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Functional Expression of Human PP2Ac in Yeast Permits the Identification of Novel C-terminal and Dominant-negative Mutant Forms

1999; Elsevier BV; Volume: 274; Issue: 34 Linguagem: Inglês

10.1074/jbc.274.34.24038

ISSN

1083-351X

Autores

David R. Evans, Timothy Myles, Jan Hofsteenge, Brian A. Hemmings,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function. The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function. protein phosphatase 2A protein phosphatase 1 PP2A catalytic subunit 5-fluoroorotic acid human PP2Ac yeast PP2Ac hemagglutinin epitope polymerase chain reaction protein A-Sepharose Tris-buffered saline temperature-sensitive kilobase pair polyacrylamide gel electrophoresis open reading frame Protein phosphatase 2A (PP2A)1 is a ubiquitous eukaryotic enzyme that is highly conserved between species (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2156) Google Scholar, 2Barton G.J. Cohen P.T.W. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar) and a member of the PPP family of protein Ser/Thr phosphatases that includes PP1 and calcineurin (3Barford D. Das A.K. Egloff M.P. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 133-164Crossref PubMed Scopus (570) Google Scholar). PP2A is implicated in diverse cellular processes (4Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (601) Google Scholar) and coordinates signal transduction through direct, regulatory interaction with protein kinases (5Evans D.R.H. Hemmings B.A. Nature. 1998; 394: 23-24Crossref PubMed Scopus (21) Google Scholar, 6Millward T.A. Zolnierwicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). PP2A exists as a holoenzyme in which the catalytic subunit (PP2Ac) binds a regulatory PR65/A subunit to form a core dimer, which associates with a large number of variable B subunits encoded by at least three gene families (7Mayer 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, 8Hendrix P. Mayer-Jaekel R.E. Cron P. Goris J. Hofsteenge J. Merlevede Hemmings B.A. J. Biol. Chem. 1993; 268: 15267-15276Abstract Full Text PDF PubMed Google Scholar, 9Hendrix P. Turowski P. Mayer-Jaekel R.E. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 7330-7337Abstract Full Text PDF PubMed Google Scholar, 10McCright B. Virshup D.M. J. Biol. Chem. 1995; 270: 26123-26128Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 11Csortos C. Zolnierowicz S. Bako E. Durbin S.D. DePaoli-Roach A.A. J. Biol. Chem. 1996; 271: 2578-2588Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Regulatory subunits of PP2A influence its substrate specificity and intracellular targeting (12Mayer-Jaekel R.E. Ohkura H. Ferrigno P. Andjelkovic N. Shiomi K. Uemura T. Glover D.M. Hemmings B.A. J. Cell Sci. 1994; 107: 2609-2616Crossref PubMed Google Scholar, 13McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 14Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar), and enzyme activity is modulated further by post-translational modification of the PP2Ac C terminus (4Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (601) Google Scholar). The budding yeast Saccharomyces cerevisiae possesses two proteins Pph21p and Pph22p similar to mammalian PP2Ac (15Sneddon A.A. Cohen P.T.W. Stark M.J.R. EMBO J. 1990; 9: 4339-4346Crossref PubMed Scopus (136) Google Scholar). The homologous PPH21 and PPH22 genes encoding yeast PP2Ac are functionally redundant because either can be deleted without effect. However, doubly deleting PPH21 and PPH22 causes severe growth inhibition and is lethal in the absence of PPH3, encoding a distantly related protein phosphatase that provides overlapping function (16Ronne H. Carlberg M. Hu G-Z. Nehlin J.O. Mol. Cell. Biol. 1991; 11: 4876-4884Crossref PubMed Scopus (166) Google Scholar). Conservation of PP2A extends to the holoenzyme because the S. cerevisiae Tpd3p (17van Zyl W Huang W. Sneddon A.A. Stark M. Camier S. Werner M. Marck C. Sentenac A. Broach J.R. Mol. Cell. Biol. 1992; 12: 4946-4959Crossref PubMed Scopus (146) Google Scholar), Cdc55p (18Healy 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 (228) Google Scholar), and Rts1p (19Evangelista C.C. Rodriguez-Torres A.M. Limbach M.P. Zitomer R.S. Genetics. 