From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600
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ABSTRACT |
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The catalytic subunit of protein serine/threonine
phosphatase 4 (PP4C) has greater than 65% amino acid
identity to the catalytic subunit of protein phosphatase 2A
(PP2AC). Despite this high homology, PP4 does not appear to
associate with known PP2A regulatory subunits. As a first step toward
characterization of PP4 holoenzymes and identification of putative PP4
regulatory subunits, PP4 was purified from bovine testis soluble
extracts. PP4 existed in two complexes of approximately 270-300 and
400-450 kDa as determined by gel filtration chromatography. The
smaller PP4 complex was purified by sequential phenyl-Sepharose, Source
15Q, DEAE2, and Superdex 200 gel filtration chromatographies. The final
product contained two major proteins: the PP4 catalytic subunit
plus a protein that migrated as a doublet of 120-125 kDa on
SDS-polyacrylamide gel electrophoresis. The associated protein, termed
PP4R1, and PP4C also bound to
microcystin-Sepharose. Mass spectrometry analysis of the purified
complex revealed two major peaks, at 35 (PP4C) and 105 kDa
(PP4R1). Amino acid sequence information of several peptides derived from the 105 kDa protein was utilized to isolate a
human cDNA clone. Analysis of the predicted amino acid sequence revealed 13 nonidentical repeats similar to repeats found in the A
subunit of PP2A (PP2AA). The PP4R1 cDNA
clone engineered with an N-terminal Myc tag was expressed in COS M6
cells and PP4C co-immunoprecipitated with Myc-tagged
PP4R1. These data indicate that one form of PP4 is similar
to the core complex of PP2A in that it consists of a catalytic subunit
and a "PP2AA-like" structural subunit.
The phosphorylation of proteins on serine or threonine residues by
protein kinases is responsible for the communication of many
intracellular signals (1). The dephosphorylation of proteins by
serine/threonine phosphatases is equally important for the occurrence
of this signaling phenomenon. Four major cellular protein serine/threonine phosphatase activities (PP1, PP2A, PP2B, and PP2C)1 have been reported and
classified according to substrate selectivity, inhibitor sensitivity,
and requirement for divalent cations (2-6). Various molecular biology
techniques have led to the identification of several additional
phosphatases including protein phosphatase 4, formerly known as protein
phosphatase X (7, 8).
PP2A is one of the best studied protein serine/threonine phosphatases.
Several reports have demonstrated an important regulatory role for PP2A
in a variety of cellular processes. Because PP2A is involved in many
cellular events, its activity must be tightly controlled in
vivo. Two major mechanisms of regulation of PP2A have been
described in the literature. The first involves post-translational modifications of the catalytic subunit of PP2A (PP2AC).
Phosphorylation of a tyrosine residue near the C terminus (9) or
phosphorylation of an unidentified threonine residue (10) decreases
catalytic activity in vitro. Conversely, carboxymethylation
of the C-terminal leucine causes an increase in phosphatase activity
(11). The second major mechanism of regulation of PP2AC is
through its association with other proteins. PP2A exists as a
heterotrimeric complex consisting of a catalytic subunit (C)
associated with a structural subunit (A) and a variable subunit (B).
The A subunit is thought to act as a scaffold for the B and C subunits
(5, 12, 13). Three families of B subunits have been identified and
include B, B', and B" (2-6, 12). The B subunit can modulate substrate
selectivity in vitro (14, 15) and is hypothesized to target
PP2A to discrete compartments within the cell (2-6, 13). In addition
to the regulation of PP2AC by associated subunits and
post-translational modification, the PP2A holoenzyme forms complexes
with other cellular proteins, such as Tau (16) and
Ca+2/calmodulin-dependent protein kinase IV
(17).
The catalytic subunit of protein phosphatase 4 (PP4C) is
65% identical to PP2AC at the amino acid level (18) and
has been placed in the type 2A family of phosphatases. PP4C
has been highly conserved between species sharing 91% amino acid
identity between human and Drosophila (19). PP4C
is predominantly localized in the nucleus in rat brain and liver but is
most highly expressed in testis (20). Additionally, PP4C
was demonstrated to be an essential enzyme in the development of
Drosophila embryos (21). The expression of PP4C
was reduced to 25% of the normal protein level in a mutant strain of
Drosophila termed centrosomes minus microtubules
(cmm) (21). An interesting characteristic of the cmm phenotype is the presence of regions of cells that are
unable to complete mitosis because no microtubules exist to connect
chromatin and centrosomes. This phenotype implicates PP4C
in the regulation of the nucleation and/or stabilization of
microtubules. These data taken together indicate that PP4 has a crucial
cellular function, although a physiological substrate for PP4 has not
yet been identified.
Appropriate regulation of PP4 is likely critical to its normal
physiological function. The close homology of PP2AC and
PP4C sequence suggests that they may share certain
regulatory properties. Indeed, it was recently shown that
PP4C, like PP2AC, is carboxymethylated (20).
Furthermore, by analogy with both PP1 and PP2A, it is unlikely that PP4
exists as free catalytic subunits in the cell (5). However,
PP4C does not associate with the A subunit of PP2A in
vitro (18).2 In order to
better understand and characterize the physiologically relevant forms
of PP4, we now report the purification of a heterodimeric form of PP4
and identification of the first PP4C-associated protein, termed PP4R1.
