Important Role for Phylogenetically Invariant PP2Ac Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Corresponding author: David R. H. Evans, Program in Molecular Pharmacology, Mailstop D2-100, Fred Hutchinson Cancer Research Ctr., 1100 Fairview Ave. N., Seattle, WA 98109., drhevans{at}usa.net (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Ac functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Ac Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Ac catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Ac C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Ac catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

PROTEIN phosphatase 2A (PP2A) is ubiquitous among eukaryotes and is implicated in multiple cellular processes including signal transduction, cell cycle regulation, and protein synthesis (GOLDBERG 1999 ). It exists as a family of holoenzymes in which the catalytic subunit (PP2Ac) binds a diverse array of regulatory subunits thought to modulate substrate specificity and intracellular targeting. PP2Ac binds a structural subunit PR65/A (RUEDIGER et al. 1992 , RUEDIGER et al. 1994 ; GROVES et al. 1999 ), forming a core dimer that in turn binds a PR55/B, PR61/B', or PR72/B'' subunit (reviewed in WERA and HEMMINGS 1995 ). In addition, PP2Ac binds other proteins, notably the novel 4 subunit (MURATA et al. 1997 ), in alternative heterodimers. Moreover, recent evidence indicates that PP2A forms stable complexes with protein kinase signaling molecules (MILLWARD et al. 1999 ), indicating that it plays a central, regulatory role in signal transduction mediated by reversible protein phosphorylation.

Additional regulation of PP2Ac activity is mediated by post-translational modification of the C terminus. The highly conserved Tyr-307 residue may be targeted for phosphorylation, leading to an inhibition of PP2Ac activity (CHEN et al. 1992 , CHEN et al. 1994 ), while the equally conserved C-terminal Leu-309 residue undergoes reversible methylation catalyzed by a competing methyltransferase and methylesterase enzyme (LEE and STOCK 1993 ; FAVRE et al. 1994 ; XIE and CLARKE 1994A , XIE and CLARKE 1994B ; DE BAERE et al. 1999 ; OGRIS et al. 1999A ). Mutation of Leu-309 inhibits PP2Ac carboxymethylation (BRYANT et al. 1999 ) while mutation of either Tyr-307 or Leu-309 disrupts PP2A subunit interactions (OGRIS et al. 1997 ; CHUNG et al. 1999 ). Additionally, PP2Ac can be phosphorylated on threonine in vitro, leading to a decrease in catalytic activity (GUO and DAMUNI 1993 ), although the site of modification is unknown.

PP2A is a member of the PPP family of protein Ser/Thr phosphatases that includes protein phosphatase 1 (PP1) and PP2B (calcineurin). These protein phosphatases share many invariant amino acids, especially active site residues believed to be involved in metal ion and/or substrate binding and catalysis (BARTON et al. 1994 ). The crystal structure of PP11 suggests a model for the catalytic mechanism of the PPP family (EGLOFF et al. 1995 ; GOLDBERG et al. 1995 ), supported by mutagenesis studies and kinetic analysis of purified enzymes (ZHUO et al. 1994 ; ZHANG et al. 1996 ; HUANG et al. 1997 ). Thus, catalysis is believed to involve a one-step, metal-ion-mediated hydrolysis of the substrate by a metal-activated water molecule. Notably, the invariant active site residue PP11 His-125 performs a critical role in the dephosphorylation reaction as a general acid that protonates the Ser/Thr leaving group oxygen, accelerating phos-phoester hydrolysis. Computer modeling based on the crystal structure of PP11 suggests that PP2Ac and PP1c possess a similar overall fold (EVANS et al. 1999 ) and an important catalytic role for PP2Ac His-118 (equivalent to PP11 His-125) has been demonstrated biochemically (OGRIS et al. 1999A , OGRIS et al. 1999B ; CHUNG and BRAUTIGAN 1999 ).

