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• W2065231473 abstract "Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. The ability of this widely distributed heterotrimeric enzyme to act on a diverse array of substrates is largely controlled by the nature of its regulatory B subunit. Only two gene families encoding endogenous B subunits have been cloned to date, although the existence of several additional regulatory subunits is likely. We have identified by two-hybrid interaction a new human gene family encoding PP2A B subunits. This family, denoted B56, contains three distinct genes, one of which is differentially spliced. B56 polypeptides co-immunoprecipitate with PP2A A and C subunits and with an okadaic acid-inhibitable, heparin-stimulated phosphatase activity. The three B56 family members are 70% identical to each other but share no obvious homology with previously identified B subunits. These phosphatase regulators are differentially expressed, with B56α and B56g highly expressed in heart and skeletal muscle and B56β highly expressed in brain. The identification of this novel phosphatase regulator gene family will facilitate future studies on the control of protein dephosphorylation and the role of PP2A in cellular function. Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. The ability of this widely distributed heterotrimeric enzyme to act on a diverse array of substrates is largely controlled by the nature of its regulatory B subunit. Only two gene families encoding endogenous B subunits have been cloned to date, although the existence of several additional regulatory subunits is likely. We have identified by two-hybrid interaction a new human gene family encoding PP2A B subunits. This family, denoted B56, contains three distinct genes, one of which is differentially spliced. B56 polypeptides co-immunoprecipitate with PP2A A and C subunits and with an okadaic acid-inhibitable, heparin-stimulated phosphatase activity. The three B56 family members are 70% identical to each other but share no obvious homology with previously identified B subunits. These phosphatase regulators are differentially expressed, with B56α and B56g highly expressed in heart and skeletal muscle and B56β highly expressed in brain. The identification of this novel phosphatase regulator gene family will facilitate future studies on the control of protein dephosphorylation and the role of PP2A in cellular function. Protein phosphatase 2A (PP2A) 1The abbreviations used are: PP2Aprotein phosphatase 2Abpbase pairPAGEpolyacrylamide gel electrophoresisHAinfluenza virus hemagglutinin epitopeHis-media lacking the amino acid histidineCMVcytomegalovirusmAbmonoclonal antibodykbkilobase(s). is a major intracellular phosphatase that regulates such diverse cellular processes as DNA replication, transcription, signal transduction, and intermediary metabolism(1Mumby M.C. Walter G. Physiol. Rev. 1993; 73: 673-699Crossref PubMed Scopus (629) Google Scholar, 2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar, 3Virshup D.M. Cegielska A. Russo A. Kelly T.J. Shaffer S. Adv. Protein Phosphatases. 1993; 7: 271-293Google Scholar). PP2A is a heterotrimer, containing A, B, and C subunits. The catalytic activity of PP2A resides in the C subunit, a 36-kDa protein encoded by two 97% identical genes. The C subunit binds stably to the carboxyl-terminal region of the A subunit, a 65-kDa rod-shaped polypeptide consisting of 15 imperfect repeats. The B subunits bind to the amino-terminal region of the A subunit (Fig. 1A) and determine the substrate specificity of the complex (4Ruediger R. Hentz M. Fait J. Mumby M. Walter G. J. Virol. 1994; 68: 123-129Crossref PubMed Google Scholar, 5Ruediger R. Roeckel D. Fait J. Bergqvist A. Magnusson G. Walter G. Mol. Cell. Biol. 1992; 12: 4872-4882Crossref PubMed Scopus (122) Google Scholar, 6Cegielska A. Shaffer S. Derua R. Goris J. Virshup D.M. Mol. Cell. Biol. 1994; 14: 4616-4623Crossref PubMed Scopus (100) Google Scholar, 7Ferrigno P. Langan T.A. Cohen P. Mol. Biol. Cell. 1993; 4: 669-677Crossref PubMed Scopus (91) Google Scholar, 8Agostinis P. Derua R. Sarno S. Goris J. Merlevede W. Eur. J. Biochem. 1992; 205: 241-248Crossref PubMed Scopus (85) Google Scholar). Three distinct B subunits have been biochemically isolated from a variety of mammalian tissues(9Mumby M.C. Russell K.L. Garrard L.J. Green D.D. J. Biol. Chem. 1987; 262: 6257-6265Abstract Full Text PDF PubMed Google Scholar, 10Mayer R.E. Hendrix P. Cron P. Matthies R. Stone S.R. Goris J. Merlevede W. Hofsteenge J. Hemmings B.A. Biochemistry. 1991; 30: 3589-3597Crossref PubMed Scopus (171) Google Scholar, 11Healy A.M. Zolnierowicz S. Stapleton A.E. Goebl M. DePaoli-Roach A.A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 5767-5780Crossref PubMed Scopus (230) Google Scholar, 12Hendrix P. Mayer-Jaekel R.E. Cron P. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 15267-15276Abstract Full Text PDF PubMed Google Scholar, 13Zolnierowicz S. Csortos C. Bondor J. Verin A. Mumby M.C. DePaoli-Roach A.A. Biochemistry. 1994; 33: 11858-11867Crossref PubMed Scopus (95) Google Scholar, 14DePaoli-Roach A.A. Park I.K. Cerovsky V. Csortos C. Durbin S.D. Kuntz M.J. Sitikov A. Tang P.M. Verin A. Zolnierowicz S. Adv. Enzyme Regul. 1994; 34: 199-224Crossref PubMed Scopus (125) Google Scholar), and several studies have suggested the existence of additional B subunits(15Dobrowsky R. Kamibayashi C. Mumby M. Hannun Y. J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar, 16Wadzinski B.E. Wheat W.H. Jaspers S. Peruski Jr., L.F.J. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (287) Google Scholar). Additionally, several DNA tumor viruses encode polypeptides that can function as PP2A B subunits(17Walter G. Ferre F. Espiritu O. Carbone-Wiley A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8669-8672Crossref PubMed Scopus (57) Google Scholar, 18Walter G. Ruediger R. Slaughter C. Mumby M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2521-2525Crossref PubMed Scopus (159) Google Scholar, 19Pallas D.C. Shahrik L.K. Martin B.L. Jaspers S. Miller T.B. Brautigan D.L. Roberts T.M. Cell. 1990; 60: 167-176Abstract Full Text PDF PubMed Scopus (459) Google Scholar). The B subunits purified to date migrate in SDS-PAGE with the apparent molecular masses of 54 kDa (B54), 55 kDa (B55), and 72 kDa (B72). Three cDNAs encoding 55-kDa B subunits have been identified(10Mayer R.E. Hendrix P. Cron P. Matthies R. Stone S.R. Goris J. Merlevede W. Hofsteenge J. Hemmings B.A. Biochemistry. 1991; 30: 3589-3597Crossref PubMed Scopus (171) Google Scholar, 11Healy A.M. Zolnierowicz S. Stapleton A.E. Goebl M. DePaoli-Roach A.A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 5767-5780Crossref PubMed Scopus (230) Google Scholar, 20Pallas D.C. Weller W. Jaspers S. Miller T.B. Lane W.S. Roberts T.M. J. Virol. 1992; 66: 886-893Crossref PubMed Google Scholar); the B55 family members are 80-90% identical, and their level of expression varies by tissue type. A cDNA encoding the 72-kDa B subunit has also been cloned, and a splice variant encoding a 130-kDa protein has been identified (12Hendrix P. Mayer-Jaekel R.E. Cron P. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 15267-15276Abstract Full Text PDF PubMed Google Scholar). The sequence of the 54-kDa B subunit cDNA has not yet been reported. Interestingly, the amino acid sequences of the B55 and B72 subunits and the viral PP2A binding proteins show little homology to each other; thus, no common motif mediating the interaction of the B subunit with the PP2A A and C subunits has been discovered. protein phosphatase 2A base pair polyacrylamide gel electrophoresis influenza virus hemagglutinin epitope media lacking the amino acid histidine cytomegalovirus monoclonal antibody kilobase(s). Heterotrimeric PP2A enzymes with different B subunits have distinct substrate specificities(7Ferrigno P. Langan T.A. Cohen P. Mol. Biol. Cell. 1993; 4: 669-677Crossref PubMed Scopus (91) Google Scholar, 8Agostinis P. Derua R. Sarno S. Goris J. Merlevede W. Eur. J. Biochem. 1992; 205: 241-248Crossref PubMed Scopus (85) Google Scholar, 21Agostinis P. Goris J. Pinna L.A. Marchiori F. Perich J.W. Meyer H.E. Merlevede W. Eur. J. Biochem. 1990; 189: 235-241Crossref PubMed Scopus (60) Google Scholar), a mode of phosphatase regulation that has important functional effects. For example, PP2A can turn SV40 DNA replication on or off, depending on the type of B subunit in the holoenzyme(6Cegielska A. Shaffer S. Derua R. Goris J. Virshup D.M. Mol. Cell. Biol. 1994; 14: 4616-4623Crossref PubMed Scopus (100) Google Scholar, 22Virshup D.M. Kauffman M.G. Kelly T.J. EMBO J. 1989; 8: 3891-3898Crossref PubMed Scopus (70) Google Scholar). Viral replication in vitro is controlled by the activity of viral initiator phosphoprotein, SV40 large T antigen. The heterotrimeric form of PP2A containing B55 removes a phosphoryl group from threonine 124 (a cyclin-dependent kinase site) (23McVey D. Ray S. Gluzman Y. Berger L. Wildeman A. Marshak D. Tegtmeyer P. J. Virol. 1993; 67: 5206-5215Crossref PubMed Google Scholar) and inactivates T antigen's ability to initiate SV40 DNA replication, while the PP2A heterotrimer containing B72 removes inhibitory phosphoryl groups from serines 120 and 123 (casein kinase I sites) (24Cegielska A. Virshup D.M. Mol. Cell. Biol. 1993; 13: 1202-1211Crossref PubMed Scopus (50) Google Scholar, 25Cegielska A. Moarefi I. Fanning E. Virshup D.M. J. Virol. 