Materials--
Goat anti-rabbit IgG-alkaline phosphatase
conjugate, Bio-Scale DEAE2 column, and prestained SDS-polyacrylamide
gel electrophoresis (PAGE) molecular mass standards were obtained from
Bio-Rad. Phenyl-Sepharose, Source 15Q, Superdex 200, and GammaBind Plus
Sepharose resins and gel filtration standards were from Amersham
Pharmacia Biotech. Microcystin-Sepharose and microcystin were obtained
from Upstate Biotechnology, Inc. (Lake Placid, NY) and Alexis Corp.
(San Diego, CA), respectively. Bovine testes were purchased from
Pel-Freeze (Rogers, AR) and stored at Preparation of Bovine Testis Soluble Extracts--
Bovine testes
(200 g) were homogenized (five 5-s bursts, followed by three 10-s
bursts) using a Waring blender in 600 ml of homogenization buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 6.3 µg/ml aprotinin, 2 µg/ml
leupeptin, 5 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, and 2 µg/ml pepstatin A) and filtered
through four layers of cheesecloth. All purification steps were
performed at 4 °C unless otherwise indicated. The homogenate was
centrifuged at 10,000 × g for 10 min, and the
resulting supernatant was subsequently centrifuged at 100,000 × g for 1 h. This supernatant was carefully removed and
immediately brought to 25% ammonium sulfate saturation. Proteins precipitating between 25 and 50% saturation were either stored at
Chromatography and Purification of PP4 Holoenzymes--
All
protein chromatographic steps were performed on the FPLC System
(Amersham Pharmacia Biotech). The resuspended 50% ammonium sulfate
pellets (above) were brought to at least 0.3 M ammonium sulfate by adding the appropriate volume of 4 M ammonium
sulfate. The mixture was incubated on ice for 30 min and subsequently
centrifuged at 13,000 rpm (Sorvall SS34) for 30 min. One-half of the
supernatant was applied to a phenyl-Sepharose column (34 ml)
equilibrated in 300 mM ammonium sulfate buffer X, and the
column was developed with a linear gradient (184 ml) of 300-0
mM ammonium sulfate (buffer X without phenylmethylsulfonyl
fluoride) at 4 ml/min collecting 8-ml fractions. PP4-containing
fractions from two successive phenyl-Sepharose columns were identified
by immunoblot analysis, pooled, and diluted 2-fold with buffer X. The
sample was loaded at 2 ml/min onto a Source 15Q column (8 ml)
equilibrated in buffer X containing 100 mM NaCl; proteins
were eluted with a linear gradient of 100-450 mM NaCl (320 ml) at 4 ml/min collecting 8-ml fractions. Two peaks of PP4 eluted from
Source 15Q at 250 and 300 mM NaCl (Fig. 1A). Fractions 19-21 (250-300 mM NaCl) containing PP4 pool 1 (see "Results") were combined and further purified over a Bio-Scale
DEAE2 column (2 ml, equilibrated in 350 mM NaCl buffer
X) using a linear gradient of 350-1000 mM NaCl (50 ml),
collecting 1-ml fractions at 1 ml/min. Finally, the PP4 fractions
obtained from DEAE chromatography (600-700 mM NaCl) were
concentrated to 2 ml with a Centricon-30 concentrator and purified to
near homogeneity by Superdex 200 gel filtration chromatography. The gel
filtration column (120 ml) was equilibrated in buffer X containing 150 mM NaCl and developed at 1 ml/min collecting 2-ml
fractions. Protein standards utilized for calibration of the gel
filtration column included thyroglobulin (669 kDa), ferritin (440 kDa),
catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa),
ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa). Molecular weights of PP4 complexes were estimated from a graph of
the logarithm of molecular weight as a function of elution volume of
the standards.
Gel Electrophoresis and Immunoblotting--
Samples were boiled
for 10 min in Laemmli sample buffer (22) and then subjected to SDS-PAGE
(10% gel, 0.75 mm, unless otherwise indicated). After electrophoresis,
proteins were either visualized by silver stain (23) or transferred to
a nitrocellulose membrane in 10 mM CAPS buffer, pH 11.0, containing 10% methanol. The blots were incubated with the appropriate
affinity-purified anti-peptide antibodies (20, 24), followed by
incubation with goat anti-rabbit IgG-alkaline phosphatase conjugate.
Immunocomplexes were visualized with 5-bromo-4-chloro-3-indolyl
phosphate and nitro blue tetrazolium as substrates for alkaline phosphatase.
Quantification of PP4C--
The amount of
PP4C in bovine testis extracts was estimated by preparing a
standard curve of purified PP4C (protocol to be published
elsewhere) and comparing the immunoblot intensities with samples from
bovine testis soluble extracts. To visualize and quantitate
PP4C immunoreactivity, an 125I-labeled donkey
anti-rabbit IgG (Amersham Pharmacia Biotech) was utilized as the
secondary antibody. The proteins corresponding to PP4C were
excised and counted using a Mass Spectrometry Analysis--
The purified PP4 holoenzyme was
concentrated to approximately 1 pmol/µl using a Centrex UF-0.5
concentrator. Glutaraldehyde was added to the sample to achieve a 0.5%
(v/v) concentration, and aliquots were removed and analyzed over a time
period of 240 min. The reaction was quenched by the addition of the
matrix, as described below. The sample was mass-analyzed with a
PerSeptive Voyager Elite matrix-assisted laser desorption/ionization
mass spectrometer. A nitrogen laser (337 nm) was used to irradiate the
sample with sinapinic acid as the matrix. Approximately 0.5 µl of
sample was mixed with 0.5 µl of a saturated solution of sinapinic
acid in a 1:1 acetonitrile/water solvent containing 0.5%
trifluoracetic acid on a target sample plate. After drying in air for
several minutes, the target plate was placed in the mass spectrometer
and then analyzed. An accelerating voltage of 25 kV was used to
accelerate the ions in the time-of-flight analysis.
