and in μl of buffer (2% TX-100, 10 pH 7.5, mm 1 mm EE at for 30 the protein G-Sepharose was and the was to a of and using 20 μl of anti-KT3 protein G-Sepharose μg of and 100 μl of buffer KT3 The protein complex in 100 μl of buffer was to the with 10 μl of 1 h of μl of protein G-Sepharose was and the was for a with TX-100 the with holoenzymes was of it was for the phosphatase and the other was in SDS-PAGE sample buffer for Western and To purification and subunit from each were on SDS-PAGE by Western blotting as described (17Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Google Scholar). basic protein was with protein following the To histone 1 of histone H1 (Roche Molecular was at 30 in 1 of buffer of B mm and The was by the of to a of and on for 30 The was at × at for 10 The histone H1 was with in μl of buffer mm pH 1 mm mm 0.01% and 1 of buffer mm pH 1 mm mm 0.01% to The of for was and for histone The of the were to 50 for and for histone H1 with to the activity was determined by the of the one of the PP2A holoenzymes to the protein G-Sepharose was in μl of buffer and with 10 μl of to a of 10 with to the The phosphatase was carried out at 30 for 10 by of the with 10% The was in a The activity of PP2A was to the of the of holoenzyme in the phosphatase was by Western blotting of the of the purified holoenzymes with 6G3 and the A subunit after the purification to a of A. The of the purified holoenzyme was to the of The phosphatase with histone H1 were carried out that the of histone H1 substrate was with to the and was by at a of the consecutive of two monoclonal anti-peptide antibodies, anti-EE and with monoclonal antibodies a form of PP2A a for the enzyme we from 293 cells transfected with vectors encoding EE-tagged Aα B“/PR72 and Cα The of the was to purify holoenzyme of other form of PP2A. in the cell a of core and holoenzymes composed of and subunits. In the with anti-EE and the of the with EE all forms of enzyme not are whereas as as core and holoenzymes composed of with or tagged B or C are In the with anti-KT3 and with KT3 holoenzymes composed of and or the C subunits (Cα or are In the with the holoenzyme C is and only the this approach highly purified enzyme is in 293 cells transfected with vectors were with from h was as described The numerous proteins including which is 1) when compared with and were with anti-EE and with EE purification that be with was in a large because of the of “free” the holoenzyme in the a large number of cellular proteins is after a 10 and The with anti-KT3 and the with KT3 the of the with the was only than after the and with and One difference was the of two and that might represent forms of C subunit to be at this that is suggesting that it might be modified by the high of of the some proteins are present at they were not found in the they might represent proteins associated with the The for and were by using a Molecular and and were for the number of in each subunit, the of cell and the of protein for each of The of that in the of the of the A, B, and C subunits not The large of after the purification is caused by high The higher of Aα in the from higher as compared with Cα and B“/PR72. The of holoenzyme after the was compared with the not both during the immunoprecipitations and but the could be by each the present one 10-cm with 1.5 × 106 293 cells μg of total protein and of purified holoenzyme after the with more of protein the described in the present has several It is and very of and be carried out in to purify at six forms of holoenzymes. the Cα and Cβ differ only by eight amino acids, seven of which are located within the 30 amino acids (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Google Scholar). We whether the differences between the two isoforms might result from differences in phosphatase activity or from differences in to with the other subunits. We previously demonstrated that Cα and Cβ to Aα (17Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Google Scholar). They to Aβ, binding to Aβ is than to Aα (17Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Google Scholar). Cα- and Cβ-containing core enzymes differ in B subunit binding has not been studied. To address this we expressed Aα with Cα or Cβ and Bα, B′α1, or B“/PR72 in 293 cells and carried out purification of holoenzymes. described Aα was Cα and Cβ were and the B subunits were tagged with Each was by Western blotting with 6G3 recognizing tagged and Aα and Aβ, anti-KT3 recognizing tagged Bα, B′α1, and and recognizing tagged Cα and a the purification was carried out with from cells transfected with vector The were to of Cα and in similar of Cα or Cβ were associated with Aα and Bα, B′α1, or B“/PR72 at all of For in the with similar of Cα and Cβ were with and and In the with similar of Bα, B′α1, or B“/PR72 were with Cα and Cβ that in cell was the most highly expressed B subunit by B′α1 and and 1 and the of holoenzymes after the and were the and The A subunit signal from A subunit by the 6G3 this signal after with anti-EE antibodies To signal fractions of the were for Western of the of the of the and of the The were to (see the to We Cα or Cβ with Aβ and B′α1 or B“/PR72 and purified the and holoenzymes. in