1996; 142: 1083-1093Crossref PubMed Google Scholar) are similar to the regulatory PR65/A, PR55/B, and PR61/B′ subunits, respectively, of mammalian PP2A, and Tpd3p and Cdc55p bind Pph21p/Pph22p in vivo (20Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (440) Google Scholar). This suggests that PP2A regulation is conserved between species, and consistent with this, a novel methyltransferase that targets the PP2Ac C-terminal leucine residue and modulates PP2A activity (21Favre B. Zolnierowicz S. Turowski P. Hemmings B.A. J. Biol. Chem. 1994; 269: 16311-16317Abstract Full Text PDF PubMed Google Scholar) is present in both higher eukaryotes and yeast (22Xie H. Clarke S. J. Biol. Chem. 1994; 269: 1981-1984Abstract Full Text PDF PubMed Google Scholar, 23Xie H. Clarke S. Biochem. Biophys. Res. Commun. 1994; 203: 1710-1715Crossref PubMed Scopus (34) Google Scholar). The role of PP2A has been studied biochemically, through the use of inhibitors and viral tumor antigens that target the enzyme specifically, and genetically, via inactivating subunit mutations. Moreover, the crystal structure of the PR65/A subunit of PP2A was recently solved (24Groves M.R. Hanlon N. Turowski P. Hemmings B.A. Barford D. Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Together these studies have implicated PP2A in processes including cell cycle regulation, cellular morphogenesis, protein synthesis, and viral replication (4Wera S. Hemmings B.A. Biochem. J. 1995; 311: 17-29Crossref PubMed Scopus (601) Google Scholar). Although the biochemical and functional properties of PP2A differ from those of protein phosphatase type 1 (PP1), the catalytic subunits of these enzymes share ∼46% amino acid sequence identity. The crystal structure of PP1γ1 has been solved (25Egloff M.-P. Cohen P.T.W. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (379) Google Scholar, 26Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (751) Google Scholar) and provides a framework for predicting the role of specific PP2Ac residues and interpreting the effect of changes generated by mutagenesis. In this study, we have functionally expressed human PP2Ac in S. cerevisiae and used this as a convenient system to generate novel mutations in human PP2Ac and analyze their effect on PP2A function in vivo. We have identified a number of novel, interfering mutant forms of PP2Ac and rationalized their effects using a model of PP2Ac structure based on that of PP1γ1. Yeast strains are described in Table I. Rich (yeast peptone medium, YPD), minimal (synthetic dextrose, SD), and 5-FOA media were described (27Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). Plasmids pBluescript II (pBS; Stratagene), pYES2 (Invitrogen), pYPGE2 (28Brunelli J.P. Pall M.L. Yeast. 1993; 9: 1299-1308Crossref PubMed Scopus (80) Google Scholar), pASZ11 (29Stotz A. Linder P. Gene (Amst.). 1990; 95: 91-98Crossref PubMed Scopus (197) Google Scholar), pRS314, pRS316, YEp351, YEp352, YCpDE1, and YCpAS6 (30Evans D.R.H. Stark M.J.R. Genetics. 