From the Department of Biochemistry and Winship
Cancer Center, Emory University School of Medicine, Atlanta, Georgia
30322 and the § Institute of Molecular Biology, Vienna
Biocenter, University of Vienna, A-1030 Vienna, Austria
Received for publication, September 22, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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Protein phosphatase 2A (PP2A) is an essential
eukaryotic serine/threonine phosphatase known to play important roles
in cell cycle regulation. Association of different B-type targeting
subunits with the heterodimeric core (A/C) enzyme is known to be an
important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits
to the A/C heterodimer might be regulated is unknown. We have used the
budding yeast Saccharomyces cerevisiae as a model system to
investigate the hypothesis that covalent modification of the C subunit
(Pph21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two
approaches were taken. First, S. cerevisiae cells were
generated whose survival depended on the expression of different
carboxyl-terminal Pph21p mutants. Second, the major S. cerevisiae methyltransferase (Ppm1p) that catalyzes the
methylation of the PP2A C subunit carboxyl-terminal leucine was
identified, and cells deleted for this methyltransferase were utilized
for our studies. Our results demonstrate that binding of the yeast B
subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites on Pph21p or by
deletion of the gene for Ppm1p. Loss of Cdc55p association was
accompanied in each case by a large reduction in binding of the yeast A
subunit, Tpd3p, to Pph21p. Moreover, decreased Cdc55p and Tpd3p binding
invariably resulted in nocodazole sensitivity, a known phenotype of
CDC55 or TPD3 deletion. Furthermore, loss of
methylation also greatly reduced the association of another yeast
B-type subunit, Rts1p. Thus, methylation of Pph21p is important for
formation of PP2A trimeric and dimeric complexes, and consequently, for
PP2A function. Taken together, our results indicate that methylation and phosphorylation may be mechanisms by which the cell dynamically regulates PP2A complex formation and function.
Protein phosphatase 2A
(PP2A)1 is a highly conserved
eukaryotic serine/threonine phosphatase known to play important roles in many cellular events including regulation of the cell cycle (reviewed in Refs. 1-3). PP2A often exists as a heterotrimeric enzyme
composed of a catalytic (C) subunit, a structural (A) subunit, and a
variable regulatory/targeting (B-type) subunit (1, 4). In mammalian
cells, three major families of B-type subunits, B (or B55), B' (or
B56), and B" (or PR72/130), have been described (1). Binding of B-type
subunits to the A/C core heterodimer modulates PP2A activity (5-11)
and localizes PP2A to specific cellular microenvironments and/or
signaling pathways (for examples, see Refs. 12-15).
Although it is clear that binding of B-type targeting subunits is a
major mechanism by which cells regulate PP2A, how the binding of these
targeting subunits to the A/C heterodimer might be regulated is
unknown. Recently, we showed in mammalian cells that deletion of nine C
subunit carboxyl-terminal residues (amino acids 301-309) or
nonconservative substitution of threonine 304 or tyrosine 307 abolished
the ability of the C subunit to form complexes with the B subunit (10).
Based on this finding, we postulated that reversible covalent
modification at the C subunit carboxyl terminus might regulate B
subunit binding.
Previous studies showed that both phosphorylation and methylation occur
on the C subunit carboxyl terminus. Phosphorylation of tyrosine 307 in vitro by pp60c-src results in 90% inhibition
of PP2A activity (16). Substitution of this same tyrosine with an
acidic residue abolishes binding of the A/C heterodimer to B subunit
in vivo, suggesting that phosphorylation of this residue
might do the same (10). PP2A is also known to be inhibited by
phosphorylation of an unknown threonine residue (17), and
interestingly, substitution of threonine 304, located in the carboxyl
terminus of the C subunit, also abolishes B subunit binding (10).
The effects of reversible methylation of leucine 309 (18-22) on PP2A
activity is unclear (20, 23, 24). C subunit methylation appears to
occur in vivo in a cell cycle-regulated manner (25) and,
thus, could be a mechanism for modulating PP2A function in a cell
cycle-specific manner. Because PP2A methylation is reversible and the
methyltransferase (23) and methylesterase (26) enzymes have recently
been cloned, PP2A methylation represents an attractive model system for
the study of the effects of reversible protein methylation on
protein-protein interactions.
The Saccharomyces cerevisiae genome contains two genes
(PPH21 and PPH22) encoding PP2A catalytic
subunits (27) and a single A subunit gene, TPD3 (28).
Deletion of either PPH21 or PPH22 alone has no
effect on yeast growth, although PP2A activity is reduced by 51 and
33%, respectively (27). Double deletion of both genes results in
severe slow growth and a temperature-sensitive phenotype (29).
Additional deletion of the PPH3 gene, which encodes a
nonessential protein phosphatase that has residual overlapping function
with PP2A, leads to cell death (29). S. cerevisiae has two
B-type subunits, Cdc55p and Rts1p, that are, respectively, homologous
to the B and B' mammalian B-type subunit families (30, 31). Cells
lacking Cdc55p or Tpd3p are sensitive to drugs such as nocodazole and
benomyl, which perturb microtubule stability, presumably because forms
of PP2A containing these subunits are important for normal mitotic
spindle checkpoint function (32, 33).