The budding yeast Saccharomyces cerevisiae possesses the proteins Pph22p/Pph21p (SNEDDON et al. 1990 ; RONNE et al. 1991 ), Tpd3p (VAN ZYL et al. 1992 ), Cdc55p (HEALY et al. 1991 ), Rts1p (EVANGELISTA et al. 1996 ; SHU et al. 1997 ), and Tap42p (DI COMO and ARNDT 1996 ; JIANG and BROACH 1999 ), similar to the mammalian PP2Ac, PR65/A, PR55/B, PR61/B', and 4 subunits, respectively. Moreover, the mammalian PP2Ac (EVANS et al. 1999 ; LIZOTTE et al. 1999 ) and PR61/B' (ZHAO et al. 1997 ) subunits functionally substitute for the equivalent proteins in yeast, indicating an exquisite conservation of PP2A structure/function. Recently, we used yeast to identify novel, dominant-negative forms of human PP2Ac that interfere with wild-type PP2A function in vivo (EVANS et al. 1999 ). In this study, we have employed yeast as a convenient system to explore the functional role of invariant PP2Ac active site and C-terminal residues in vivo and in vitro.

	MATERIALS AND METHODS

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Strains, media, vectors, and sequence analysis:
Yeast strains are described in Table 1. Rich (YPD), minimal (SD), and 5-fluoroorotic acid (5-FOA) media and nucleotide sequence analysis were described (EVANS et al. 1999 ). Plasmids YIplac128, YCplac22 (GIETZ and SUGINO 1988 ), pYES2 (Invitrogen, Carlsbad, CA), pYPGE2 (BRUNELLI and PALL 1993 ), YEp352, and YCpDE1 (EVANS and STARK 1997 ) were described.

Construction of strain DEY103HA:
DEY103HA was constructed by replacing the genomic PPH22::URA3 allele in strain DEY213 (EVANS and STARK 1997 ) with the PPH22^HA allele by two-step gene transplacement. Thus, a PPH22^HA 1.8-kb XbaI/EcoRI fragment was inserted into the LEU2 vector YIplac128. The plasmid was linearized at a KpnI site upstream of the PPH22^HA open reading frame (ORF) and introduced into DEY213. Ura⁺ Leu⁺ merodiploid transformants were selected and subjected to a period of nonselective growth in YPD medium to promote recombination and excision of one of the duplicated PPH22 loci. Ura^- recombinants were selected on medium containing 5-FOA and screened for a Leu^- phenotype on SD lacking leucine. One Ura^- Leu^- recombinant strain, DEY103HA, in which the chromosomal PPH22::URA3 allele was replaced by PPH22^HA, was identified by PCR analysis of genomic DNA and Western blot analysis.

DNA manipulations:
Epitope-tagged yeast PP2Ac, encoded by PPH22^HA with a double HA-tag inserted at the NsiI site, was a gift from Mike Stark. Plasmid YCpDE22HA is a PPH22^HA 1.8-kb KpnI/EcoRI fragment in vector YCplac22. Plasmid YCpDE22-12HA was constructed by first inserting the PPH22^HA 1.8-kb KpnI/EcoRI fragment into vector YEp352. The 5-kb BstXI/EcoRI fragment from the resulting plasmid (YEpDE2e) was ligated to the 0.9-kb BstXI/EcoRI fragment of plasmid pDE22-12 (EVANS and STARK 1997 ). From the resulting plasmid, the pph22-12^HA 1.8-kb XbaI/EcoRI fragment was finally inserted into YCplac22. Plasmid YCpDE22-172HA was constructed by first inserting a pph22-172 1.8-kb XbaI/EcoRI fragment (EVANS and STARK 1997 ) into vector YIplac128. The 4.9-kb BstXI/KpnI fragment from the resulting plasmid was ligated to the 0.7-kb BstXI/KpnI fragment of YEpDE2e. Finally the pph22-172^HA 1.8-kb XbaI/EcoRI fragment was inserted into YCplac22. Human PP2Ac alleles were tagged with the hemagglutinin (HA) epitope and expressed from the PGK1 promoter of vector pYPGE2 or the GAL1 promoter of vector pYES2 as described (EVANS et al. 1999 ). C-terminal mutations were generated in HA-PP2Ac by PCR using a reverse primer encoding the relevant substitution.