1997; 145: 227-241Crossref PubMed Google Scholar) were described. DNA constructs were sequenced with dRhodamine dye terminators using Perkin-Elmer GeneAmp PCR system 2400, 9700 thermocycler and analyzed using an Applied Biosystems PRISM 377 sequencer. Pairwise alignment of human PP2Acα and yeast Pph22p was performed using ALIGN (Baylor College of Medicine).Table ISaccharomyces cerevisiae strains used in this studyStrainGenotypeDEY11-aEvans and Stark (30).MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YCpAS6: URA3 PPH22] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1-Cα1-cGenerated by plasmid shuffling in strain DEY1.MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YEpDE-PGK-Cα: TRP1 HA-PP2Acα] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1-P2MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YCpDE8: URA3 PPH22] [pYPGE2: TRP1] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1071-aEvans and Stark (30).MAT a pph22–172 pph21Δ1::HIS3 pph3Δ1::LYS2 lys2–952 W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1-Δ521-cGenerated by plasmid shuffling in strain DEY1.MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YEpDE-PGK-Δ52: TRP1 PPH22Δ52] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY111-aEvans and Stark (30).MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YCpDE1: TRP1 PPH22] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1-A3091-cGenerated by plasmid shuffling in strain DEY1.MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YEpDE-PGK-A309: TRP1 HA-PP2Ac-A309] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1-Δ3091-cGenerated by plasmid shuffling in strain DEY1.MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YEpDE-PGK-Δ309: TRP1 HsPP2Ac-Δ309] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1-c42m1-cGenerated by plasmid shuffling in strain DEY1.MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YEpDE-PGK-CHA: TRP1 HA-PP2Acα][YEpDE4–2m: URA3 myc-TPD3] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1-c3521-cGenerated by plasmid shuffling in strain DEY1.MAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2[YEpDE-PGK-CHA: TRP1 HA-PP2Acα] [YEp352:URA3] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY11–42mMAT a pph21::LEU2 pph22Δ1::HIS3 pph3Δ1::LYS2 [YCpDE1: TRP1 PPH22] [YEpDE4–2m: URA3 myc-TPD3] W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.Y1661-dFrom Steve Elledge, Baylor College of Medicine.ura3–52 leu2–3,112 his3Δ-200 ade2–101 trp1–901 gal4D LYS2::GAL::UAS::HIS3 RNR::GAL1::URA3 GAL1::laczDEY1031-aEvans and Stark (30).MAT a pph21Δ1::HIS3 pph3Δ::LYS2 lys2–952 W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEY1001-aEvans and Stark (30).MAT a pph22–12 pph21Δ1::HIS3 pph3Δ::LYS2 lys2–952 W3031-bIsogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.DEN2–3m[YEpDE2CαHA: URA3 HA-PP2Acα] [YEpDE3–2m: LEU2 myc-TPD3] INVSC11-eIsogenic with INVSC1 MAT α ura3–52 his3Δ1 trp1–289 leu2 (Invitrogen).DEN2512–3m[YEpDE-GAL-2512HA: URA3 HA-PP2Ac-2512][YEpDE3–2m: LEU2 myc-TPD3] INVSC11-eIsogenic with INVSC1 MAT α ura3–52 his3Δ1 trp1–289 leu2 (Invitrogen).DEN2–11[YEpDE2CαHA: URA3 HA-PP2Acα][YEpDE11: LEU2 TPD3] INVSC11-eIsogenic with INVSC1 MAT α ura3–52 his3Δ1 trp1–289 leu2 (Invitrogen).DEN2512–11[YEpDE-GAL-2512HA: URA3 HA-PP2Ac-2512][YEpDE11: LEU2 TPD3] INVSC11-eIsogenic with INVSC1 MAT α ura3–52 his3Δ1 trp1–289 leu2 (Invitrogen).DEY23m[pYES2: URA3] [YEpDE3–2m:LEU2 myc-TPD3] INVSC11-eIsogenic with INVSC1 MAT α ura3–52 his3Δ1 trp1–289 leu2 (Invitrogen).DEY211[pYES2:URA3] [YEpDE11: LEU2 TPD3] INVSC11-eIsogenic with INVSC1 MAT α ura3–52 his3Δ1 trp1–289 leu2 (Invitrogen).1-a Evans and Stark (30Evans D.R.H. Stark M.J.R. Genetics. 1997; 145: 227-241Crossref PubMed Google Scholar).1-b Isogenic with W303 ade2–1 ura3–1 his3–11 trp1–1 leu2–3,112, can1–100 ssd1-d2.1-c Generated by plasmid shuffling in strain DEY1.1-d From Steve Elledge, Baylor College of Medicine.1-e Isogenic with INVSC1 MAT α ura3–52 his3Δ1 trp1–289 leu2 (Invitrogen). Open table in a new tab Two forms of HsPP2Ac tagged with the HA epitope were used. A single HA tag was inserted downstream of the PP2Acα initiation codon, by amplifying the human cDNA (31Khew-Goodall Y. Mayer R.E. Maurer F. Stone S.R. Hemmings B.A. Biochemistry. 