Although only one S. cerevisiae methylesterase homolog
(Ppe1p) exists that was found to be nonessential for growth under
normal conditions (26), two putative PP2A methyltransferase homologs (Ppm1p and Ppm2p) exist (23) whose importance has not yet been determined. The first, Ppm1p, is highly homologous to the mammalian PP2A methyltransferase (PMT1) that was recently cloned, expressed, and
shown to have activity by De Baere et al. (23). These
authors also reported the existence of a second homologous protein in both mammalian cells (PMT2) and S. cerevisiae (Ppm2p) that
has ~350 additional amino acids containing significant similarity to
the kelch domain (23), which has been implicated in actin binding. To
date, Ppm1p, Ppm2p, and the human Ppm2p homolog have not been shown to
have PP2A methyltransferase activity, and therefore, the relative
contributions of these enzymes toward methylating PP2A in
vivo are not known.
We previously created a set of mutants targeting highly conserved
residues in the carboxyl terminus of the mammalian PP2A catalytic (C)
subunit. Several of these mutants showed decreased binding of B subunit
and, in some cases, decreased C subunit methylation (10).2 Expression of these
mutants in mammalian cells showed no consistent phenotype3 perhaps because of
the high level of endogenous wild-type (wt) C subunit. In this study,
we have generated S. cerevisiae cells whose survival depends
on the expression of the corresponding yeast (Pph21p) mutants. Analysis
of these cells together with strains deleted for the putative PP2A
methyltransferase genes, PPM1 and PPM2, reveals
that carboxymethylation of the yeast C subunit carboxyl terminus is
required for the efficient assembly of PP2A heterotrimers containing
Cdc55p or Rts1p. Given that C subunit methylation is known to be
reversible and to oscillate in a cell cycle-dependent
manner in mammalian cells, these results suggest that the cell may
regulate B-type subunit binding and, consequently, PP2A function by
modulating the methylation of the PP2A catalytic subunit.
Plasmids and PPH21 Mutant cDNAs--
A 2.5-kilobase
polymerase chain reaction product (forward primer,
CGGGATCCGAGAGCAAATCGTTAAGTTCAGG; reverse primer,
ACGCGTCGACGCTCAATACTCGAGTTATTCGTGTG) encoding the
PPH21 gene was cut with BamHI and SalI
and ligated into pBluescript SK+ vector (Stratagene). The
two nucleotides preceding the ATG start codon were then mutated to CC
by site-directed polymerase chain reaction mutagenesis to create an
NcoI site (CCATGG) that includes the start codon.
Subsequently, site-directed polymerase chain reaction mutagenesis was
used to create the T364A (codon change: ACG to GCT), T364D (codon
change: ACG to GAC), Y367E (codon change: TAC to GAA), Y367F (codon
change: TAC to TTC), and L369 Growth Media and Yeast Strains--
S. cerevisiae
strain W303a (MATa ura3-1 leu2-3, 112 trp1-1
his3-11, 15 ade2-1 can1-100) and its isogenic derivative ADR496 (MATa ura3-1 leu2-3, 112 trp1-1 his3-11, 15 ade2-1 can1-100 CDC55::HIS3) were obtained from A. Murray (32). Strain H328 (MATa ade2-1 can1-100
his3-11, 15 leu2-3, 112 trp1-1 ura 3-1
PPH21 Antibodies--
Rabbit anti-Cdc55p polyclonal antibody was
raised to a fusion protein containing a 6×His tag fused to the first
193 amino acids of Cdc55p. Rabbit anti-Tpd3p antibody was raised to a
fusion protein containing a amino-terminally 6×His-tagged Tpd3p
protein lacking 64 amino-terminal amino acids and 38 carboxyl-terminal residues. Histidine tag (His probe; H-15) antibody was obtained from
Santa Cruz Biotechnology. Methylation-sensitive (binding inhibited by
methylation of the C subunit) anti-PP2A C subunit monoclonal antibody
clone 1d6 was generated against a 15-residue unmethylated
carboxyl-terminal peptide with an additional amino-terminal cysteine
added for coupling to keyhole limpet hemocyanin2 and is now
carried by Upstate Biotechnology, Inc. Anti-HA tag antibodies, 12CA5
and 16B12, were obtained from Berkeley Antibody Co. (BAbCo).
Preparation of Cellular Extracts Immunoprecipitations and
Immunoblotting--
Cells were harvested and lysed as described (39),
except that vortexing of cells with glass beads was done for 45 s
intervals. Immunoprecipitation of PP2A complexes via HA-tagged wt or
mutant C subunits (Pph21p) was performed using anti-HA tag monoclonal antibody (12CA5) plus protein A-Sepharose beads (Amersham Pharmacia Biotech) as described (39), except that 300 µl of lysate was immunoprecipitated with 2 µg of antibody for 90 min at 4 °C. In some immunoblots, anti-mouse Nocodazole Sensitivity Assay--
Yeast cells were grown at
30 °C overnight. Cell numbers were counted, and cultures were
diluted to ~2 × 105 cells/ml and grown to log phase
in medium containing 10 or 40 µg/ml nocodazole. At 0 (before
nocodazole addition), 4, and 6 h, cells were removed and diluted
10-fold, and 50 µl was spread on each of three YPD plates. After
incubation of these plates at 30 °C for 2 days, the percent survival
of each sample was calculated by dividing the mean of the colony
numbers at each time point by the mean of the colony numbers at time 0 and multiplying by 100.