Preparation of yeast cell extracts and Western blot analysis:
Extracts were prepared and Western blotting was performed as described (EVANS et al. 1999 ) except that cell lysis and TNPT blocking buffer contained NP-40 at 0.1% (v/v) and 0.5% (v/v), respectively.

Immunoprecipitations and protein phosphatase activity measurements:
Precipitations were performed as described (EVANS et al. 1999 ) except that, following incubation of 12CA5-protein A Sepharose with cell extract, the beads were washed with cell lysis buffer (4 times in 20 volumes) and Tris-buffered saline (TBS; 2 times in 20 volumes). Prior to Western blotting, HA-tagged Pph22 proteins were eluted from 12CA5-Protein A Sepharose beads by incubation (2 times for 30 min) with the HA-epitope peptide (50 µl, 1 mg/ml). Protein phosphatase assays were performed using an RRA(pT)VA phosphopeptide substrate as described (EVANS et al. 1999 ) and activity was expressed as units (1 unit = 1 µmol of phosphate hydrolyzed per min).

Galactose-induced expression of PP2Ac alleles in liquid medium:
PP2Ac alleles were expressed from the GAL1 promoter of pYES2. Transformant cells (strain INVSC1) were grown to a density of 5.0 x 10⁶ per ml at 30° in selective S-medium containing raffinose (2.0% w/v), glycerol (3.0% v/v), and cassamino acids (0.2% w/v), whereupon an equal volume of SG medium, containing galactose at 4.0%, was added.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Analysis of highly conserved PP2Ac active site residues:
PP2Ac contains residues that are highly conserved between species and implicated in catalysis. The PP2Ac-Y127N mutant protein was identified in a screen for mutant forms that cause a dominant inhibition of cell growth when overexpressed in yeast (EVANS et al. 1999 ). PP2Ac-Y127N is of particular interest because it is mutated for a tyrosine residue invariant between species and conserved in PP1. Computer modeling of PP2Ac structure predicts that the side chain of Tyr-127 is in van der Waal's contact with the active site residues, His-118 and Asp-88, that apparently are critical for PP2Ac- catalytic function (EGLOFF et al. 1995 ; CHUNG and BRAUTIGAN 1999 ; EVANS et al. 1999 ; OGRIS et al. 1999A , OGRIS et al. 1999B ). The PP2Ac mutant allele encoding Y127N was originally generated by random mutagenesis and encoded a second substitution, V159E. Surprisingly, site-directed mutagenesis revealed that each of these substitutions causes a dominant-negative effect individually (EVANS et al. 1999 ). To investigate the interfering effect of the PP2Ac-Y127N and V159E substituted forms we analyzed them further and found that each was expressed stably in soluble yeast extracts at the level of wild-type PP2Ac (Fig 1A). However, unlike the wild-type protein, both the Y127N and V159E substituted form was severely impaired for catalytic activity in vitro (Fig 1B) and, consistent with this, each failed to provide essential PP2Ac function in yeast when expressed at a low level from the PGK1 promoter (Fig 1C). These results demonstrate that the conserved Tyr-127 and Val-159 residues are important for PP2Ac catalytic function. Furthermore, they support the notion that the inhibition of cell growth observed during PP2Ac-Y127N or PP2Ac-V159E overexpression is caused by native folding, but catalytic impairment, of the mutant proteins.