1994; 68: 269-275Crossref PubMed Google Scholar) and activates SV40 DNA replication(6Cegielska A. Shaffer S. Derua R. Goris J. Virshup D.M. Mol. Cell. Biol. 1994; 14: 4616-4623Crossref PubMed Scopus (100) Google Scholar). An additional role of B subunits may be to act as targeting subunits that direct PP2A to specific subcellular locations. This method of regulation has been most clearly demonstrated for protein phosphatase 1, where the catalytic subunit is localized to its substrates, e.g. phosphorylase, phosphorylase kinase, and glycogen synthase, by association with a specific glycogen-binding subunit(2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar). Similarly, PP2A-B55α has recently been shown to associate with microtubules(26Sontag E. Nunbhakdi-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar). While PP2A activity has also been found in membrane and nuclear fractions, the B subunits of PP2A that direct the heterotrimer to these sites have not yet been identified. This current study was designed to identify novel PP2A B subunits. Using the yeast two-hybrid method (27Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Abstract Full Text PDF PubMed Scopus (526) Google Scholar) with the PP2A A subunit as bait, we identified a novel gene family encoding three polypeptides with a predicted size of approximately 56 kDa that were 70% identical to each other but with no significant similarity to either B55 or B72. Full-length polypeptides expressed in 293 cells bound to PP2A A and C subunits and co-immunoprecipitated with a heparin-stimulated, okadaic acid-inhibited phosphorylase phosphatase activity. Northern blots of human tissue showed that these genes have tissue-specific expression patterns, with two isoforms highly expressed in heart and skeletal muscle and one highly expressed in the brain. These results are further evidence that protein serine/threonine phosphatase diversity is generated in large part by association of a common catalytic subunit with an increasing array of regulatory or targeting subunits. A two-hybrid screen based on the method of Fields and Song (28Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4880) Google Scholar) (Fig. 1) was performed using the Saccharomyces cerevisiae strain L40(MATa, his3Δ200, trp1-901, leu2-3, 112, ade2, LYS::(lexAop)4-HIS, URA3::(lexAop)8-lacZ (constructed by S. Hollenberg). AMR70 (MATα, his3, lys2, trp1, leu2, URA::(lexAop)8-lacZ) (constructed by R. Sternglanz, the gift of S. Hollenberg) was used in mating assays to test for specific interactions. Untransformed yeast were grown in YPD, and transformed yeast were grown in minimal media with necessary supplements(29Rose M.D. Winston F. Heiter P. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). The HeLa cDNA library in pGAD-GH (with the selectable marker LEU2) contains inserts averaging 1500 bp in length and was the gift of Greg Hannon. pGAD-GH expresses inserts as fusion proteins with the transcriptional activation domain of GAL4 (Fig. 1A)(30Hannon G.J. Demetrick D. Beach D. Genes & Dev. 1993; 7: 2378-2391Crossref PubMed Scopus (405) Google Scholar). All the baits and positive controls used in the two-hybrid method were cloned by use of polymerase chain reaction using Taq polymerase. High levels of substrate (~100 ng) and low numbers of cycles(10Mayer R.E. Hendrix P. Cron P. Matthies R. Stone S.R. Goris J. Merlevede W. Hofsteenge J. Hemmings B.A. Biochemistry. 1991; 30: 3589-3597Crossref PubMed Scopus (171) Google Scholar, 11Healy A.M. Zolnierowicz S. Stapleton A.E. Goebl M. DePaoli-Roach A.A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 5767-5780Crossref PubMed Scopus (230) Google Scholar, 12Hendrix P. Mayer-Jaekel R.E. Cron P. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 15267-15276Abstract Full Text PDF PubMed Google Scholar, 13Zolnierowicz S. Csortos C. Bondor J. Verin A. Mumby M.C. DePaoli-Roach A.A. Biochemistry. 1994; 33: 11858-11867Crossref PubMed Scopus (95) Google Scholar, 14DePaoli-Roach A.A. Park I.K. Cerovsky V. Csortos C. Durbin S.D. Kuntz M.J. Sitikov A. Tang P.M. Verin A. Zolnierowicz S. Adv. Enzyme Regul. 1994; 34: 199-224Crossref PubMed Scopus (125) Google Scholar, 15Dobrowsky R. Kamibayashi C. Mumby M. Hannun Y. J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar) were used to minimize inadvertent mutagenesis. Polymerase chain reaction primers (Table 1) added appropriate restriction sites for in-frame insertion into the polycloning sites of pBTM116 or pGAD-GH.Tabled 1 Open table in a new tab The bait plasmid was based on pBTM116 (originally constructed by P. Bartel and S. Fields; with the selectable marker TRP1 and modified by the insertion of the ADE2 gene (31Stotz A. Linder P. Gene (Amst.). 