Use of Methylation-sensitive C Subunit Monoclonal Antibody, 1d6,
to Assay C Subunit (Pph21p/Pph22p) Methylation--
This
assay2 uses a methylation-sensitive mouse monoclonal
antibody (1d6) rather than a polyclonal antibody (20, 25) to evaluate
PP2A methylation. 1d6 was generated against an unmethylated PP2A
carboxyl-terminal peptide and almost exclusively detects unmethylated
PP2A. Fig. 5A shows that its binding to a mammalian C
subunit carboxyl-terminal nonapeptide is inhibited by methylation of
the carboxyl-terminal leucine. The nonapeptide has only one conservative difference from the Pph22p sequence: a lysine instead of
an arginine at its second position. 1d6 recognizes both unmethylated Pph21p and unmethylated Pph22p. 1d6 immunoblotting plus and minus base
treatment can be used to determine the C subunit methylation status as
described in the legend to Fig. 5B. This method has the
advantage over radiolabeling methylation assays in that it measures the
steady-state methylation level of C subunit directly, avoiding
potential problems with label uptake and incorporation.
Carboxy-terminal Pph21p Mutants Are Functional--
To investigate
the possibility that PP2A might be regulated by reversible covalent
modification of its carboxyl terminus, we previously created and
analyzed a set of mutants targeting highly conserved residues in the
carboxyl terminus of the mammalian PP2A catalytic (C) subunit (10) (see
examples in Table I). Several of these
mutants showed decreased binding to B subunit (10) and, in some cases,
decreased C subunit methylation.2 The lack of a consistent
phenotype in mammalian cells prompted us to move to a model system that
was more amenable to genetic manipulation. Because the carboxyl
terminus of PP2A is highly conserved from yeast to humans and PP2A
methylation is conserved in yeast (40), we chose to construct cDNAs
expressing S. cerevisiae Pph21p versions of a subset of
these mutants (T364A, T364D, Y367E, Y367F, and L369
Ronne et al. (29) previously created an S. cerevisiae strain (H328) deleted for PPH21 and
PPH3 that expresses Pph22p under control of the
GAL promoter. H328 is viable when grown in the presence of
galactose but is inviable on glucose, indicating that these cells are
dependent on production of Pph22p from the galactose-inducible promoter
for viability. To determine whether our Pph21p mutants could support
viability, H328 cells were transformed with plasmids expressing wt or
mutant C subunits or vector only and then grown on galactose or
glucose. Fig. 1 shows that although H328
cells transformed with empty vector are inviable on glucose, all the mutants could support growth of these cells on glucose, indicating that
these mutant proteins are functional. Because leucine 369 is the site
of PP2A carboxymethylation in S. cerevisiae, this result
also suggests that methylation of Pph21p is not essential for cell
viability.
The Importance of C Subunit Carboxy-terminal Residues for
Interaction with B Subunit Is Highly Conserved between Mammals and
Yeast--
We next analyzed amino-terminal HA-tagged versions of all
the Pph21p carboxyl-terminal point mutants except L369
Fig. 2C shows the results of analyzing Cdc55p binding to all
the Pph21p mutants except L369
When these same immunoprecipitates were probed with anti-Tpd3p antibody
(characterized in Fig. 2B), T364D and Y367E were found to
bind greatly reduced levels of Tpd3p compared with wt Pph21p, T364A,
and Y367F (Fig. 2C). However, the effect of acidic
substitution of Thr-364 and Tyr-367 on Tpd3p binding was less dramatic
than on the association of Cdc55p. Decreased A subunit binding was also
found previously for the corresponding mammalian C subunit tyrosine
mutant (Y307E) but not for the corresponding mammalian threonine mutant
(T304D) (10).
To determine whether loss of Cdc55p and Tpd3p binding affects the
levels of these proteins in cells, lysates from cells expressing wt or
mutant Pph21p proteins were probed with antibodies to these proteins.
Similar levels of these two proteins were found in all samples (Fig.
2D), indicating that decreased binding did not impact protein stability in vivo.
Mutations in Tyrosine 367 Cause a Temperature-sensitive
Phenotype--
Previously, a strain deleted for CDC55 was
reported to be cold-sensitive (30) but not temperature-sensitive (28).
Because the strain expressing our mutants, H328, has a different
parental background (W303a) than the strain for which
To determine whether any of the C subunit mutants were
temperature-sensitive or cold-sensitive for growth, H328 cells
expressing untagged wt or mutant Pph21p proteins were tested for growth
at 14, 25, and 37 °C. Although none of the mutants displayed a
cold-sensitive phenotype (data not shown), several mutants were
temperature-sensitive (Fig. 3B). Y367E showed the most
severe temperature-sensitive phenotype, whereas Y367F and L369 Cells Expressing C subunit Mutants Defective in Cdc55p and Tpd3p
Binding Are Nocodazole-sensitive--
Several laboratories have shown
that deletion of CDC55 or TPD3 results in
sensitivity to the microtubule depolymerizing drug, nocodazole (32,
33). To determine whether decreased binding of Cdc55p and Tpd3p to C
subunit results in the same phenotype in cells expressing wt Cdc55p and
Tpd3p, we tested whether H328 cells expressing Pph21p mutants unable to
stably bind Cdc55p were more sensitive to nocodazole than H328 cells
expressing wt Pph21p. Fig. 4A
shows that cells expressing T364D, Y367E, or L369 PPM1 but Not PPM2 Encodes the Major PP2A Methyltransferase--
We
hypothesized that mutation of certain C subunit carboxyl-terminal
residues may affect Cdc55p and Tpd3p binding indirectly by altering
methylation of leucine 369. In particular, the fact that loss of
leucine 369 causes sensitivity to nocodazole suggested that methylation
of this residue might affect Cdc55p and Tpd3p function via regulation
of PP2A complex formation. We therefore used a combined genetic and
biochemical approach in S. cerevisiae to determine whether
methylation of Pph21p is required for Cdc55p and/or Tpd3p association
with C subunit and, consequently, for Cdc55p and/or Tpd3p function as
assayed by resistance to nocodazole.