View larger version (30K): In this window In a new window Download PPT slide   Figure 1. Analysis of the human PP2Ac-Y127N and V159E substituted proteins. (A) Western blot analysis of PP2Ac wild-type and mutant forms expressed in yeast. HA-tagged proteins were inducibly expressed from the GAL1-promoter by incubating cells in SG medium for 4.5 hr. Soluble extracts were resolved by SDS-PAGE and analyzed by Western blotting using a 12CA5 probe. Molecular weight marker is shown on the right. (B) Catalytic activity of PP2Ac proteins in vitro. HA-tagged wild-type PP2Ac, or the Y127N or V159E substituted form, was inducibly expressed from the GAL1 promoter in cells grown in SG for 6.5 hr. Cells containing empty vector were the negative control. HA-tagged proteins, precipitated with the 12CA5 monoclonal antibody from 200 µg of yeast cell extract, were assayed for phosphatase activity in the absence (open bars) or presence (solid bars) of 10 nM okadaic acid. Numbers represent the mean values (±SD) from three experiments. The inset shows a representative Western blot analysis of immune complexes prepared in parallel with those used for phosphatase assays; HA-tagged PP2Ac protein (36 kD) was absent from complexes prepared from the (vector) control extract. (C) Functional analysis of PP2Ac forms in yeast by plasmid shuffling. Wild-type PP2Ac, the Y127N or V159E substituted form, or no insert DNA (empty vector) was expressed from the TRP1 plasmid pYPGE2 in cells of strain DEY3 and tested for essential function in vivo (ability to replace Pph22p expressed from the URA3 plasmid, YCpDE8). Transformant cells were grown to saturation in liquid SD with uracil, whereupon cell suspensions were spotted onto agar medium containing 5-FOA or nonselective SD and incubated at 30° for 3 days.

Two active site substitution mutations have been identified in the yeast PP2Ac protein, Pph22p, that cause a recessive and conditional loss of function in vivo (EVANS and STARK 1997 ). Thus, temperature-sensitive (ts) mutant cells containing the Pph22 F232S or P240H substituted form are viable at 30° (the permissive temperature) but inviable at 37° (restrictive temperature) when the wild-type protein is absent. Computer modeling of human PP2Ac structure predicts that the residues Phe-164 and Pro-172 (equivalent to Pph22 Phe-232 and Pro-240, respectively) pack closely to the highly conserved residue His-167 (Pph22 His-235), which may be important for catalysis through a role in metal binding (EGLOFF et al. 1995 ; GOLDBERG et al. 1995 ; EVANS et al. 1999 ). To test the Pph22 F232S and P240H substitutions for a temperature-dependent effect on catalysis, we measured PP2Ac activity in vitro. The wild-type and substituted forms of Pph22p were precipitated from a yeast cell extract (Fig 2A) and preincubated at 4° or 42° for 22 min before measurement of PP2A activity (Fig 2B); 42° was chosen arbitrarily as a temperature likely to reveal a difference between the wild-type and mutant proteins. Following preincubation at 4°, each mutant protein displayed reduced activity at 30° relative to wild-type Pph22p, suggesting that each is partially impaired for catalysis even at the permissive temperature for cell growth. Strikingly, however, both the F232S and P240H substituted forms displayed a dramatic loss of activity (7.9- and 6.8-fold, respectively) following incubation at 42°, while wild-type Pph22p displayed a 1.6-fold decrease under identical conditions. This supports the notion that PP2Ac loss of function at 37°, in cells containing the Pph22 F232S or P240H substituted protein, is caused by a reduction of PP2A activity in vivo.

View larger version (27K): In this window In a new window Download PPT slide   Figure 2. Biochemical analysis of wild-type and ts mutant forms of Pph22p. (A) Immunoprecipitation of HA-tagged forms of Pph22p. Immune complexes containing an HA-tagged wild-type or mutant Pph22p were prepared from yeast cell extracts (900 µg) using the 12CA5 monoclonal antibody bound to protein-A Sepharose (30 µl). Lane 1, strain DEY-103HA (expressing HA-Pph22p); lane 2, DEY22-12HA (HA-Pph22-F232S); lane 3, DEY22-172HA (HA-Pph22-P240H); lane 4, strain DEY3 (untagged Pph22p). Ig, immunoglobulin. (B) PP2A activity of wild-type and mutant Pph22 proteins in vitro. Immune complexes prepared at 4° from the cell extracts (250 µg) in A were preincubated at 4° or 42° for 22 min, then assayed at 30° for protein phosphatase activity in the absence or presence of okadaic acid (10 nM). Numbers represent the mean values from two experiments (with assays performed in duplicate) corrected both for PP2A-specific (okadaic acid-sensitive) activity and background levels of activity (untagged Pph22p, see lane 4 in A). Average error was ±14.9 milliunits (4°) and ±17.7 milliunits (42°).