1990; 95: 91-98Crossref PubMed Scopus (197) Google Scholar) in the PvuII site) and expresses LexA fused to the full-length human PP2A Aα subunit(5Ruediger R. Roeckel D. Fait J. Bergqvist A. Magnusson G. Walter G. Mol. Cell. Biol. 1992; 12: 4872-4882Crossref PubMed Scopus (122) Google Scholar, 18Walter G. Ruediger R. Slaughter C. Mumby M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2521-2525Crossref PubMed Scopus (159) Google Scholar). Expression of this construct, LexA-65A, was confirmed by immunoprecipitation of the fusion protein from [35S]methionine-labeled yeast extracts with an antibody to the PP2A A subunit. Constructs encoding mutant A subunits were also prepared to facilitate classification of isolated clones by the mating assay. These included COOH-terminal truncations A397 (LexA-A397) and A315 (LexA-A315) and the 12/5 loop substitution mutant (LexA-SUB), all described by Ruediger et al.(4Ruediger R. Hentz M. Fait J. Mumby M. Walter G. J. Virol. 1994; 68: 123-129Crossref PubMed Google Scholar, 5Ruediger R. Roeckel D. Fait J. Bergqvist A. Magnusson G. Walter G. Mol. Cell. Biol. 1992; 12: 4872-4882Crossref PubMed Scopus (122) Google Scholar) (Fig. 1A). The 12/5 (LexA-SUB) loop mutant was generated by site-directed mutagenesis; this mutation has been previously shown to eliminate B55 and SV40 small t antigen binding to the A subunit. Human B72 and C subunit genes were cloned into pGAD-GH in frame with the activation domain of GAL4 for use in control experiments (GAL4-B72, GAL4-C). Rat B55α cDNA (20Pallas D.C. Weller W. Jaspers S. Miller T.B. Lane W.S. Roberts T.M. J. Virol. 1992; 66: 886-893Crossref PubMed Google Scholar) was cloned into the two-hybrid expression vector pVP16 (32Vojtek A. Hollenberg S. Cooper J. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar) for use in control experiments (pVP16-B55). 500 μg of HeLa cDNA plasmid was used for the large scale transformation of the L40 strain carrying the LexA-65A bait plasmid(33Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1776) Google Scholar), and interacting cDNA clones were selected by rapid growth on His- plates. Positive colonies were further screened for lacZ expression using the colony lift method (29Rose M.D. Winston F. Heiter P. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). lacZ+ yeast were then allowed to lose the bait plasmid by removal of selection (Trp+ Leu- media) and were identified by accumulation of a red metabolite due to loss of the ADE2 gene also carried on the bait plasmid. Yeast containing only the library plasmids encoding putative interacting proteins were then tested for specific interaction by mating to AMR70 yeast-carrying plasmids encoding LexA-65A, LexA-SUB, LexA-A315, LexA-397, or LexA-lamin (Fig. 1)(32Vojtek A. Hollenberg S. Cooper J. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar, 34Bartel P. Chien C.T. Sternglanz R. Fields S. BioTechniques. 1993; 14: 920-924PubMed Google Scholar). Full-length clones were isolated from a human fetal brain cDNA library in bacteriophage lambda by standard methods (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning; A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and sequenced on both strands using Sequenase 2.0 according to the manufacturer's instructions. The protein coding sequences of B55α(20Pallas D.C. Weller W. Jaspers S. Miller T.B. Lane W.S. Roberts T.M. J. Virol. 1992; 66: 886-893Crossref PubMed Google Scholar), B56α, and B56β were cloned using the indicated polymerase chain reaction primers into pCEP-4/Lerner, a CMV promoter-driven expression vector based on pCEP4 (Invitrogen) that encodes a hemagglutinin epitope tag (36Wilson I.A. Niman H.L. Houghton R.A. Cherenson A.R. Connolly M.L. Lerner R.A. Cell. 1984; 37: 767-778Abstract Full Text PDF PubMed Scopus (658) Google Scholar) at the amino terminus of the expressed polypeptide. 293 cells (human embryonic kidney cells transformed with adenovirus) grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) plus 10% supplemented calf serum were transfected using lipofectamine (Life Sciences) following the manufacturer's instructions. Using a plasmid that expresses lacZ from the CMV promoter, the transfection efficiency was determined to be about 40%. Cells were lysed 24-36 h after transfection on ice in 20 mM Tris-HCl, pH 7.5, 0.2% Nonidet P-40, 10% glycerol, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 1 mM benzamidine, and 2 μg/ml pepstatin. Lysates were then centrifuged at 14,000 × g for 3 min, and complexes were immunoprecipitated from the supernatant by the addition of 12CA5 mAb (a mouse monoclonal antibody that recognizes the hemagglutinin epitope) (36Wilson I.A. Niman H.L. Houghton R.A. Cherenson A.R. Connolly M.L. Lerner R.A. Cell. 1984; 37: 767-778Abstract Full Text PDF PubMed Scopus (658) Google Scholar) and protein A-agarose. For immunoblotting, washed immunoprecipitates were solubilized in 1 × SDS-PAGE loading buffer, separated by SDS-PAGE on 10% gels, transferred to nitrocellulose, then probed with rabbit anti-PP2A-A and PP2A-C antibodies and visualized by enhanced chemiluminescence (Amersham). Phosphatase activity in the immunoprecipitate was determined by quantitating the release of trichloroacetic acid-soluble 32P from [32P]phosphorylase(37Brautigan D.L. Shriner C.L. Methods Enzymol. 