Preparation of Phosphorylated Substrates and Phosphatase
Assay--
Phosphorylated substrates were prepared as described
previously for casein (25) and phosphorylase a and histone
H1 (26). Purified proteins (approximately 0.14 ng of PP2AC
and approximately 11.3 ng of PP4C contained in PP4) were
assayed for phosphatase activity in a 100-µl reaction volume
containing 25 mM Tris-HCl, pH 7.4, 1 mM
dithiothreitol, 1 mM EDTA, 0.5 mg/ml bovine serum albumin,
and 32P-labeled substrate. Following a 10-min incubation at
37 °C, the reaction was terminated by the addition of
trichloroacetic acid (20% final concentration), and proteins were
pelleted by centrifugation at 13,000 × g for 10 min.
Supernatants were collected and quantitated for
[32P]i, released by scintillation counting. Less
than 20% of the total phosphorylated substrate was dephosphorylated
during the assays, thus assuring that substrate was not limiting.
Determination of Peptide Amino Acid Sequences--
PP4 obtained
from the final gel filtration column was concentrated using a
Centricon-30 concentrator, separated on a 6% (for analysis of
120-125-kDa doublet) or 10% (analysis of 29-kDa protein) polyacrylamide gel and transferred to polyvinylidene fluoride membrane.
The membrane was briefly stained with Coomassie R-250. Individual
proteins were excised and digested in situ with lysyl endopeptidase alone or with lysyl endopeptidase followed by
endoproteinase Asp-N. The resulting peptides were separated by high
performance liquid chromatography and subjected to automated Edman
degradation on a Perkin Elmer/Applied Biosystems Procise 492 protein
sequencer. Sequencing was performed by Vanderbilt University Peptide
Sequencing and Amino Acid Analysis Shared Resource.
Cloning and Sequencing of PP4R1--
The cDNA
for PP4R1 was identified by using the peptide sequences
(derived from the purified proteins) to search the expressed sequence
tag data base with the BLAST
protocol.3 The cDNA
clones were obtained from The Institute for Genomic Research/American
Type Culture Collection Human cDNA Special Collection (Rockville,
MD) and sequenced in their entirety by fluorescent dideoxy
terminator-based DNA sequencing (performed on an ABI310 sequencer in
the Center for Molecular Neuroscience DNA Sequencing Facility,
Vanderbilt University).
PP4R1 Antibody Generation and Immunoaffinity
Purification--
A peptide (CASTHPASTRISED) identical to an amino
acid sequence found in one of the bovine PP4R1 peptides was
synthesized with an amino-terminal cysteine residue. The peptide was
coupled to keyhole limpet hemocyanin using
m-maleimidobenzoyl-N-hydroxysuccinimide ester as
described previously (27). The peptide-keyhole limpet hemocyanin (250 µg) conjugates were injected subcutaneously into rabbits, first in
complete Freund's adjuvant followed by several boosts in incomplete
adjuvant. The antiserum was characterized by immunoblotting bovine
testes soluble extracts in the absence and presence of the peptide.
PP4R1 anti-peptide antibodies (designated
A second peptide (CASENIFNRQMVARS) corresponding to amino acid residues
39-52 in the predicted human sequence was synthesized with an
amino-terminal cysteine residue. This peptide was utilized to generate
a second rabbit polyclonal antiserum (designated
Microcystin-Sepharose Affinity Isolation--
Bovine testis
soluble extracts (5 mg) were incubated with 30 µl of
microcystin-Sepharose (50% slurry) while rotating overnight at
4 °C. The beads were pelleted and washed six times with 1 ml of
buffer X containing 150 mM NaCl, and bound proteins were
eluted with Laemmli sample buffer. Applied, unbound, and bound samples were analyzed by immunoblot.
Construction of Myc-tagged PP4R1 cDNA--
A
cDNA fragment encoding the NH2-terminal Myc-tagged
(MEQKLISEEDL) PP4R1 was produced by successive polymerase
chain reactions (PCRs) and restriction enzyme digestion. The first PCR
product was made using sense oligonucleotide 1 (5'-CTC ATC TCA GAA GAG GAT CTG GCG GAC CTC TCG CTG CT-3'), antisense oligonucleotide 2 (5'-AAA
AAG CAT ATG GTA TTG-3'), and human PP4R1 cDNA as
template. Successive PCR using sense oligonucleotide 3 (5'-GGC CGA ATT
CAG GAT GGA ACA AAA ACT CAT CTC AGA AGA GGA TCT GGC-3'), antisense oligonucleotide 2, and the first PCR product as template generated a
PCR product that was digested with the restriction enzymes
EcoRI and NdeI. The resulting cDNA fragment
and the cDNA fragment of PP4R1 from the NdeI
site to the XhoI site (3'-end of PP4R1) were ligated into the EcoRI/XhoI cloning site of the
mammalian expression vector pcDNA3.1 to produce the
NH2-terminal epitope-tagged PP4R1 cDNA
under the control of the cytomegalovirus promoter. The construct was
verified by restriction enzyme analysis and DNA sequencing.