Functional analysis of invariant PP2Ac C-terminal residues:
The C terminus of PP2Ac is highly conserved between species and is a site of post-translational modification in the mammalian protein. The Tyr-307 residue of PP2Ac is invariant between species and believed to be a site of inhibitory phosphorylation (CHEN et al. 1992 , CHEN et al. 1994 ) while Thr-304 is equally conserved and is a candidate for inhibitory threonine phosphorylation (GUO and DAMUNI 1993 ). To investigate the functional role of highly conserved PP2Ac C-terminal residues in vivo, we tested PP2Ac mutant proteins for rescue of the ts growth defect caused by the F232S or P240H substitution in Pph22p. This revealed that substituting an aspartate for the conserved Tyr-307 or Thr-304 residue impaired PP2Ac function severely in vivo, while the Y307F and T304A substitutions caused partial and no detectable impairment, respectively (Table 2). This assay required incubation of cells at 37°, an extreme temperature for yeast cell growth, raising the possibility that under stress conditions, partial PP2Ac function might be obscured. To address this, we performed parallel experiments testing the ability of PP2Ac to substitute for wild-type Pph22p in vivo at 30° (EVANS et al. 1999 ). The results were in accordance with those obtained by the former assay (Fig 3A); the Y307D and T304D substitutions abolished essential PP2Ac function in vivo while the Y307F and T304A substituted proteins supported impaired and largely wild-type yeast cell growth, respectively, in the absence of Pph22p. A Y307A substituted form similarly supported impaired cell growth, indicating that alanine at this position does not abolish PP2Ac function in vivo. Growth rate analysis revealed that the Y307F substitution impaired protein function severely in vivo while T304A had little inhibitory effect (Table 3). Furthermore, the growth rate of cells functionally expressing PP2Ac-Y307F varied between experiments, suggesting the accumulation of suppressor mutations. Western blot analysis of cell extracts (Fig 3B) revealed that all human PP2Ac proteins tested were stably expressed and soluble in yeast and, typical of the wild-type protein (EVANS et al. 1999 ), migrated as a prominent doublet during SDS-PAGE. These results indicate that PP2Ac function is sensitive to mutation of evolutionarily invariant residues at its C terminus, especially replacement of Tyr-307 or Thr-304 by a charged aspartate residue. Nevertheless, replacement of Thr-304 with alanine is largely tolerated, while uncharged residues replacing Tyr-307 cause partial impairment of protein function.

View larger version (53K): In this window In a new window Download PPT slide   Figure 3. Analysis of PP2Ac mutant forms in yeast. (A) Functional replacement of yeast PP2Ac (Pph22p) by wild-type and C-terminal mutant forms of human PP2Ac was tested by plasmid shuffling as in Fig 1C, except that cell suspensions were diluted (1:1, 1:10, or 1:100) prior to spotting onto agar medium containing 5-FOA. (B) Western blot analysis of HA-tagged forms of human PP2Ac expressed in strain DEY3. Yeast cell extracts (30 µg) containing wild-type PP2Ac (wt), a mutant form of PP2Ac, containing the substitution mutation indicated, or the empty expression vector were resolved by SDS-PAGE and probed with the 12CA5 monoclonal antibody. Molecular mass marker shown on the right.

View this table: In this window In a new window   Table 2. Growth of pph22 ts mutant cells containing a PP2Ac wild-type or mutant protein

View this table: In this window In a new window   Table 3. Growth rate of cells functionally expressing a PP2Ac wild-type or mutant protein