First, the steady-state in vivo C subunit methylation levels
of wt cells and of cells deleted for one or both of the putative S. cerevisiae methyltransferase genes ( Deletion of PPM1 Greatly Reduces Cdc55p, Tpd3p, and Rts1p
Binding--
If C subunit methylation is necessary for stable
formation of PP2A heterotrimers containing Cdc55p, then deletion of
PPM1 should result in a decrease of Cdc55p binding to C
subunit. To test this hypothesis, lysates from wt,
To assess whether decreased binding of Cdc55p and Tpd3p to C subunit
affects the levels of these proteins in cells, lysates from vector
only, wt,
To determine whether methylation is important for binding of the yeast
B' subunit, Rts1p, lysates from wt,
To determine whether Rts1p-6×His was expressed at similar levels in
these cells, lysates were probed with Tpd3p and anti-6×His tag
antibodies. Relative to Tpd3p, Rts1p-6×His was expressed at a lower
level in Deletion of PPM1 Leads to Nocodazole Sensitivity--
Wt,
PP2A plays important roles in many cellular processes, and
consequently, the cell controls its activity, localization, and substrate specificity through a complex set of covalent and noncovalent mechanisms. We have used the budding yeast S. cerevisiae as
a model system to investigate the hypothesis that covalent modification of the C subunit (Pph21p) carboxyl terminus modulates PP2A complex formation. Our results demonstrate that Cdc55p binding to Pph21p was
disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites or by deletion of the gene for the yeast PP2A
methyltransferase homolog, Ppm1p, which resulted in almost complete
loss of Pph21p methylation. Loss of Cdc55p association was accompanied
in each case by a large reduction in Tpd3p binding to C subunit.
Moreover, decreased Cdc55p and Tpd3p binding invariably resulted in
nocodazole sensitivity, a known phenotype of CDC55 or
TPD3 deletion. Furthermore, our data show that loss of
methylation also greatly reduces Rts1p association. Thus, methylation
of Pph21p is important for formation of PP2A trimeric and dimeric
complexes and, consequently, for PP2A function. These results provide
the first example of the physiological importance of reversible
carboxymethylation of PP2A C subunit.
Very little is known about the potential effects of reversible protein
methylation at a single residue. Our findings provide evidence that
this modification is capable of regulating protein-protein interactions
in a manner similar to phosphorylation. The total amount of C subunit
in mammalian cells is known to be tightly regulated and does not appear
to vary over the course of the cell cycle (41). Because methylation of
PP2A C subunit has been shown to fluctuate during the cell cycle in
mammalian cells (25), methylation may be a key mechanism for regulating
PP2A function in a cell cycle-dependent manner.
Our results show that Ppm1p is the major PP2A methyltransferase in
S. cerevisiae. Although deletion of PPM2 did not
lead to significant change of the methylation level of Pph21p, we could not rule out that Ppm2p might have some PP2A methyltransferase activity. Instead, in repeated experiments, we found that the level of
unmethylated Pph21p in Deletion of PPM1 led not only to decreased Cdc55p, Tpd3p,
and Rts1p binding but also to nocodazole sensitivity. The nocodazole sensitivity may be due to loss of Cdc55p and/or Tpd3p binding because
previous studies have demonstrated that deletion of CDC55 or
TPD3 leads to nocodazole sensitivity (32, 33). However, we
cannot rule out an effect of Rts1p or another, unknown regulatory subunit whose binding may be affected by methylation. In any case, our
results clearly show that changes in C subunit methylation can affect
the function of PP2A in vivo.
The effects of mutation or methylation on the binding of one or more of
the PP2A subunits could be indirect. Tpd3p has been shown to be
required for Rts1p binding to C subunit (31), and Cdc55p presumably has
a similar requirement based on mammalian in vitro data.
However, the fact that mutation or loss of methylation leads to a more
severe effect on Cdc55p binding than Tpd3p association suggests that
loss of Cdc55p binding is not solely due to loss of Tpd3p association.
Instead, decreased Cdc55p association may be directly affected by
mutation or loss of C subunit methylation and cause a destabilization
of A(Tpd3p)/C subunit heterodimeric complexes.
Deletion of the CDC55 gene has previously been shown to
result in a cold-sensitive phenotype (30). However, our findings have
shown that ADR496, a CDC55 deletion strain, displays a
temperature-sensitive phenotype, suggesting that Cdc55p may be
important in this strain for response to stress induced by elevated
temperature. RTS1 or TPD3 deletion is also known
to cause a temperature-sensitive growth phenotype. H328 cells
expressing the T364D mutant, which does not interact stably with
Cdc55p, are not temperature-sensitive. Furthermore, both
The PP2A residues threonine 364 and tyrosine 367 are completely
conserved in all organisms, suggesting that they have important functions. In both yeast and mammalian cells (10), substitution of a
negatively charged amino acid for either of these residues abolishes
Cdc55p (B subunit) binding, whereas conservative substitution with
alanine or phenylalanine, respectively, does not. Moreover, we have
shown in the present study that acidic substitution of these residues
leads to a defect in PP2A function as assayed by nocodazole
sensitivity. Together, these results suggest that phosphorylation of
these residues might regulate B subunit-directed PP2A functions by
dissociating Cdc55p (B subunit) or by modulating PP2A activity directly
(16). Although we have not been able to detect threonine or tyrosine
phosphorylation of yeast C subunits using either phosphoamino acid-specific antibodies or
radiolabeling,4 it is possible that
these modifications are transient and triggered by specific signals, as
appears to be the case for tyrosine phosphorylation of mammalian C
subunit (42).