1991; 30: 89-97Crossref PubMed Scopus (83) Google Scholar) in the PCR using an appropriate forward primer. A Sal I/Kpn I HA-PP2Acα clone, or a frameshifted allele, HA-PP2Acα-FS lacking the second nucleotide of the ninth codon, was introduced between the PGK1 promoter/CYC1 terminator of vector pYPGE2 generating plasmids YEpDE-PGK-Cα and YEpDE-PGK-FS. A similar Nco I/Bam HI fragment was introduced into vector pACTII (P. Legrain, Pasteur Institute) fusing the HA-PP2Acα ORF to the Gal4 activation domain. A second sequence encoding an extended HA-PP2Acα ORF was used for HsPP2Ac immunoprecipitation experiments because it encodes native HsPP2Ac with an epitope accessible to the 12CA5 antibody (32Voorhoeve P.M. Hijmans E.M. Bernards R. Oncogene. 1999; 18: 515-524Crossref PubMed Scopus (89) Google Scholar).PP2Acα containing this tag was introduced into vectors pYPGE2 (YEpDE-PGK-CHA) and pYES2 (YEpDE2-CαHA), and similarly tagged PP2Ac-2512 and PP2Ac-118 alleles were introduced into pYES2 (YEpDE-GAL-2512HA and YEpDE-GAL-118HA). Mutations were generated in HA-PP2Acα by PCR amplification using QuickChange (Stratagene). Truncations of the HA-PP2Acα ORF were generated in vitro by PCR using a reverse primer that introduced a stop at codon 309 (PP2Ac-Δ309), 301 (PP2Ac-Δ300), or 67 (PP2Ac-Δ67) (codon numbering for untagged PP2Acα). PP2Acα mutant alleles were inserted between the Hin dIII/Bam HI sites of pYES2 and downstream of Sal I in pYPGE2. Plasmid DNA was prepared from strain DEY1-Cα using a Kristal extraction kit (Cambridge Molecular Technologies); six plasmids, recovered from independent ampr Escherichia coli colonies, were tested for a restriction pattern (Sal I/Kpn I or Xba I) identical to that of YEpDE-PGK-Cα. The nucleotide sequence of one plasmid was analyzed, and it encoded YEpDE-PGK-Cα. Cell extracts were prepared as described (33Cohen P. Schelling D.L. Stark M.J.R. FEBS Lett. 1989; 250: 601-606Crossref PubMed Scopus (90) Google Scholar). Protein transfer and Western blotting were as described (9Hendrix P. Turowski P. Mayer-Jaekel R.E. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 7330-7337Abstract Full Text PDF PubMed Google Scholar). Antibodies were diluted in TNPT (1.0% Nonidet P-40, 0.3% Triton X-100 in Tris-buffered saline (TBS, 150 mm NaCl, 50 mmTris-HCl, pH 7.5)) containing 7.5% powdered milk. Membranes were washed with TNPT. Detection was by goat anti-mouse IgG-HRP and ECL (Amersham Pharmacia Biotech). The PPH22 gene was PCR-amplified using a forward primer appropriate to generate pph22Δ52 encoding an initiation codon followed by codons 53–377 of PPH22 and a stop codon TAA. A pph22Δ52 1.0-kb Sal I/Kpn I fragment was fused to the PGK1 promoter in pYPGE2. A 3.4-kb genomic fragment encompassing the yeast TPD3 gene (17van Zyl W Huang W. Sneddon A.A. Stark M. Camier S. Werner M. Marck C. Sentenac A. Broach J.R. Mol. Cell. Biol. 1992; 12: 4946-4959Crossref PubMed Scopus (146) Google Scholar) was inserted into YEp351 (YEpDE11) and was template for a PCR generating a TPD3 ORF, flanked by its native promoter/terminator, with a double c-myc tag downstream of the initiator codon (myc-TPD3). A 3169-base pair myc-TPD3 Sac I/Sal I fragment was inserted into vectors pRS314, YEp351, and YEp352 (YCpDE3-2m, YEpDE3-2m, and YEpDE4-2m, respectively). The myc-TPD3 allele is functional because YCpDE3-2m rescued the ts growth defect of haploid tpd3Δ1::URA3 mutant cells (not shown). To precipitate HA-tagged proteins, PAS CL-4B (Amersham Pharmacia Biotech) equilibrated in 1× TBS was incubated at 4 °C with 12CA5 