PP2Ac-Y307D is enzymatically active and causes recessive PP2Ac impairment:
It has been reported that phosphorylation on Tyr-307 inhibits PP2Ac activity (CHEN et al. 1992 , CHEN et al. 1994 ). Therefore, we asked whether an acidic substitution mutation at this position mimics the inhibitory effect of phosphorylation by testing the PP2Ac-Y307D mutant protein for catalytic activity in vitro. Surprisingly, PP2Ac-Y307D displayed approximately wild-type activity (Fig 4), indicating that its inability to provide essential PP2Ac function in vivo is not due to severe unfolding or catalytic impairment. We demonstrated recently that similarly nonfunctional PP2Ac mutant proteins, truncated at the C terminus, cause a severe dominant-negative inhibition of cell growth when expressed in yeast from a strong promoter (EVANS et al. 1999 and see PP2Ac-Y218stop, Table 4). In contrast, when the noncomplementing Y307D and T304D C-terminally substituted forms of PP2Ac were expressed from the GAL1 promoter, neither inhibited cell growth severely, though a weak dominant-negative effect was observed initially (see colony formation after 5 days in Table 4). Together, these results suggest that, unlike truncated PP2Ac proteins, the Y307D and T304D C-terminally substituted forms retain some property that prevents severe interference with wild-type PP2Ac function.

View larger version (34K): In this window In a new window Download PPT slide   Figure 4. Catalytic activity of the PP2Ac-Y307D substituted form. HA-tagged wild-type PP2Ac, or the Y307D-substituted form, was inducibly expressed from the GAL1 promoter in cells grown in SG for 14 hr. Cells containing no HA-tagged protein (vector) were the negative control. HA-tagged proteins, precipitated by the 12CA5 monoclonal antibody from 250 µg of yeast cell extract, were assayed for phosphatase activity in the absence (open bars) or presence (solid bars) of 10 nM okadaic acid to identify PP2A activity. Numbers represent the mean from two experiments. (Top) Phosphatase activity measured in immune complexes. (Middle) Western blot analysis of HA-tagged Pph22 proteins present in yeast cell extracts. (Bottom) Representative Western blot analysis of immune complexes prepared (from 1.0 mg of cell extract) in parallel with those used for phosphatase assays; HA-tagged PP2Ac protein (36 kD) was absent from complexes prepared from the (vector) control extract. Ig, immunoglobulin. Blots were probed with the 12CA5 monoclonal. Molecular mass marker shown on the right.

View this table: In this window In a new window   Table 4. Effect on yeast cell growth of PP2Ac proteins inducibly expressed from the GAL1 promoter

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Human PP2Ac provides an essential function in yeast (EVANS et al. 1999 ; LIZOTTE et al. 1999 ), consistent with the phylogenetic conservation of PP2A holoenzyme structure and biochemistry (COHEN et al. 1989 ; MAYER-JAEKEL and HEMMINGS 1994 ). Thus, we have used yeast as a convenient system to explore the role of highly conserved PP2Ac active site and C-terminal residues that are invariant between species. Our results demonstrate the importance of the PP2Ac C terminus for protein function in vivo and identify active site residues important for enzyme activity. Moreover, this study characterizes a number of human and yeast PP2Ac mutant forms that will be useful for further study of PP2A function in vivo.