The results of our findings suggest that the PP2A methyltransferase
could be an attractive chemotherapeutic drug target for viruses such as
human immunodeficiency virus that target trimeric PP2A via the B
subunit. Recently, E. Cohen and co-workers (43) demonstrated that the
human immunodeficiency virus Vpr protein associates with PP2A via the B
subunit (43). Furthermore, they showed that this association is
important for the cell cycle arrest function of Vpr. Drugs or other
approaches that inhibit PMT1 or decrease its expression could have the
net effect of interfering with human immunodeficiency Vpr function and
result in decreased human immunodeficiency virus titers in AIDS
patients (44, 45).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(deleted codon) mutations. Wt and
mutant PPH21 cDNAs were then subcloned into pRS316 (CEN6
URA3 amp) (35), pPS310 (CEN6 URA3 GAL1-10 amp)
(36), pRS425 (2µ URA3 amp) (35), or pRS423 (2µ
TRP1 amp) (35) vectors. Amino-terminal epitope-tagged
versions of the mutants in pRS316 and pRS425 vectors were also made by
inserting double-stranded oligonucleotides encoding the influenza
hemagglutinin (HA) epitope (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala)
followed by the thrombin recognition site (Leu-Val-Pro-Arg-Gly-Ser)
just before the start ATGs, making use of the NcoI site
created previously. A pRS315 construct
(RTS1::his6) expressing Rts1p with a
carboxyl-terminal 6×His epitope tag (Rts1p-6×His) was obtained from
R. Hallberg (37).
::HIS3 GAL1/10:PPH22 PPH3::LEU2) was obtained from J. Ariño (29,
46). Strains BY4741 (MATa his3 leu2 met15 ura3),
YDP16650 (MAT
PPM2
::KAN his3 leu2 lys2 ura3), and YDP4271 (MAT
PPM1
::KAN his3 leu2 met15 ura3) were
obtained from Research Genetics (Huntsville, AL). Strain YDP5D
(MATa PPM1
::KAN
PPM2
::KAN his3 leu2 lys2 ura3 met15) was made by mating YDP16650 and YDP4271, sporulating, and selecting the
appropriate spore. Growth media were prepared according to standard
recipes (38) or obtained from BIO101 (Carlsbad, CA).
light chain secondary antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL) were used to
reduce background at the position of HA-tagged Pph21p.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
; see
"Materials and Methods" and Table I) and to study these mutants in
S. cerevisiae cells whose viability depends on their
expression.
B subunit binding of selected mammalian C subunit mutants and
properties of the corresponding S. cerevisiae C subunit (Pph21p)
mutants used in this study
View larger version (24K):
[in a new window]
Fig. 1.
Carboxyl-terminal C subunit mutants are
functional. Cells were transformed with pRS316 plasmid encoding
the various carboxyl-terminal Pph21p mutants and grown on media
containing galactose or glucose. WT-CC and all mutants have
the two nucleotides just upstream of the ATG start codon changed to CC
to create a NcoI site for additional constructs (see
"Materials and Methods"). As can be seen, this change did not
affect the ability of wt Pph21p to support growth of these cells on
glucose.
for their ability to bind Cdc55p by immunoprecipitating them via their epitope tag and immunoblotting for coimmunoprecipitated Cdc55p. For unknown reasons, the addition of an amino-terminal HA tag to L369
resulted in undetectable expression of this mutant using several different constructs, and HA-tagged L369
was unable to support the growth of
H328 cells. As described under "Materials and Methods," we raised a
polyclonal antibody to Cdc55p to use in this assay. Fig. 2A shows the characterization
of this antibody and the validation of the coimmunoprecipitation assay.
As expected, the antibody detected a strong Cdc55p band in lysates from
wt cells (lane 1) that was missing in lysates from cells
deleted for CDC55 (
CDC55; lane 2).
Preimmune serum from the same rabbit did not detect the Cdc55p band
(not shown). In addition, the antibody easily detected Cdc55p
coimmunoprecipitated with HA-tagged wt C subunit (lane 4),
whereas a parallel immunoprecipitate (lane 3) from cells
expressing untagged wt Pph21p had no detectable Cdc55p.
View larger version (32K):
[in a new window]
Fig. 2.
Substitution of threonine 364 or tyrosine 367 with an acidic residue abrogates C subunit interaction with Cdc55p and
decreases its interaction with Tpd3p, whereas substitution with more
conservative residues does not. A, characterization of
the Cdc55p antibody. 12CA5 immunoprecipitates were prepared from H328
cells expressing untagged Pph21p (Pph21p) or HA-tagged
Pph21p (HA-Pph21p) and analyzed along with cell lysates from
W303a (wt) and ADR496 ( CDC55) by
immunoblotting with anti-Cdc55p rabbit polyclonal antibody (1:5000).