Cell Transfection--
COS M6 cells maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum were
transiently transfected (10-cm tissue culture dishes) using the
DEAE-dextran procedure as described previously (28, 29). Transfected
COS cells (one 10-cm dish) were dislodged by scraping in 1 ml of lysis
buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5% Triton X-100, 6.3 µg/ml
aprotinin, 4 µg/ml leupeptin, 10 mM benzamidine, 4 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl
fluoride. Cells were sonicated with two 10-s bursts followed by
centrifugation for 15 min. The protein concentration of clarified
supernatants was estimated by the BioRad protein assay using bovine
serum albumin as the standard.
Immunoprecipitation of Myc-PP4R1 and
PP4C--
Approximately 1 mg of protein from cell lysates
(in 1 ml of lysis buffer) was incubated with 40 µl of anti-Myc
ascites for 6 h while rotating at 4 °C. Immune complexes were
isolated by adding 40 µl of a 50% slurry of GammaBind Plus
Sepharose. After washing the beads six times with lysis buffer, bound
proteins were eluted with 20 µl of Laemmli sample buffer. The
proteins were analyzed by immunoblot analysis with affinity-purified
antibodies directed against the C terminus of PP4C.
Observation of Multiple PP4 Complexes in Bovine Testis Soluble
Extracts--
During the initial stages of purification of PP4
holoenzymes from various tissues, a broad elution profile of PP4
immunoreactivity was observed on several chromatography columns
including anion exchange and gel filtration (data not shown). One
possible explanation of this behavior is that PP4, like PP2A, exists in
multiple oligomeric complexes. To address this possibility, the elution
of distinct partially purified PP4 species from gel filtration
chromatography were compared. Because PP4C protein is
expressed in greatest quantity in the testis (20), this tissue was
utilized for subsequent biochemical manipulations and holoenzyme
purification. Bovine testis soluble proteins recovered from 25-50%
ammonium sulfate precipitation were separated by sequential hydrophobic
interaction and anion exchange chromatography. Fractions containing PP4
were identified by immunoblot analysis using a PP4C
antibody previously described (20). PP4 eluted over several fractions
with two noticeable peaks from Source 15Q (fractions 20 and 25; Fig.
1A). Pools of each PP4 peak
were individually applied to a gel filtration column (Fig.
1B). Immunoblot analysis of select gel filtration fractions indicated that PP4 in pool 1 exhibited an apparent molecular mass of
approximately 270-300 kDa (fractions 31-34), whereas PP4 in pool 2 was slightly larger, approximately 400-450 kDa (fractions 29-32).
These data suggest that PP4C exists in at least two
distinct complexes in bovine testis. Immunoblot analysis of the gel
filtration fractions using affinity-purified antibodies to the A and
B Purification of One PP4 Holoenzyme--
As a first step toward
identifying PP4-associated subunits, the purification of a PP4
holoenzyme from bovine testis was undertaken. PP4 (270-300 kDa) was
purified by sequential chromatography on hydrophobic, strong anion
exchange, weak anion exchange, and gel filtration columns. Fig.
2 shows fractions from the gel filtration column as analyzed by immunoblot and silver stain. Silver stain analysis revealed a protein doublet (120-125 kDa) co-fractionating with the catalytic subunit of PP4. The molecular mass of the
co-purifying protein doublet was carefully determined by comparing its
migration with the migration of SDS-PAGE standards (data not shown).
Taking into account the differences in molecular masses of these two proteins (see mass spectrometry analysis below), one might predict a
3:1 protein staining ratio if the complex existed in a 1:1 molar stoichiometry. The protein silver staining ratio of the 120-125-kDa co-purifying proteins to PP4C ranged from 1.4 to 2.7 as
quantitated by NIH Image, suggesting a near stoichiometric complex.
Extracts from 200 g of bovine testis (1200 mg of total protein)
contained approximately 400 µg of PP4C, as determined by
quantitative immunoblot analysis (see "Materials and Methods"). The
final PP4 holoenzyme contained 7 µg of PP4C (based on the
intensity of the silver-stained protein; see "Materials and
Methods"), indicating an overall yield of approximately 2%.
PP2AC was not detected in the silver-stained gels of the
purified PP4 preparation, but very low levels of PP2AC immunoreactivity were observed. The purified PP4 holoenzyme was further
characterized by FPLC on Mono Q and phenyl-Superose columns. Silver
stain analysis of these column fractions confirmed that the
120-125-kDa protein doublet also co-purified with PP4C on these analytical resins (data not shown). Moreover, when partially purified PP4 holoenzyme was incubated with microcystin-Sepharose (an
affinity resin frequently used to identify PP1- and PP2A-associated proteins Refs. 30-32), both PP4C and the 120-125-kDa
protein doublet bound to microcystin (data not shown; see below).
Together, these data provide compelling evidence that a
PP4C-associated protein, termed PP4R1, had been
isolated.
To obtain a more accurate determination of the size of the individual
components of the PP4 complex, the sample was analyzed by mass
spectrometry. This technique has recently been introduced as an
excellent method to dissect the size of multiprotein complexes (33).