1988; 159: 339-346Crossref PubMed Scopus (72) Google Scholar). All phosphatase assays were performed in duplicate, and the amount of PP2A activity was calculated by subtracting radioactivity released from immunoprecipitates of untransfected cells or cells transfected with a HA-ERK1 construct. A multiple tissue human Northern blot (Clontech) was probed sequentially with 32P-labeled cDNAs encoding B56β (800-bp NcoI-EcoRI fragment), B56α (605-bp EcoRI fragment), and B56g (entire prey plasmid isolated by the two-hybrid method) and human β-actin (supplied by Clontech). Sequence analysis was performed with the Wisconsin package (38Program Manual for the Wisconsin Package 8. Genetics Computer Group, Inc., Madison, WI1994Google Scholar) and GenBank searches using the BLAST algorithm(39Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar). The A subunit of PP2A was expressed in S. cerevisiae strain L40 as a fusion protein with the DNA binding protein LexA (LexA-65A) and used as the bait for a two-hybrid screen (Fig. 1). Two-hybrid interactions in this strain activate transcription of the HIS3 and lacZ genes. In pilot experiments, plasmids expressing the fusion proteins VP16-B55, GAL4-B72, and GAL4-C subunit were constructed and tested for interaction with LexA-65A. The GAL4-B72 fusion protein interacted strongly and specifically with LexA-65A (Fig. 1B), demonstrating the feasibility of identifying PP2A B subunits with the two-hybrid method. A HeLa cell cDNA library in pGAD-GH was then screened for proteins that interact with the LexA-65A bait (Fig. 1). 17 × 106 transformants were plated on His- plates, and rapidly growing colonies were further screened by blue/white assay for lacZ expression and for specific interactions in a mating assay. 212 clones were isolated that interacted strongly and specifically with the A subunit. These 212 clones were separated into 16 groups based on dot blot hybridization and restriction digests of polymerase chain reaction-amplified inserts. The 5′- and 3′-ends of representative clones from each of the 16 groups were then sequenced. The three groups that are the subject of this report encoded polypeptides that are 70% identical to each other and are referred to as B56α, -β, and -g1. Five clones encoded B56α, two clones encoded B56β, and one clone encoded B56g1. To identify regions of the PP2A A subunit bait required for interaction with these putative B subunits, yeast strain AMR70 carrying mutant bait constructs (as described under “Experimental Procedures”) were mated to L40 yeast carrying only the prey plasmid; interaction was assessed by growth on His- media (Fig. 1). The B56 family of B subunits isolated in this screen did not interact with either the carboxyl-terminal truncated A subunits or the 12/5 loop mutant. This is similar to the result reported when B55 binding to PP2A AC complex was assessed in an in vitro assay(4Ruediger R. Hentz M. Fait J. Mumby M. Walter G. J. Virol. 1994; 68: 123-129Crossref PubMed Google Scholar, 5Ruediger R. Roeckel D. Fait J. Bergqvist A. Magnusson G. Walter G. Mol. Cell. Biol. 1992; 12: 4872-4882Crossref PubMed Scopus (122) Google Scholar). In contrast, GAL4-B72 interacted with a carboxyl-truncated A subunit, suggesting a different mode of interaction with the A subunit. None of the clones isolated in this two-hybrid screen encoded the PP2A C subunit, B55, or B72. This is consistent with the inability of the GAL4-C and VP16-B55 fusions to interact strongly with LexA-65A in control experiments and the reported absence of B72 mRNA in HeLa cells(12Hendrix P. Mayer-Jaekel R.E. Cron P. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 15267-15276Abstract Full Text PDF PubMed Google Scholar). Ruediger and co-workers (4Ruediger R. Hentz M. Fait J. Mumby M. Walter G. J. Virol. 1994; 68: 123-129Crossref PubMed Google Scholar, 5Ruediger R. Roeckel D. Fait J. Bergqvist A. Magnusson G. Walter G. Mol. Cell. Biol. 1992; 12: 4872-4882Crossref PubMed Scopus (122) Google Scholar) have demonstrated in in vitro studies that B55 binding to PP2A A subunit requires a C subunit binding site, suggesting that B55•C contacts are essential. Thus, the absence of human C subunit may have prevented formation of a stable two-hybrid complex between B55 and LexA-65A. Full-length cDNA clones were obtained for B56α and B56β by screening a human fetal brain cDNA library with 32P-labeled probes from the 5′-end of clones from the HeLa two-hybrid library. Two independent phage clones containing similar amounts of additional 5′-sequence were obtained for B56α, and 4 phage clones with additional 