All lanes are from the same gel but were not all originally
adjacent. The dark exposure shown reveals no trace of Cdc55p signal in
the deletion strain. B, characterization of the Tpd3p
antibody. Immunoblots of cell lysates from W303a cells lacking Tpd3p
(
TPD3) and from W303a cells (wt) were probed with
anti-Tpd3p rabbit polyclonal antibody (1:5000). C, Cdc55p
coimmunoprecipitates with T364A and Y367F but not with T364D or Y367E.
H328 cells expressing the indicated Pph21p proteins were grown in
glucose-containing medium. 12CA5 immunoprecipitates prepared from
lysates of these cells were immunoblotted with anti-Cdc55p and
anti-Tpd3p antibodies (IPs). Even on long exposures, no
Cdc55p could be seen coimmunoprecipitating with Y364D and Y367E,
whereas a small amount of Tpd3p was detected. Pph21p mutants migrate as
doublets in these gels, but whether double or single bands are seen can
vary. This pattern of migration in SDS-polyacrylamide gel
electrophoresis has been noted previously for endogenous and
epitope-tagged mammalian PP2A C subunits (10, 25, 34) and does not
appear to be due to degradation. D, Cdc55p and Tpd3p are
still present in H328 cells expressing mutant Pph21p proteins. Lysates
from H328 cells expressing the various wt and mutant Pph21p proteins
(same order of lanes as in panel C) were
immunoblotted for Cdc55p and Tpd3p.
. Although the control
immunoprecipitate from cells expressing untagged Pph21p (lane
1) again contained no detectable Cdc55p, Cdc55p was specifically
coimmunoprecipitated with wt HA-Pph21p and with the HA-tagged T364A and
Y367F mutants (lanes 2, 3, and 6,
respectively). In contrast, Cdc55p could not be detected in
immunoprecipitates of T364D (lane 4) or Y367E (lane 5) even on long exposures. These results parallel those obtained previously with the corresponding mammalian mutants (Table I), indicating that the roles of these residues in B subunit binding are
highly conserved.
CDC55 cold sensitivity was reported, we first analyzed
the growth of a W303a CDC55 deletion strain (ADR496) at
different temperatures. Although we could not detect a cold-sensitive
phenotype (data not shown), this strain demonstrated a moderate
temperature-sensitive phenotype (Fig. 3A).
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Fig. 3.
A CDC55 deletion strain and
H328 cells expressing Y367E, Y367F, L369 are
temperature-sensitive. A, deletion of CDC55
in a W303a background is temperature-sensitive. W303a cells with an
intact CDC55 gene (WT) or with
CDC55 deleted (
CDC55) were grown to saturation
at 25 °C. 10-Fold serial dilutions were spotted onto YPD plates and
incubated at 25 or 37 °C. B, H328 cells expressing Y367E,
Y367F, or L369
are temperature-sensitive, whereas those
expressing T364A or T364D are not. Cells were grown to saturation at
25 °C. 10-Fold serial dilutions were then spotted onto YPD plates
and incubated at 25 or 37 °C. For presentation, data from two
different plates were photographically merged for each
panel. In repeated experiments, Y367E always showed a more
severe temperature-sensitive phenotype than T367F, L369
, and the
CDC55 deletion strain. WT-CC and all mutants have the two
nucleotides just upstream of the ATG start codon changed to CC in order
to create a NcoI site for additional constructs (see "Materials and
Methods").
showed intermediate sensitivity. wt Pph21p, T364A, and T364D did not
demonstrate a temperature-sensitive phenotype. Thus, the temperature
sensitivity of the mutants appears to be residue-specific. Furthermore,
this temperature-sensitive phenotype does not correlate with their
ability to bind Cdc55p in our assay (Table I).
were very
sensitive to nocodazole treatment, whereas cells expressing T364A
showed the same sensitivity to the drug treatment as cells expressing
wt Pph21p. T364D and Y367E do not bind Cdc55p, and from the mammalian
data (Table I), L369
would be predicted not to bind. Decreased
Cdc55p/Tpd3p binding therefore correlates well with nocodazole
sensitivity. The only exception was that cells expressing Y367F had
intermediate nocodazole sensitivity and yet bound similar amounts of
Cdc55p and Tpd3p as T364A, which had wt sensitivity. This result
suggests that Y367F has an additional defect.
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Fig. 4.
Cells expressing Pph21p mutants defective in
binding Cdc55p and Tpd3p are nocodazole-sensitive. H328 cells
expressing wt Pph21p or the indicated mutants from the pRS316 vector
were grown overnight and then treated with nocodazole (see "Materials
and Methods"). At 4 and 6 h of treatment, cells were diluted and
plated on YPD plates. After incubation, colonies were counted to
determine percent survival. Error bars indicate the standard
deviations of values obtained from triplicate plates. WT-CC and
all mutants have the two nucleotides just upstream of the ATG start
codon changed to CC in order to create a NcoI site for additional
constructs (see "Materials and Methods").
PPM1,
PPM2, and
PPM1
PPM2) were
compared using an assay employing a methylation-sensitive monoclonal
antibody, 1d6, which is specific for unmethylated C subunit (see
"Materials and Methods" and Fig. 5).