The mass spectrum of cross-linked purified PP4 holoenzyme revealed two
major peaks and several minor peaks (Fig.
3). The two major peaks are proteins of
35.1 and 104.5 kDa, presumably corresponding to PP4C and
PP4R1. The molecular mass of PP4R1 measured by
mass spectrometry (104,499 Da) was somewhat different from that
observed by SDS-PAGE (120-125 kDa). In contrast to the sharp PP4C peak, the peak corresponding to PP4R1 is
quite broad. This broad PP4R1 peak may be due to the
presence of multiple species of similar molecular weight as detected by
SDS-PAGE. Since the size of the 140-kDa peak is virtually identical to
the sum of the molecular weights of PP4C and
PP4R1, it probably represents a heterodimeric complex
containing stoichiometric amounts of the two proteins cross-linked with
glutaraldehyde.
Phosphatase Activity of PP4--
Purified PP4 was assayed for
phosphatase activity using casein, protein kinase A-phosphorylated
histone H1, protein kinase C-phosphorylated histone H1, and
phosphorylase a as substrates. The specific activity of
purified PP4 for these phosphoproteins ranged from 0.624 to 13.1 pmol/min/µg of PP4C contained in the purified PP4
holoenzyme, whereas the specific activity of purified PP2AC
toward identical substrates ranged from 67 to 1197 pmol/min/µg. An
immobilized phosphatase assay similar to the one described by Ding
et al. (34) also was developed to test PP4 phosphatase activity toward proteins phosphorylated in vitro (bovine
testis soluble extracts) or in vivo (HEK cells). Okadaic
acid-sensitive protein dephosphorylation could be detected in this
assay with purified PP2AC but not with purified PP4 (data
not shown).
Identification of the PP4R1 cDNA--
Multiple
peptides from both proteins of the putative bovine PP4R1
were subjected to amino acid sequence analysis (Fig.
4). A total of nine peptides were
sequenced; peptides 1 and 4, 3 and 9, and 5 and 6 had nearly identical
sequences. The peptide sequences were used to search the expressed
sequence tag data base to identify potential cDNA clones. Several
human cDNA clones with translated sequences highly homologous to
the sequenced peptides (Fig. 4) were identified and obtained from The
Institute for Genomic Research/American Type Culture Collection Human
cDNA Special Collection. One clone was of particular interest
because it contained a Kozak consensus start sequence (35), an open
reading frame of 933 amino acids with a predicted molecular weight of
105,189, and a poly(A) tail. Both the cDNA and predicted amino acid
sequences of this clone are shown in Fig.
5A. The predicted molecular
weight of the encoded protein is similar to the molecular weight
determined by mass spectrometry of the purified PP4R1
protein (104,500). Thus, these data indicate that the isolated cDNA
clone contains most, and perhaps all, of the PP4R1 coding
sequence. Furthermore, all of the bovine peptides could be identified
in one human cDNA sequence, and two peptides were shared by both
proteins of the doublet, suggesting that the proteins of the doublet
are very similar. Interestingly, PP4R1 has 13 repeats
similar to the nonidentical or "heat" repeats found in the A
subunit of PP2A (PP2AA) (36, 37). The repeats identified in
PP4R1 are indicated by white type on black in
Fig. 5A and are also listed in Fig. 5B. Amino acid residues in boldface type in Fig.
5B are residues identified in the consensus repeat sequence
in PP2AA (38). Seven of the PP4R1 repeats
(indicated by an asterisk) were matched to the
PP2A repeats by employing the ProfileScan
server.4 The remaining
repeats were found by manual examination of the amino acid sequence.
PP4R1 is distinct from PP2AA in that these motifs are not contiguous; instead, the repeats are separated by a
novel region of approximately 332 amino acids between the sixth and
seventh repeats. To date, this divergent region does not have homology
to any known protein.
Characterization of an Antibody Generated toward PP4R1
( Co-isolation of PP4R1 and PP4C on
Microcystin-Sepharose--
The availability of
PP4R1-specific antibodies ( Expression and Immunoprecipitation of PP4R1--
To
aid in the detection and isolation of the recombinant protein, the
PP4R1 cDNA was engineered with a Myc tag on the amino terminus. In Fig. 8A, cell
lysates prepared from vector and Myc-PP4R1-transfected COS
M6 cells were subjected to immunoblot analysis with the anti-Myc antibody. A protein migrating slightly faster than the purified bovine
PP4R1 was observed only in the lysates from cells
transfected with Myc-PP4R1 cDNA. To determine if
recombinant Myc-PP4R1 protein could associate with
endogenous PP4C, Myc-PP4R1 was
immunoprecipitated from cell lysates with the anti-Myc antibody, and
the immune complexes were analyzed for PP4C and
PP4R1 by immunoblotting. PP4C
co-immunoprecipitated with Myc-PP4R1 in lysates prepared
from Myc-PP4R1-transfected cells, but not in lysates from
vector-transfected cells (Fig. 8B). These data confirm that
the PP4R1 cDNA clone encodes a protein that can
associate with PP4C. Moreover, PP4R1 appears to
be a specific PP4C-associated protein because
PP2AC was not detected in the Myc-PP4C immune
complex (data not shown).