5′-sequence were obtained for B56β. Since the reading frame of each isolate was known from the two-hybrid screen, in-frame stop and start sites could be used to deduce putative protein coding regions. The sequence of B56α was determined using the phage clones for bases 1-456 and the HeLa two-hybrid library clone for bases 456-3120. B56β sequence was entirely determined from the phage cDNA clone. For B56g, only the 3′- and 5′-ends (350 bases) of the HeLa library clone were sequenced, and no attempt was made to find full-length clones. The full-length B56α cDNA is 3120 bp, with an open reading frame encoding a polypeptide of 56.1 kDa. The translational start was determined to be at nucleotide 572, since this was the first in-frame ATG and gives a start site offset only four amino acids from that of B56β. Additional ATG codons in the B56α sequence were either far downstream (252 bp) or out of frame. The B56β cDNA is 2450 bp, and encodes a polypeptide of 57.3 kDa. The putative initiator methionine for B56β at nucleotide 326 is the first in-frame ATG upstream of the fusion site seen in HeLa prey library clones and has an optimum context for translational initiation(40Kozak M. Annu. Rev. Cell Biol. 1992; 8: 197-225Crossref PubMed Scopus (418) Google Scholar). The only other in-frame ATG is at bp 230 but is followed by an in-frame stop codon at bp 242. We have not isolated a full-length cDNA encoding B56g; the 1447-bp clone obtained in the two-hybrid screen codes for a predicted protein of 51.5 kDa. The first 1298 bp of B56g1 are identical to a 3700-bp expressed sequence, HumORFY (GenBank accession number D26445)(41Miyajima N. Sazuka T. Tanaka A. Kawarabayashi Y. Nagase T. Ishikawa K. Seki T. Tabata S. DNA Res. 1994; 1: 27-35Crossref PubMed Scopus (272) Google Scholar). However, HumORFY, encoding a 56.5-kDa polypeptide, appears to be an alternatively spliced message, as it completely diverges from B56g1 7 codons upstream of the g1 stop site and encodes an additional 43 amino acids. This would predict that there are at least two splice variant forms of B56g, a conclusion also supported by Northern blot analysis (see Fig. 5 and below). The three B56 cDNAs encode polypeptides with 70% identity to each other, with divergence limited to the extreme amino- and carboxyl-terminal regions. The B56 genes are also homologous to the S. cerevisiae gene RTS1/SCS1 (see “Discussion”). However, the predicted B56 amino acid sequences show no obvious similarity to previously cloned B subunits (Fig. 2).Figure 2The B56 family contains closely related proteins. B56 is encoded by one yeast and three human cDNAs with 70% amino acid identity. Predicted amino acid sequences were aligned using the Genetics Computer Group PILEUP program (38Program Manual for the Wisconsin Package 8. Genetics Computer Group, Inc., Madison, WI1994Google Scholar) and displayed using SeqVu(45Gardner J. SeqVu 1.0. Garvan Institute of Medical Research, Sydney, Australia1995Google Scholar). Only residues 219-758 of RTS1 are shown. Amino acid identities are shaded, and similarities are boxed; gaps introduced to optimize the alignments are indicated by a dash.View Large Image Figure ViewerDownload Hi-res image Download (PPT) By definition, PP2A B subunits form stable heterotrimers with the A and C subunits of PP2A. To evaluate the ability of these putative B subunits to form stable complexes with PP2A A and C subunits in vivo, B56α and B56β polypeptide coding sequences were cloned with a 5′-extension encoding the hemagglutinin epitope into a CMV expression vector. To verify that these constructs indeed encoded proteins of the predicted molecular weight, cytosolic extracts from transfected cells were subject to SDS-PAGE and immunoblotting with the 12CA5 mAb (Fig. 3A). HA-B56α migrates slightly faster than HA-B55α, and HA-B56β migrates at about the same speed as HA-B55α. Having established that the constructs indeed produced soluble proteins of the expected size, extracts from transfected cells were subjected to immunoprecipitation with 12CA5 mAb. Immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with antibodies that recognize PP2A A and C subunits. As Fig. 3, B and C, demonstrates, HA-tagged B56α and B56β polypeptides co-immunoprecipitate with PP2A A and C subunits in transfected human cells, indicating that B56 forms a stable complex with PP2A in human cells. PP2A heterotrimers contain a catalytic (C) subunit whose phosphatase activity is (i) inhibited by nanomolar concentrations of okadaic acid and (ii) stimulated toward nonspecific substrates such as phosphorylase a by heparin (presumably by facilitating dissociation of the catalytic subunit from the A and B subunits(42Kamibayashi C. Estes R. Slaughter C. Mumby M. J. Biol. Chem. 1991; 266: 13251-13260Abstract Full Text PDF PubMed Google Scholar)). To