Because base treatment demethylates C subunits, a lysate with
methylated C subunits will have an increase in 1d6 signal intensity
upon treatment with base, whereas a lysate containing only unmethylated
C subunits will have no increase. Fig. 5B shows that
deletion of PPM1 caused a great decrease in C subunit
methylation in vivo, whereas deletion of PPM2 had
at best a small effect. Quantitation of four independent experiments
determined that although C subunits in the wt (BY4741) and
PPM2 strains are methylated at 58 ± 9 and 54 ± 10%, respectively, C subunits in
PPM1 and
PPM1
PPM2 strains are methylated at
9.6 ± 12.5 and <1%, respectively. These results indicate that
PPM1 encodes the major Pph21p/Pph22p methyltransferase.
Moreover, cells deleted for PPM1, PPM2, or both
PPM1 and PPM2 grow normally at room temperature
(data not shown), suggesting that C subunit methylation is not
essential for cell growth at this temperature.
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Fig. 5.
PPM1 encodes the major PP2A methyltransferase
in S. cerevisiae. A, 1d6 monoclonal
antibody specifically recognizes unmethylated PP2A carboxyl-terminal
peptide. A C subunit carboxyl-terminal nonapeptide carboxymethylated on
leucine (described under "Materials and Methods") was synthesized
and high performance liquid chromatography-purified. 0.25 µg of this
peptide was demethylated by treatment with 0.5 M NaOH (+)
for 5 min on ice and then neutralized, whereas another 0.25 µg of
peptide ( ) was treated with an equivalent amount of preneutralized
solution. The unmethylated and methylated aliquots of the peptides were
then spotted onto nitrocellulose, and the membrane was probed with 1d6
monoclonal antibody. B, deletion of PPM1 results
in nearly complete loss of PP2A methylation. Lysates were prepared from
wt (BY4741),
PPM1,
PPM2, and
PPM1
PPM2 cells in the presence of 200 nM okadaic acid to prevent further methylation and
demethylation (19). Twenty µl of each lysate (+) was placed on ice
and demethylated by the addition of 10 µl of 0.5 M NaOH.
After a 5-min incubation, the samples were neutralized with an
equivalent volume of 0.5 M HCl and one-half volume of 2 M Tris, pH 6.8. Another 20 µl of each lysate (
) was
combined with an equivalent amount of preneutralized base solution.
Then the samples were analyzed by 10% SDS-polyacrylamide gel
electrophoresis and immunoblotted with methylation-sensitive C subunit
monoclonal antibody (1d6; 1:10,000). Because 1d6 almost exclusively
recognizes unmethylated Pph21p and Pph22p, the ratio of signal
intensity between the
base lane (in vivo amount
of unmethylated C subunit) and the +base lane (100%
unmethylated) indicates the percentage of C subunit that is
unmethylated in vivo. The percent unmethylated C subunits
was subtracted from 100 to obtain the percentage of methylated C
subunits (see values under "Results"). S. cerevisiae C
subunits migrated as tight doublets in this gel, but whether double or
single bands are seen can vary.
PPM1,
PPM2, and
PPM1
PPM2 cells
expressing HA-tagged Pph21p were immunoprecipitated with HA tag
antibody, and the immunoprecipitates were probed for the presence of
Cdc55p (Fig. 6A). Although
Cdc55p could be coimmunoprecipitated with Pph21p in wt (lane
2) and
PPM2 cells (lane 4), it could not
be coimmunoprecipitated with Pph21p in
PPM1 (lane
3) and
PPM1
PPM2 (lane 5)
cells. Thus, deletion of PPM1, which results in
predominantly unmethylated Pph21p, disrupts Cdc55p binding to C
subunit, whereas loss of PPM2, which has little effect on Pph21p methylation, has no detectable effect. To determine whether binding of Tpd3p was also affected, the same immunoprecipitates were
probed for Tpd3p. Fig. 6A shows that loss of methylation also resulted in a large reduction in Tpd3p association.
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Fig. 6.
Deletion of PPM1, but not
PPM2, results in decreased Cdc55p, Tpd3p, and Rts1p binding
to Pph21p. A, Cdc55p no longer associates stably with
Pph21p, and Tpd3p binding is greatly reduced in cells deleted for
PPM1. Wt (BY4741; lane 2), PPM1
(lane 3),
PPM2 (lane 4), and
PPM1
PPM2 (lane 5) cells
expressing HA-tagged Pph21p from the GAL1-10 promoter and
vector-only wt cells (lane 1) were grown in
galactose-containing media. Anti-HA tag (12CA5) immunoprecipitates
(IPs) prepared from lysates of these cells were
probed sequentially with anti-Cdc55p, anti-Tpd3p, and anti-HA tag
(16B12) antibodies. B, Cdc55p and Tpd3p are still present at
normal levels in cells deleted for PPM1. Lysates from the
same cells used in panel A (same order of lanes)
were immunoblotted for Cdc55p and Tpd3p. The variation in amount of
shifted Cdc55p seen in this experiment was not consistently seen in all
experiments. C, deletion of PPM1 results in
reduced Rts1p binding to Pph21p. Cell lysates were prepared from wt
(BY4741; lanes 1-2),
PPM1,
PPM2, and
PPM1
PPM2 cells
expressing both 6×His-tagged Rts1p (Rts1p-6×His) and HA-tagged Pph21p
(lanes 2-5) or vector only (lane 1). Anti-HA tag
(12CA5) immunoprecipitates were prepared from these lysates and probed
for the presence of Rts1p-6×His and Tpd3p using anti-6×His tag and
anti-Tpd3p antibodies. D, levels of Rts1p and Tpd3p in the
various cells used in panel C. Lysates from the same cells
used in panel C (same order of lanes) were
immunoblotted for Rts1p-6×His and Tpd3p.