The high homology of PP2AC and PP4C led to
the hypothesis that PP4, like PP2A, may associate with regulatory
subunits to form multimeric complexes. To identify any proteins
associated with the catalytic subunit of PP4, one oligomeric form of
this phosphatase was purified. The purified complex consisted of
PP4C and a PP4C-associated protein
(PP4R1). Sequence information obtained from
PP4R1 peptides was utilized to identify a cDNA clone.
The amino acid sequence encoded by this cDNA has a predicted
molecular weight of 105,189, which is very similar to the size of
PP4R1 measured by mass spectroscopy (104,500). Furthermore,
the cDNA contains the Kozak consensus start sequence (35),
indicating that the most 5' methionine is an ideal place for the
initiation of translation. These data suggest that the cDNA clone
may indeed represent a full-length PP4R1 cDNA. Although
the Myc-tagged PP4R1 migrated slightly faster on SDS-PAGE
than the purified protein, we would like to point out that the
expressed protein was of human origin, whereas the purified protein was
obtained from bovine tissue. Thus, the differences in sizes could be
species variations of PP4R1. Alternatively, the difference
in migration on SDS-PAGE of the purified and expressed proteins could
be attributed to proteolysis of the expressed PP4R1 protein. The PP4R1 cDNA does, however, encode a
functional PP4C binding protein, because when expressed in
mammalian cells it can be immunoprecipitated with the catalytic subunit
of PP4.
The purified PP4 preparation exhibited very little phosphatase activity
toward substrates readily dephosphorylated by PP2AC. The
activity that was measured could be accounted for with as little as 1%
PP2AC contamination, and it is important to point out that
a hint of PP2AC immunoreactivity was observed in the purified PP4 holoenzyme. Two reasonable explanations for the low apparent PP4 activity include inhibition of PP4C catalysis
by PP4R1 or a narrow PP4 substrate profile (i.e.
the ideal or physiological substrate has not yet been tested).
Additionally, we cannot rule out the possibility that PP4 was
inactivated during the purification procedure. However, this
explanation seems unlikely, because PP2A catalytic activity was not
inactivated following similar purification conditions (data not shown),
and partially purified PP4 bound to microcystin-Sepharose, suggesting
that the catalytic site is intact.
PP4R1 migrated as a doublet of 120-125 kDa on SDS-PAGE and
exhibited a fairly broad peak (~105 kDa) as determined by mass spectrometry. The apparent size discrepancy may be due to aberrant migration of PP4R1 on SDS-PAGE. Several possibilities could
account for the protein doublet. First, the smaller protein may
represent a proteolytic fragment of the larger protein giving rise to
the appearance of two proteins on SDS-PAGE and a fairly broad peak on
mass spectrometry. We are aware of some proteolysis, because another
minor co-purifying protein (29 kDa) was identified as a fragment of
PP4C by amino acid sequence analysis (data not shown). A
second explanation is that the two proteins are identical except that
one is post-transcriptionally or post-translationally modified. The
mass spectrometry data is consistent with this possibility; however, at
present no direct evidence exists to support this interpretation.
Finally, the two proteins may represent highly homologous isoforms of
PP4R1 that differ slightly in molecular weight.
PP2AA and PP4R1 both contain nonidentical or
"heat" repeats. Each of the 15 PP2AA repeats is
predicted to consist of two tightly packed amphipathic helices (36). A
linear organization of these repeats would form a rodlike protein with
an axial ratio of 10.5:1 (39, 40). PP2AC has been shown to
bind to the C-terminal repeats of the A subunit, whereas the simian
virus 40 and polyomavirus T antigens, and perhaps the B subunit of
PP2A, bind to the N terminus region (36). By analogy to PP2A, it is
quite likely that PP4C binds to the C terminus of
PP4R1, since this is where PP2AA and PP4R1 share the greatest homology. PP4R1 is
distinct from PP2AA in that the nonidentical repeats are
not contiguous in PP4R1. It is difficult to interpret the
implications of this unique PP4R1 feature both structurally
and functionally. Since we did not identify any PP2A "B-like"
subunits in our purified preparation, it is conceivable that the
divergent region separating the repeats may function similarly to the B
subunit in dictating substrate specificity and subcellular
localization. However, we cannot rule out the possibility that a B-like
subunit of PP4 was lost during the purification. Finally, the larger
PP4 complex (440 kDa), which was not extensively characterized, may
contain distinct PP4 subunits because the PP4R1 to
PP4C ratio of immunoreactivity in this complex was
substantially lower (data not shown) than the ratio in the purified
270-300-kDa complex.
The data presented in this report suggest that the purified PP4 complex
is composed of one PP4 catalytic subunit and one associated protein;
however, it is possible that two dimers may combine to form a tetramer.
A complex corresponding to the predicted size of a tetramer was not
detected by mass spectrometry but would be consistent with the gel
filtration data. Since the anomalous elution of PP2AA on
gel filtration may be explained by the predicted rodlike structure (39,
40), the size discrepancy of purified PP4 estimated by gel filtration
and mass spectrometry could be reconciled by similar factors.