determine whether the complex containing HA-B56 indeed contained active PP2A-like phosphatase, 12CA5 immunoprecipitates were incubated with 32P-labeled phosphorylase a (Fig. 4). HA-B56α and HA-B56β both co-immunoprecipitate with a phosphorylase phosphatase activity that is 50-80% inhibited by the inclusion of 1 nM okadaic acid (Fig. 4A). While both PP2A and PP1 can be inhibited by okadaic acid, PP1 is 10-100-fold less sensitive than PP2Ac, with an IC50 of 10-20 nM. Thus, inhibition by 1 nM okadaic acid strongly suggests that the phosphatase activity present in these assays is PP2A. Phosphorylase phosphatase activity was also increased 2-4-fold in the presence of 15 μg/ml heparin (Fig. 4B), a result expected for PP2A complexes. Thus, the data strongly suggest that the B56 polypeptides are part of a stable and active PP2A complex. Finally, to evaluate whether the B56 polypeptides bound to PP2A heterotrimers (forming heterotetramers) or formed novel heterotrimers, soluble extracts from transfected cells were analyzed by glycerol gradient velocity sedimentation followed by immunoblot analysis with 12CA5 mAb. The majority (>75%) of HA-B56α sedimented at the same velocity as HA-B55α and as aldolase, a 158-kDa protein (data not shown). These results are most consistent with the HA-B56 polypeptides forming a heterotrimeric complex with the PP2A A and C subunits. The previously cloned PP2A regulatory subunits have shown tissue-specific and developmentally regulated patterns of gene expression, with specific isoforms preferentially expressed in muscle and brain(10Mayer R.E. Hendrix P. Cron P. Matthies R. Stone S.R. Goris J. Merlevede W. Hofsteenge J. Hemmings B.A. Biochemistry. 1991; 30: 3589-3597Crossref PubMed Scopus (171) Google Scholar, 13Zolnierowicz S. Csortos C. Bondor J. Verin A. Mumby M.C. DePaoli-Roach A.A. Biochemistry. 1994; 33: 11858-11867Crossref PubMed Scopus (95) Google Scholar). To determine the expression pattern of the B56 genes, a multiple human tissue Northern blot was probed with sequences from the three B56 genes (Fig. 5). B56α mRNA is approximately 3500 nucleotides, with expression in all tissues examined but with the highest expression in heart and skeletal muscle. B56β mRNA is 3000 nucleotides with highest expression in brain. The apparent sizes of B56α and B56β mRNAs (3.5 and 3.0 kb) are 380-550 nucleotides longer than the cDNA clones we have obtained. B56g probe hybridizes with three mRNA species of 1.8, 2.1, and 4.4 kb. The 4.4-kb transcript is most highly expressed in heart and skeletal muscle, while the shorter transcripts are present largely in heart alone. The 4.4-kb species most likely corresponds to the 3700-bp HumORFY sequence, while the smaller more cardiac-specific mRNAs most likely correspond to the B56g1 clone isolated in this study. Reversible protein phosphorylation is one of the most widely utilized mechanisms to regulate cellular processes. The activity of many cellular enzymes is determined by the net protein phosphorylation level, which is determined by the balance of specific protein kinase and protein phosphatase activities. The substrate specificity of the regulatory protein kinases is determined largely by the diversity of their catalytic subunits, as demonstrated by the estimate that over 2000 distinct protein kinase catalytic subunits are encoded by the human genome(43Hunter T. Semin. Cell Biol. 1994; 5: 367-376Crossref PubMed Scopus (89) Google Scholar). However, there appears to be a more limited number of protein serine/threonine phosphatase catalytic subunits. Their substrate specificity is determined instead by association with a variety of regulatory and targeting subunits(2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar). We now report the identification, using the two-hybrid method, of a new gene family encoding PP2A regulatory subunits. These genes encode authentic PP2A B subunits based on the following evidence. First, all members of the gene family B56 (-α, -β, -g) interacted with a LexA-A subunit bait in the two-hybrid assay and failed to interact with irrelevant baits such as LexA-lamin. Second, PP2A A and C subunits were shown to associate with epitope-tagged B56α and β polypeptides in human cells in co-immunoprecipitation assays. Third, HA-B56α and -β co-immunoprecipitated with a phosphatase activity that was inhibited by 1 nmol of okadaic acid and enhanced by heparin, the results expected for a component of a PP2A complex. It is of note that the B56 gene family has no obvious similarity to previously identified gene families encoding polypeptides that bind to the amino-terminal end of the PP2A A subunit (B72/130, B55, B56, and polyoma and SV40 small t antigens). Thus, it has not been possible to define an interaction domain that is required for binding to the A subunit of PP2A. The