PPM1,
PPM2, and
PPM1
PPM2 cells were probed with antibodies
to these proteins. Similar levels of these two proteins were found in
all cell lines (Fig. 6B), indicating that deletion of
methyltransferase did not impact protein stability in
vivo.
PPM1,
PPM2, and
PPM1
PPM2 cells
expressing both HA-tagged Pph21p (or vector only) and carboxyl-terminal
6×His-tagged Rts1p (Rts1p-6×His) were immunoprecipitated with HA tag
antibody. Probing of the immunoprecipitates for the presence of
Rts1p-6×His using an anti-6×His tag antibody (Fig. 6C)
showed that Rts1p-6×His coimmunoprecipitated specifically with
HA-tagged C subunits from wt and
PPM2 cells but was not detectable in HA-tag immunoprecipitates from cells deleted for PPM1. Moreover, Tpd3p association was again found to be
greatly reduced (Fig. 6C). Thus, methylation appears to be
important for binding of Cdc55p, Tpd3p, and Rts1p to Pph21p.
PPM1 and
PPM1
PPM2
cells than in wt and
PPM2 cells (Fig. 6D). It
is not possible to distinguish whether the reduced levels of
Rts1p-6×His expression in
PPM1 and
PPM1
PPM2 cells are a cause or an effect of
decreased association with C subunit. Although this lower level of
expression is not sufficient to account for all the reduction in
Rts1p-6×His association seen in Fig. 6C, it makes it
difficult to determine the exact amount of reduction in Rts1p-6×His
binding due to loss of C subunit methylation. When Rts1p-6×His was
immunoprecipitated from lysates of these cells with an anti-6×His
antibody, reduced but detectable levels of HA-Pph21p were found in
PPM1and
PPM1
PPM2 cells
compared with wt and
PPM2 cells (data not shown),
indicating that Rts1p-6His binding is reduced but not abolished.
PPM1,
PPM2, and
PPM1
PPM2 cells were next analyzed for
nocodazole sensitivity as a functional assay for loss of Cdc55p and
Tpd3p association.
PPM1 and
PPM1
PPM2 cells were much more sensitive
than wt cells to nocodazole treatment, indicating that deletion of
PPM1 generates a phenotype consistent with loss of Cdc55p
and Tpd3p association (Fig. 7) (32, 33).
Although in the experiment shown,
PPM2 cells appeared to
show some sensitivity to nocodazole at 4 h, they were wt in their
sensitivity at 6 h, demonstrating that they did not have a
substantially greater sensitivity to nocodazole than wt cells. Thus,
loss of PPM1, but not of PPM2, leads to loss of C
subunit methylation. This, in turn, appears to result in a large
decrease in C subunit association with Cdc55p and Tpd3p, inducing
sensitivity to nocodazole.
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Fig. 7.
Cells deleted for PPM1 are
nocodazole-sensitive. Cells were grown overnight and then treated
with nocodazole (see "Materials and Methods"). At 4 h and
6 h of treatment, cells were diluted and plated on YPD plates.
After incubation, colonies were counted to determine percent survival.
Error bars indicate the S.D. of values obtained from
triplicate plates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PPM1
PPM2 was
slightly higher than in
PPM1, suggesting that Ppm2p may
have low methyltransferase activity in vivo, perhaps on a
specific subpopulation of PP2A or at a specific time in the cell cycle.
Because Ppm2p has an ~350-amino acid extension that may be involved
in actin binding, Ppm2p might localize to actin-containing structures
(23) and be responsible for methylating a small fraction of C subunit
localized to that part of the cytoskeleton.
PPM1 and
PPM1
PPM2 strains
displayed no temperature-sensitive phenotype even though Pph21p
association with Cdc55p could not be detected.4 The
simplest explanation for these data is that in T364D and strains
deleted for PPM1, some residual Cdc55p and Rts1p binding occurs that is sufficient to prevent the temperature-sensitive phenotype but cannot be detected by our assay.
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ACKNOWLEDGEMENTS |
---|
We thank Monica McQuoid, Matthew Stark, Amanda Bauman, and Marie Kozel for excellent technical assistance, J. Ariño, A. Rudner, R. Hallberg, and A. Murray for sending reagents, and Karma Carrier for critical reading of the manuscript. Under agreements between Upstate Biotechnology Inc. and Emory University and Calbiochem and Emory University, David Pallas is entitled to a share of sales royalty received by the University from these companies. In addition, this same author serves as a consultant to Upstate Biotechnology Inc. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA57327 (to D. C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-5620; Fax: 404-727-3954; E-mail: dpallas@emory.edu.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M008694200
2 X. X. Yu, X. Du, C. S. Moreno, R. E. Green, E. Ogris, Q. Feng, L. Chou, M. J. McQuoid, and D. C. Pallas, Mol. Biol. Cell 12, in press.
3 E. Ogris, H. Wei, and D. C. Pallas, unpublished data.
4 H. Wei and D. C. Pallas, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: PP2A, protein phosphatase 2A; C subunit, catalytic subunit; HA, hemagglutinin; PMT1, protein phosphatase methyltransferase-1 (mammalian); wt, wild type; Vpr, virion-associated accessory protein; YPD, yeast extract/peptone/dextrose.
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