PP2Ac is sensitive to C-terminal mutations:
We have found that PP2Ac function is sensitive to mutation of invariant C-terminal residues, especially replacement of Tyr-307 or Thr-304 with an aspartate residue. This is consistent with the observation that the PP2Ac C terminus is an important regulatory domain, which, in mammalian cells, is targeted for multiple post-translational modifications modulating PP2Ac activity and subunit interactions. In mammalian cells, the C-terminal Tyr-307 residue may undergo phosphorylation, leading to an inhibition of PP2Ac activity (CHEN et al. 1992 , CHEN et al. 1994 ), and because PP2A participates in stable complexes with protein kinase signaling molecules, this may contribute to a mechanism permitting transient activation and feedback regulation of kinase-mediated signaling pathways (EVANS and HEMMINGS 1998 ; MILLWARD et al. 1999 ). Nevertheless, we show here that the Y307D substitution does not detectably impair the catalytic function of PP2Ac in vitro and therefore does not mimic phosphorylation of Tyr-307. This is consistent with the observations of OGRIS et al. 1997 , who introduced uncharged, basic, or acidic substitutions (including a Y307E and T304D) to the PP2Ac C terminus and reported that each mutant protein retained activity in vitro. However, the same authors reported that changing the PP2Ac Tyr-307 or Thr-304 residue to a charged (but not an uncharged) amino acid disrupts binding of the PR55/B regulatory subunit to the PP2Ac·PR65/A core dimer. This suggests that the PP2Ac-Y307D and T304D substituted proteins are unable to support yeast cell growth because of defective subunit binding. Accordingly, in an in vivo assay testing the ability of PP2Ac to rescue the ts growth of cells containing a Pph22p F232S or P240H substituted protein, neither PP2Ac-Y307D nor PP2Ac-T304D provided essential function at 37°. It is noteworthy that in the W303 yeast strain background used in this study, deletion of the CDC55 gene (encoding the yeast PR55/B subunit) confers a ts growth defect at 37° (D. EVANS, unpublished observation) in addition to the previously reported cold-sensitive growth phenotype (HEALY et al. 1991 ). Thus, PP2Ac-Y307D and PP2Ac-T304D may be inhibited for Cdc55p binding. However, each mutant protein failed additionally to provide essential PP2Ac function in an alternative plasmid-shuffling assay that tests the ability of PP2Ac to replace Pph22p at 30° (a permissive temperature for growth of cdc55 mutant cells). This indicates that the Y307D and T304D substitutions may inhibit PP2Ac function by impairing the binding of multiple proteins, possibly including the yeast PR61/B' subunit, Rts1p (the lack of which causes a ts growth defect at 37°; SHU et al. 1997 ), the essential 4-like subunit, Tap42p (DI COMO and ARNDT 1996 ), and endogenous PP2A substrates. In contrast to the observations of OGRIS et al. 1997 , it has been reported that changing Tyr-307 to an uncharged amino acid inhibits the interaction of PP2Ac with PR55/B (CHUNG et al. 1999 ). Our results are in agreement with this, because the PP2Ac-Y307F and PP2Ac-Y307A mutant proteins were impaired for PP2Ac function in vivo, possibly reflecting a partial defect in subunit binding. Consistent with this, mutation of the adjacent, highly conserved C-terminal leucine of mammalian PP2Ac similarly inhibits PR55/B binding (BRYANT et al. 1999 ; CHUNG et al. 1999 ) and impairs, but does not abolish, PP2Ac function in yeast (EVANS et al. 1999 ). Moreover, mutation of the Pph22p C-terminal leucine to alanine similarly inhibits Cdc55p binding (D. EVANS and B. HEMMINGS, unpublished results).

Identification of active site residues important for PP2Ac catalytic function:
The PP2Ac-Y127N and V159E substituted forms were identified in a screen for dominant-negative mutant proteins that interfere with wild-type PP2Ac function in vivo (EVANS et al. 1999 ). Here we show that the Y127N and V159E substitutions each cause a profound impairment of PP2Ac-mediated catalysis in vitro. Our observations provide support for the prediction, derived from structural data, that the Tyr-127 side chain is in van der Waal's contact with that of the invariant active site residues, His-118 and Asp-88 (EGLOFF et al. 1995 ). Thus, His-118 is thought to serve as a general acid that protonates the seryl/threonyl phosphate ester oxygen atom, accelerating the dephosphorylation reaction, while the acidic character of His-118 is believed to be increased by salt bridge formation between its side chain and that of Asp-88 (EGLOFF et al. 1995 ). Consistent with this, we have found that a D88N mutation reduces PP2Ac activity and abolishes essential PP2Ac function in yeast without preventing PR65/A subunit binding (T. MYLES, D. EVANS and B. HEMMINGS, unpublished results). Thus, the Y127N substitution may inhibit PP2Ac activity by disrupting the interaction between key catalytic residues. In addition, computer modeling predicts that Tyr-127 interacts with the Ser(P)/Thr(P) substrate (EVANS et al. 1999 ), suggesting that Y127N may impair PP2Ac catalysis by inhibiting substrate binding. Like Y127N, the V159E substitution impairs PP2Ac catalytic function profoundly. The residue equivalent to Val-159 is highly conserved among PP2Ac proteins, and computer modeling predicts that the V159E substitution causes a global disruption of the PP2Ac active site (EVANS et al. 1999 ), which may account for its effect on enzyme activity. However, the dominant-negative effect on cell growth conferred by both the PP2Ac-Y127N and V159E substituted forms suggests that each undergoes native folding, at least over a portion of the molecule. Thus, when overexpressed in yeast cells, these catalytically impaired, but partially folded, mutant proteins may titrate substrates or regulatory subunits into PP2A complexes that are effectively inactive, thereby interfering with the function of the wild-type enzyme. Consistent with this, we have found that each of these mutant proteins competes with wild-type PP2Ac in vivo (EVANS et al. 1999 ). Surprisingly however, we have found that yeast cells induced to overexpress the catalytically impaired PP2Ac-H118N mutant protein (EVANS et al. 1999 ; LIZOTTE et al. 1999 ), which similarly inhibits yeast colony formation on agar medium, fail to arrest proliferation completely in liquid medium, as determined by measurement of growth rate and DNA content (D. EVANS and B. HEMMINGS, unpublished results). Thus, the precise physiological effect of PP2Ac interfering proteins in vivo remains to be uncovered, but we are currently testing for effects of PP2Ac wild-type and mutant forms expressed in mammalian cells. Interestingly, we have found that the PP2Ac-Y307D mutant protein confers a weak, dominant-negative effect despite retaining catalytic activity. This supports the notion that proper folding of mutant PP2Ac forms facilitates interference with the wild-type protein, but that catalytic impairment greatly exacerbates the dominant-negative effect.