overnight and then washed extensively (six times with 10 volumes of TBS). Antibody-saturated beads (PAS-12CA5, 35 μl) were added to ∼100 μl of yeast extract, incubated at 4 °C for 2 h, and washed extensively. Phosphatase assays (50 μl) were performed on immune complexes according to Promega (kit V2460) measuring phosphate release over 10 min from a chemically synthesized phosphopeptide (RRA(pT)VA, where pT indicates phosphothreonine) with MnCl2 present at 1 mm. Assays were performed in duplicate on immune complexes prepared independently, and activity is expressed as units (1 unit = 1 μmol of phosphate hydrolyzed per min). For co-immunoprecipitations, cell extracts were prepared in Buffer A (100 mm Tris-HCl pH 7.5, 200 mm NaCl, 1.0 mm EDTA, pH 8.0, 0.5 mm dithiothreitol, 0.1% Nonidet P-40) with protease inhibitors. PAS-12CA5 (35 μl) was added to 1.0 mg of extract, incubated at 4 °C for 2–5 h, washed extensively with Buffer A, and boiled in 50 μl of Laemmli Buffer. A 970-base pair Hin dIII/Bam HI HA-PP2Acα fragment was inserted into the GAL1/CYC1 promoter/terminator cassette of pYES2, and this plasmid, YEpDE-CαHA, was cleaved with Afl II/Bst EII generating a gap within the PP2Acα ORF. In parallel, the intact insert in YEpDECαHA was amplified under mutagenic conditions for the PCR (0.2 ng/μl template DNA, 1× reaction buffer (Perkin-Elmer), 0.5 pmol/μl primer T7, 0.5 pmol/μl primer 20622 (5′-AGAGGATCCTTACAGGAAGTAGTCTGGGGTACGACGAGTAAC), 0.5 mm each dCTP and dTTP, 0.1 mm each dATP and dGTP, 3 mm MgCl2, 0.1 or 0.2 mmMnCl2, 5 units of Taq polymerase, 33 cycles of 94 °C for 1.5 min, 55 °C for 2.0 min, 72 °C for 3.0 min).PP2Acα mutant alleles were recovered by co-transforming (34Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (416) Google Scholar) cells (strain DEY1-Cα) with gapped plasmid DNA (0.5 μg) and mutagenic PCR product (200 ng). Ura+ transformants were selected on SD lacking uracil and then replica-plated to synthetic dextrose (SD) and synthetic galactose (SG) medium to identify plasmids containing dominant-negative alleles expressed from the GAL1 promoter. Plasmids recovered from cells capable of growth on glucose but not galactose were checked for gap repair by restriction analysis and reintroduced into DEY1-Cα to confirm growth inhibition on galactose. To test competition between PP2Acα wild type and mutant alleles, the 2.3-kb Sna BI/Eco RI fragment from YEpDE-CαHA, containing HA-PP2ACα fused to the GAL1 promoter, was inserted between the Sma I/Eco RI sites of vector pASZ11[CEN.ARS ADE2] and named YCpDE11-GAL-Cα. The HA-PP2Acα-FS allele was similarly inserted into pASZ11 (YCpDE11-GAL-FS) as a negative control for PP2Acα function. To express PP2Ac-2512 from the GAL1 promoter in pASZ11 the 0.75-kb Nco I/Sac I fragment of YCpDE11-GAL-Cα was replaced by the equivalent DNA from YEpDE-GAL-2512 (PP2Ac-2512 in pYES2) and tested (plasmid YCpDE11-GAL-2512) for inducible growth inhibition in DEY1-Cα as above. PP2Acα wild type and mutant alleles were expressed from the GAL1 promoter of pYES2 in yeast strain INVSC1. Cells were grown to a density of 5.0 × 106 per ml at 30 °C in selective (synthetic raffinose, SR) medium containing raffinose (2.0%), glycerol (3.0%), and casamino acids (0.2%) followed by the addition of an equal volume of SG medium containing galactose at 4.0%. A homology-based model was obtained using the coordinates of PP1γ1 (a gift from Dr. David Barford, University of Oxford). Residues 1–309 of human PP2Acα and 8–316 of human PP1γ1 were aligned using "GAP" (GCG Wisconsin Package, Madison, WI). The optimal alignment (46% identity with four 1-amino acid gaps) was used by "MODELER" (Ref.35Sali A. Blundell T.T. Mol. Biol. 1993; 2324: 779-815Crossref Scopus (10563) Google Scholar; BIOSYM/Molecular Simulations, San Diego, CA) to build and refine five PP2Ac models. PP1γ1 appears unstructured between residues 299 and 316 (25Egloff M.