both the AβCα and AβCβ core enzymes to B′α1 or B“/PR72. The subunit was not in this as previously (17Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Google Scholar), it not to core The from these is that the core enzymes AαCα and AαCβ on the one and AβCα and AβCβ on the other are in to B subunits. We whether AαCα and AαCβ differ in phosphatase activity when associated with Bα, B′α1, or B“/PR72 subunits. were carried out with two different by protein and histone H1 by B. These have been in the past to between the activities of different forms of PP2A R. K. Walter G. Mumby Mol. Cell. Biol. 1991; Scholar). in the purified holoenzymes and have the same phosphatase activities with or histone H1 as substrates. and have the same activities or histone H1 Furthermore, and have activities both substrates. The with histone H1 and that the for activity are Bα, B′α1, and with histone H1 the holoenzyme the activity by the and holoenzymes We compared with and with and found no differences in activity between the Cα- and Cβ-containing However, holoenzymes are because of the affinity of Aβ for C and B subunits (17Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Google Scholar), and they in the of Therefore, the after the C subunit and core and the phosphatase activities obtained were not of the holoenzymes not The of our was to the marked functional differences between Cα and Cβ (9Götz J. Probst A. Ehler E. Hemmings B. Kues W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12370-12375Google Scholar, J. Probst A. Mistl C. Nitsch R.M. Ehler E. Mech. Dev. 2000; 93: 83-93Google Scholar) could be in of the differences in We Cα and Cβ might have different catalytic catalytic are It is that the sequence of 30 amino acids, which seven of the eight differences between Cα and Cβ and is located the catalytic (7Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Google Scholar, 8Egloff M.P. Cohen P.T. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Google Scholar), the catalytic activity in a similar as the C terminus of activity (see Ref. B. Cell. Scholar for review). Cα and Cβ might with A or B subunits. For the N of Cα and Cβ are involved in A subunit Cα and Cβ might differ in core enzyme This in might the of holoenzymes, which on the of core Cα and Cβ to the A subunits, they might differ in B binding that the 30 amino acids a in B Cα and Cβ with A and B subunits in identical they might differ in to other regulatory In the present we that Cα and Cβ have identical phosphatase activities when associated with the same A and the same type of B subunit. We that Cα and Cβ are identical in to with A and B subunits. These the two above They are with the for one amino Cα and Cβ are identical in catalytic (7Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Google Scholar, 8Egloff M.P. Cohen P.T. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Google Scholar). We that the activities of Cα and Cβ might differ we other substrates. It is possible that other regulatory subunits Bα, B′α1, and B“/PR72 exist that to Cα and we the that the 30 amino acids Cα- and Cβ-containing holoenzymes to different subcellular by with distinct For Cα may Cα-containing holoenzymes to the plasma membrane in early embryonic development (10Götz J. Probst A. Mistl C. Nitsch R.M. Ehler E. Mech. Dev. 2000; 93: 83-93Google Scholar). the other Cβ may to a or protein with (10Götz J. Probst A. Mistl C. Nitsch R.M. Ehler E. Mech. Dev. 2000; 93: 83-93Google Scholar). In a study, two holoenzymes either B or B“/PR72 were isolated from both enzymes Aα and it was that the isoforms of A or C not which B subunit with the core enzyme P. P. R.E. Goris J. J. W. Hemmings B.A. J. Biol. Scholar). However, the that both enzymes Aα and Cα is not because most cells and including at 10-fold more Aα and Cα than Aβ and Cβ (6Khew-Goodall Y. Mayer R.E. Maurer F. Stone S.R. Hemmings B.A. Biochemistry. 1991; 30: 89-97Google Scholar, B.A. C. Maurer F. P. Goris J. W. J. Stone S.R. Biochemistry. 1990; Scholar). Therefore, no and of AαCα with AβCα or of AαCβ with AβCβ in binding B subunits was In addition, as we Aβ is in binding B and C subunits than Aα (17Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Google Scholar). that the Cα subunit with proteins other than A and B subunits (see 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Google Scholar and 3Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Google Scholar for review). Cα to and might be involved in Wnt signaling by the of β-catenin W. F. J. Biol. Scholar). Cα to B cell protein K. J. Brautigan D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). Interestingly, not with the core suggesting that with the A subunit for binding to of Cα subunit binding are the C M.A. J. Biol. 2000; Scholar), the protein T. Nature. 1997; Scholar), and the N. S. C. Goris J. Hemmings B.A. J. Scholar). The is of because it was demonstrated that the binding is located within the 50 amino acids of In all other the binding have not been identified. It be of to out whether they are located within the 30 amino In addition, binding for Cβ to be identified. We Ruediger for and the We Mumby for Bα, B′α1, and Cα subunit and Hemmings for B“/PR72