Several studies had indicated that PP4 existed in a high molecular
weight complex with proteins distinct from the regulatory subunits of
PP2A (18).2 The biochemical purification of one PP4
holoenzyme has provided direct evidence to support this hypothesis and
has resulted in identification of the first PP4C-associated
protein (PP4R1). Interestingly, the predicted amino acid
sequence of PP4R1 has homology to the A subunit of PP2A and
suggests similar functions. The PP4R1 cDNA clone and
encoded protein have led to the development of polyclonal antibodies
that can be employed to examine tissue distribution and subcellular
localization of the endogenous protein. Additionally, the purification
and characterization of other PP4 complexes may reveal more
similarities with and/or differences from PP2A complexes. These studies
will contribute to the overall understanding of the regulation of the
type 2A-like phosphatase family.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
80 °C. Centricon-30
concentrators were from Amicon (Beverly, MA). Aprotinin,
phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and SDS-PAGE
molecular mass standards were from Sigma. Nylon-reinforced
nitrocellulose membrane and Centrex UF-0.5 concentrators were
manufactured by Schleicher & Schuell. Polyvinylidene fluoride membrane
was obtained from Millipore Corp. (Bedford, MA). The sequencing
reagents BigDye and Half-TERM were purchased from Perkin-Elmer and
GenPak (Stony Brook, NY), respectively. NdeI was from New
England Biolabs (Beverly, MA); EcoRI and XhoI
were from Promega (Madison, WI). Plasmid purification kits and
pcDNA3.1 were obtained from Qiagen (Valencia, CA) and Invitrogen
(Carlsbad, CA), respectively. SulfoLink Gel was purchased from Pierce.
The monoclonal anti-Myc antibody was a generous gift from Dr. Lee
Limbird (Vanderbilt University).
20 °C or immediately resuspended in 80 ml of buffer X (20 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 10%
glycerol, 6.3 µg/ml aprotinin, 4 µg/ml leupeptin, 10 mM
benzamidine, 4 µg/ml pepstatin A, and 1 mM
phenylmethylsulfonyl fluoride) and utilized for chromatographic
analyses and PP4 purification.
-counter. The amount of PP4C
in the purified PP4 holoenzyme was measured by quantifying the
intensity of silver-stained proteins using NIH Image. Purified PP2AC was used as the standard, and its concentration was
determined by the Bio-Rad protein assay.
PP4R1-1) were purified from rabbit antiserum by affinity
chromatography (27). Briefly, the synthetic peptide was attached
covalently to SulfoLink Gel, and the slurry was transferred to a
column. Rabbit antiserum diluted 1:2 with Tris buffer (10 mM Tris-HCl, pH 7.5) was passed over the peptide-Sepharose
conjugate three times. The resin was washed with Tris buffer and then
with Tris buffer containing 500 mM NaCl. Bound antibodies
were eluted by sequential addition of 100 mM glycine, pH
2.5, followed by 100 mM triethylamine, pH 11.5. The eluates
were pooled, and the antibodies were concentrated and dialyzed against
Tris-buffered saline containing 0.02% NaN3 using
Centriprep-30 concentrators.
PP4R1-2) using similar methodology as described above.
RESULTS
subunits of PP2A revealed that the PP2A subunits did not
co-fractionate with PP4C (data not shown).
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Fig. 1.
PP4 exists in two complexes in bovine testis
soluble extracts. A, Source Q profile of PP4
holoenzymes. Bovine testis soluble extracts were fractionated by
phenyl-Sepharose chromatography, followed by Source 15Q chromatography. Select fractions of the Source 15Q column were analyzed by
immunoblot using a mixture of antibodies specific for PP4C
and PP2AC. B, elution of pool 1 and pool 2 on
gel filtration. Fractions 18-23 and fractions 24-27 from Source 15Q
were pooled and individually subjected to gel filtration chromatography
followed by immunoblot analysis with PP2AC- and
PP4C-specific antibodies. This experiment is representative
of at least three independent fractionations. The elution of gel
filtration standards is indicated by the arrows.
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Fig. 2.
Immunoblot (top) and silver
stain analysis (bottom) of the purified PP4
holoenzyme. PP4 was purified from bovine testis soluble extracts
by sequential FPLC on phenyl-Sepharose, Source 15Q, and DEAE. Pooled
PP4 fractions from DEAE Sephacel were concentrated to 2 ml and further
purified by gel filtration chromatography. The gel filtration fractions
were analyzed by immunoblot with both PP2AC and
PP4C antibodies. The data shown are representative of five
separate purifications. The elution of gel filtration standards is
indicated by the arrows.
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Fig. 3.
Mass spectrometry analysis of purified PP4
holoenzyme. Purified PP4 holoenzyme was concentrated to
approximately 1 pmol/µl, cross-linked with glutaraldehyde for 1 min,
and analyzed by mass spectrometry. The peaks at 35143.8, 104499, and
140692 presumably correspond to PP4C, PP4R1,
and the PP4 complex (i.e. PP4R1-PP4C
dimer), respectively.
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Fig. 4.
Amino acid sequence of peptides isolated from
the purified PP4-associated protein. The first peptide of each
pair was obtained from the purified bovine protein, and the second
peptide corresponds to the homologous region identified in the human
sequence. The last column indicates whether the peptide was derived
from the upper or lower protein of the PP4C-associated
doublet.
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Fig. 5.
Sequence of PP4R1
(A) and identification of "heat" repeats
(B). A, the peptides obtained from
purified bovine PP4R1 are underlined. Regions
homologous to the repeats found in the A subunit of PP2A are printed in
white type on black. B, amino acid
sequence alignment of the PP4R1 repeats. Amino acid
residues in boldface type are loosely conserved
amino acids previously defined in the repeats of PP2AA. The
repeats with asterisks were identified by ProfileScan as
having homology to the PP2AA "heat" repeats.