B56 family has a very highly conserved (80% identical) central region, while both the carboxyl terminus and the amino terminus are significantly more divergent. This suggests that the conserved region is required for interaction with the A and possibly the C subunit, whereas the ends may perform different functions such as regulation of substrate specificity or intracellular location of PP2A. The lack of sequence similarity between the different B subunit families suggests that they each bind differently to the PP2A core A•C complex and exert their effects on substrate specificity in this manner. In support of this hypothesis, we note that test preys encoding B55, B72, and B56 fusion proteins each interacted differently in the two-hybrid screen when tested for association with the mutant and wild type A subunits (Fig. 1B). Thus, B72, unlike B55 and B56, interacted with carboxyl-truncated A subunit A397, and the B56 preys interacted with full-length A subunit significantly more strongly than did the B55 preys. B55 interaction with the A subunit is reportedly stabilized by the presence of the C subunit(4Ruediger R. Hentz M. Fait J. Mumby M. Walter G. J. Virol. 1994; 68: 123-129Crossref PubMed Google Scholar); this is supported by our finding that the B55 prey construct interacted only weakly with the A bait in yeast, where no mammalian C subunit exists. B72 and B56, like SV40 small t antigen, may bind more tightly to the A subunit and thus have less of a requirement for a B-C interaction. Additionally, within each B subunit family there are non-conserved sequences that may contribute to unique interactions with the PP2A A and C subunits or contribute to interactions with additional cellular proteins. For example, PP2A heterotrimers containing either B55α or B55β differ in their response to effector molecules such as protamine and heparin (44Kamibayashi C. Estes R. Lickteig R.L. Yang S.I. Craft C. Mumby M.C. J. Biol. Chem. 1994; 269: 20139-20148Abstract Full Text PDF PubMed Google Scholar). Differential expression in diverse tissues of B56 and B55 family members also implies that each isoform performs a specific function. A yeast gene 68% similar to the B56 gene family has been independently identified by two groups (Fig. 2)(24Cegielska A. Virshup D.M. Mol. Cell. Biol. 1993; 13: 1202-1211Crossref PubMed Scopus (50) Google Scholar, 25Cegielska A. Moarefi I. Fanning E. Virshup D.M. J. Virol. 1994; 68: 269-275Crossref PubMed Google Scholar). SCS1 (suppressor of chaperonin sixty-1), was isolated as a high copy suppressor of several temperature-sensitive alleles of hsp60 (a mitochondrial chaperonin)(24Cegielska A. Virshup D.M. Mol. Cell. Biol. 1993; 13: 1202-1211Crossref PubMed Scopus (50) Google Scholar). SCS1 in budding yeast is a cytosolic protein that when overexpressed appears to positively regulate transcription of additional chaperonin genes. The identical gene, termed RTS1, was cloned as a multicopy suppressor of ROX3, which encodes a transcriptional regulator involved in the response to anaerobic conditions. Thus, one function of the B56 homologues in yeast appears to be as regulators of the transcriptional response to environmental stress. The PP2A regulatory subunits we have identified are similar in size and tissue distribution to a previously purified regulator of PP2A with an Mr of 54,000 as assessed by SDS-PAGE(13Zolnierowicz S. Csortos C. Bondor J. Verin A. Mumby M.C. DePaoli-Roach A.A. Biochemistry. 1994; 33: 11858-11867Crossref PubMed Scopus (95) Google Scholar). If the B56 genes indeed encodes the 54-kDa protein, we would predict based on Northern blot analysis that the cardiac B subunit, also known as B‘, is encoded by B56α or B56g. PP2A has been implicated in the control of the cell cycle and the initiation of DNA replication. Since the PP2A A and C subunits are distributed in multiple cellular compartments, one function of a B subunit may be to target the heterotrimer to the nucleus, where it can participate in the regulation of these processes. The expression of the B55, B56, and B72 genes largely in terminally differentiated tissues such as heart and brain suggests that the PP2A B subunit that has direct involvement in cell cycle regulation or DNA replication remains to be identified. We thank Rolf Rüediger, Gernot Walter, David Pallas, Jozef Goris, and Brian Hemmings for plasmids encoding wild type and mutant PP2A subunits; Stan Hollenberg and Graeme Bolger for plasmids and yeast strains; Kimberly Fish for assistance with methods; and John Phillips, David Stillman, and Andrew Thorburn for helpful discussions." @default.
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• W2065231473 title "Identification of a New Family of Protein Phosphatase 2A Regulatory Subunits" @default.
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