-P. Cohen P.T.W. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (379) Google Scholar), and no models were obtained for PP2Ac in this region (the C terminus of the models is Pro-291). The best model, selected on the basis of the lowest violations of the probability density functions, was evaluated using "Profiles-3D" (Ref. 36Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2586) Google Scholar; BIOSYM/Molecular Simulations, San Diego, CA). Its overall self-compatibility score, 139.2, was close to that expected for a polypeptide chain of this length, 140.6, indicating reliability of the model. The primary structure of PP2Ac is highly conserved from yeast to humans (2Barton G.J. Cohen P.T.W. Barford D. Eur. J. Biochem. 1994; 220: 225-237Crossref PubMed Scopus (154) Google Scholar); excluding an N-terminal extension (∼50 residues) to the S. cerevisiae protein, the yeast and human PP2Ac polypeptides display 71% amino acid sequence identity. To investigate whether the conservation of PP2Ac structure reflects a conservation of function, we tested the ability of human PP2Ac (HsPP2Ac) to functionally replace the corresponding protein from S. cerevisiae (ScPP2Ac). We used haploid yeast cells (strain DEY1) triply deleted for the chromosomal PPH21, PPH22, and PPH3 genes and supported by a plasmid-borne PPH22 gene encoding ScPP2Ac (30Evans D.R.H. Stark M.J.R. Genetics. 1997; 145: 227-241Crossref PubMed Google Scholar). A second plasmid, carrying the human PP2Acα clone fused to the yeast PGK promoter and the hemagglutinin epitope, was introduced, and heterologous expression of HA-tagged HsPP2Ac was analyzed by immunoblot analysis (see Fig.1A). Two forms of HsPP2Ac were detected, suggesting that the human protein undergoes post-translational modification in yeast, and both forms migrated at approximately 36 kDa, faster than the endogenous ScPP2Ac at 43 kDa (not shown). HsPP2Ac was analyzed for function in yeast using a plasmid shuffling assay, testing for the ability of cells to grow in the absence of ScPP2Ac (Fig. 1B); this assay tests for the ability of yeast cells to grow on medium containing 5-FOA (37Boeke D.P. Trueheart J. Natsoulis G. Fink G.R. Methods Enzymol. 1987; 154: 164-175Crossref PubMed Scopus (1075) Google Scholar) which negatively selects against the essential URA3 PPH22 plasmid encoding ScPP2Ac in strain DEY1. Cells containing PP2Acα lost PPH22 at high frequency and grew on 5-FOA medium, whereas cells containing a non-functional PP2Acα clone with a 5′-frameshift (PP2Acα-FS) or the empty vector failed to grow without PPH22. An incoming TRP1 PPH22 plasmid also substituted for the resident URA3 PPH22 plasmid as expected. This indicates that HsPP2Ac is functional in yeast and that it supports cell growth in the absence of ScPP2Ac. Consistent with this, cells cured of PPH22 by plasmid shuffling contained plasmid DNA carrying the PP2Acα clone (see "Experimental Procedures") and expressed the 36-kDa HsPP2Ac (Fig. 1 A). Moreover, in an alternative test of function (Fig. 1 C), PP2Acα rescued the ts growth defect of pph22-172 mutant yeast cells that undergo loss of endogenous ScPP2Ac function at 37 °C because of a conditional-lethal point mutation in PPH22 (30Evans D.R.H. Stark M.J.R. Genetics. 