PP4R1-1)--
Polyclonal anti-peptide antibodies
(designated
PP4R1-1) directed against a bovine sequence
homologous to residues 912-924 of the expected human protein were
produced in rabbits. The antiserum recognized a doublet of identical
molecular weight in both bovine testis soluble extracts and in the
purified PP4 holoenzyme (Fig. 6A). Antibody binding could be
blocked in the presence of excess peptide. The anti-peptide antibodies
were affinity-purified from the rabbit antiserum and used to monitor
the purification of the PP4 holoenzyme. Both PP4R1 and
PP4C were significantly enriched throughout the
purification protocol as shown in Fig. 6B.
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Fig. 6.
Characterization of PP4R1
antibodies ( PP4R1-1)
(A) and assessment of PP4 purification
(B). A, bovine testis soluble extracts
and purified PP4 protein were analyzed by immunoblotting with a
polyclonal antibody directed toward PP4R1 (in the absence
and presence of the synthetic peptide). B, pools of the peak
PP4 fractions from each step of the purification were analysed by
silver stain (right panel) or subjected to immunoblot
analysis with specific PP4C and PP4R1
antibodies (left panel) (5 µg of protein at each step,
except 0.5 µg in the final gel filtration step).
PP4R1-1) allowed
examination of PP4 binding to microcystin-Sepharose. This affinity
resin previously has been utilized to isolate PP1 and PP2A complexes
from crude tissue extracts (30-32). Soluble proteins from bovine
testis were incubated with the microcystin-Sepharose in the absence or
presence of excess unconjugated microcystin, and bound proteins were
eluted by Laemmli sample buffer. Immunoblot analysis of the applied,
flow-through, and bound samples revealed that PP4C and
PP4R1 co-purified on microcystin-Sepharose (Fig. 7). The interaction of both proteins with
the resin was blocked by preincubation of bovine testis soluble
extracts with 5 µM unconjugated microcystin.
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Fig. 7.
Microcystin affinity resin binding.
Bovine testis soluble extracts (5 mg) were incubated with
microcystin-Sepharose in the presence and absence of 5 µM
unconjugated microcystin. The beads were washed six times with buffer
containing 150 mM NaCl. Bound proteins were eluted with
Laemmli sample buffer, and the immunoblot was analyzed with antibodies
to PP4C and PP4R1 ( PP4R1-1). The
data shown are representative of three experiments.
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Fig. 8.
Expression and association of
PP4R1 with PP4C in mammalian cells.
A, Myc-PP4R1 expression. COS M6 cells were
transfected with 10 µg of either empty vector or vector containing
Myc-PP4R1 cDNA. Cell lysates were prepared and analyzed
by immunoblot with an antibody directed toward the Myc epitope. The
third lane contains purified PP4 holoenzyme;
PP4R1 in the purified preparation was visualized with
affinity-purified PP4R1 antibodies
( PP4R1-1). B,
-Myc immunoprecipitation.
Anti-Myc immune complexes from cellular lysates obtained in
A were analyzed by immunoblot with PP4R1
(
PP4R1-2) antiserum and affinity-purified
PP4C antibodies. The top half of the
immunoblot was probed with
PP4R1-2 antiserum because the
affinity-purified
PP4R1-1 antibodies fail to recognize
the human protein. The data are representative of three
experiments.
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Carol Ann Bonner for the supply and maintenance of the COS M6 cells and Drs. Terry Farmer and Richard Caprioli for assistance and interpretation of mass spectrometry analysis. We acknowledge the Vanderbilt University Medical Center Cell Imaging Resource and Eric Howard and Dr. Masaaki Tamura from the Peptide Sequencing and Amino Acid Analysis Shared Resource. We also are grateful to Drs. Roger Colbran and Ryan Westphal for critical evaluation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM51366 (to B. E. W.), Vanderbilt Diabetes Research and Training Center Grant DK20593, Cancer Center Grant CA68485, and Center for Molecular Neurosciences Grant MH19732.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF111106.
Supported by Pharmacology Training Grant GM07628.
§ To whom correspondence should be addressed: Dept. of Pharmacology, Rm. 424 MRBI, Vanderbilt University Medical Center, Nashville, TN 37232-6600. Tel.: 615-343-2080; Fax: 615-343-6532; E-mail: brian.wadzinski{at}mcmail.vanderbilt.edu.
2 S. Kloeker and B. Wadzinski, unpublished observations.
3 This protocol can be found on the World Wide Web at http://www.ncbi.nlm.nih.gov/BLAST/.
4 The ProfileScan server can be found on the World Wide Web at http://www.isrec.isb-sib.ch/software/PFSCAN_form.html.
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ABBREVIATIONS |
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The abbreviations used are: PP1, -2A, -2B, -2C, and -4, protein serine/threonine phosphatase 1, 2A, 2B, 2C, and 4, respectively; PP4C, PP4 catalytic subunit; PP2AC, PP2A catalytic subunit; PP4R1, PP4-associated protein; PP2AA, A or PR65 subunit of PP2A; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; CAPS, 3-(cyclohexylamino)propanesulfonic acid; FPLC, fast protein liquid chromatography.
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REFERENCES |
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