The Pph22 F232S and P240H substitutions were shown by genetic analysis to cause a conditional-lethal loss of PP2Ac function in vivo (EVANS and STARK 1997 ). In this study, we show that the loss of function caused by these substitution mutations reflects a ts reduction in PP2Ac enzyme activity in vitro. This supports the notion that the mitotic and cell lysis defects caused by these mutations (EVANS and STARK 1997 ) are caused by loss of PP2A activity in vivo. The Phe-232 and Pro-240 residues flank a His-Gly-Gly motif invariant in PP2Ac proteins and in which the histidine residue is implicated in metal binding (EGLOFF et al. 1995 ; GOLDBERG et al. 1995 ). Computer modeling (EVANS et al. 1999 ) predicts that the equivalent residue in human PP2Ac, His-167, lies at the base of a hydrophobic pocket (encompassing ß-sheet 6) and that its packing may be disrupted by a F164S or P172H substitution (equivalent to Pph22 Phe-232 and Pro-240, respectively), possibly leading to impairment of metal-ion-mediated catalysis. Nevertheless, the ts growth defect at 37°, of cells containing the Pph22 F232S or P240H substituted form, is rescued by wild-type Pph22p (EVANS and STARK 1997 ) even when the mutant proteins are expressed from a high copy (2µ) vector and the wild-type protein is expressed from a low copy (CEN) vector (D. EVANS, unpublished observations). Thus, although we have not expressed the Pph22 F232S or P240H proteins from the same GAL1-based expression system from which the human PP2Ac Y127N and V159E dominant-negative forms were overexpressed, the Pph22 F232S and P240H mutations apparently do not cause a severe dominant-negative effect. This suggests that, at 37°, these mutant proteins may undergo ts denaturation over a large portion of the molecule, preventing interference with wild-type Pph22p. However, these conditionally defective mutant proteins will be useful tools for studying PP2A function in haploid yeast cells. Moreover, because PP2A function is likely to regulate the basal and stimulated activity of protein kinase-mediated signaling pathways, the recessive and dominant-negative mutant forms of PP2Ac characterized here may help uncover important mechanisms and components of eukaryotic signal transduction.

	ACKNOWLEDGMENTS

We thank Mike Stark (Dundee University) for HA-tagged Pph22p; Herbert Angliker for DNA sequence analysis; and Timothy Myles, Thomas Millward, Mirjana Andjelkovic, and Pat Dennis for comments on the work. This work was supported by a Human Frontiers program grant (B.A.H).

Manuscript received February 4, 2000; Accepted for publication May 4, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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