1997; 145: 227-241Crossref PubMed Google Scholar). In addition, a truncated allele of PPH22 lacking the first 52 codons (pph22Δ52) functionally replaced full-length PPH22 in strain DEY1 (Fig. 1 D) even at 37 °C (not shown) indicating that the N-terminal extension to ScPP2Ac is dispensable for cell viability, consistent with the ability of HsPP2Ac to replace the endogenous yeast enzyme. Because PP2Ac heterodimerizes with a regulatory PR65/A subunit (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2156) Google Scholar), we tested whether HsPP2Ac binds the endogenous yeast PR65/A subunit Tpd3p in vivo. We tagged Tpd3p with the Myc epitope (Myc-Tpd3p) and asked whether it co-precipitates with HA-tagged HsPP2Ac from a yeast extract (Fig. 2 A). When proteins were precipitated using the 12CA5 antibody, Myc-Tpd3p but not untagged Tpd3p was detected in an immune complex prepared from cells expressing HA-HsPP2Ac, whereas Myc-Tpd3 was absent from a similar complex prepared from cells expressing ScPP2Ac lacking the HA epitope. This indicates HsPP2Ac binds the yeast PR65/A subunit and that, consistently, HsPP2Ac interacted with Tpd3p in the yeast two-hybrid system (Fig.2 B). This supports the conclusion that HsPP2Ac is functional in yeast. Moreover, HsPP2Ac prepared from yeast was catalytically active (see below). We used yeast cells functionally expressing HsPP2Ac to identify mutant forms of the human enzyme that interfere with wild type PP2Ac function. A library of human PP2Acα mutant alleles, fused to the yeast GAL1 promoter, was generated by random mutagenesis and screened for those that inducibly inhibited cell growth on galactose medium. Eleven mutant alleles were identified (Fig. 3) that dominantly inhibited the growth of cells expressing wild type PP2Acα constitutively from the PGK1 promoter (strain DEY1-Cα). The PP2Ac-2512 allele inhibited growth even when transferred to an alternative GAL plasmid (see "Experimental Procedures") demonstrating that its dominant-negative effect was caused by mutation of PP2Acα and not vector DNA (not shown). Five mutant alleles contained nonsense mutations within the PP2Acα ORF and encoded C-terminally truncated forms of HsPP2Ac. Of these, the PP2Ac-182 allele encoded the largest truncation deleting the C-terminal 145 amino acids. A further dominant-negative allele, PP2Ac-144-2, encoded a −1 frameshift mutation within codon 307 of PP2Acα, extending the ORF by 57 codons. Together this indicates that an intact C terminus is required for wild type HsPP2Ac function, and its modification may cause an interfering effect. However, the PP2Ac-Δ67 allele, encoding HsPP2Ac lacking residues 68–309 (Fig. 3), did not cause a dominant-negative effect (see "Discussion"). Another five PP2Acα mutant alleles encoded missense mutations, and in each case (excepting PP2Ac-2446) a single amino acid substitution was sufficient for dominant inhibition of cell growth (Fig. 3). Immunoblot analysis of HA-tagged HsPP2Ac proteins revealed that wild type PP2Acα and dominant-negative missense alleles were expressed from the GAL1 promoter to a similar level, whereas nonsense alleles were expressed to a lower level (data not shown). Because GAL-expressed wild type PP2Acα did not inhibit cell growth, the interfering effect of PP2Acα dominant-negative alleles must be caused by an intrinsic property of their products and not by toxic, high levels of expression. The screen for PP2Acα dominant-negative alleles was performed in yeast cells containing human, and lacking yeast, PP2Ac. To test for growth inhibition in the presence of ScPP2Ac, we introduced PP2Acα mutant alleles into PPH22 pph21Δ pph3Δ cells, in which Pph22p provides ScPP2Ac function. When expressed from the GAL1 promoter, each PP2Acα dominant-negative allele tested inhibited cell growth (Table II) suggesting that its product interfered with ScPP2Ac function. Moreover, when PP2Acα dominant-negative alleles were expressed in pph22-12 pph21Δ pph3Δ cells, containing a mutant Pph22p functionally impaired by a single amino acid substitution

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