(Received for publication, September 29, 1995; and in revised form, November 28, 1995)
From the
Association of the catalytic subunit (C2) with a variety of
regulatory subunits is believed to modulate the activity and
specificity of protein phosphatase 2A (PP2A). In this study we report
the cloning and expression of a new family of B-subunit, the B`,
associated with the PP2A form. Polymerase chain reactions
and cDNA library screening have identified at least seven cDNA
isotypes, designated
,
1,
2,
3,
4,
, and
. The different
subtypes appear to be generated by
alternative splicing. The deduced amino acid sequences of the
,
2,
3,
4 and
isoforms predict molecular weights of
57,600, 56,500, 60,900, 52,500, and 68,000, respectively. The proteins
are 60-80% identical and differ mostly at their termini. Two of
the isoforms, B`
3 and B`
, contain a bipartite nuclear
localization signal in their COOH terminus. No homology was found with
other B- or Brelated subunits. Northern analyses indicate a
tissuespecific expression of the isoforms. Expression of B`
protein in Escherichia coli generated a polypeptide of
53
kDa, similar to the size of the B` subunit present in the purified
PP2A
. The recombinant protein was recognized by antibody
raised against native B` and interacted with the dimeric PP2A
(A
C2) to generate a trimeric phosphatase. The deduced amino acid
sequences of the B` isoforms show significant homology to mammalian,
fungal, and plant nucleotide sequences of unknown function present in
the data bases. Notably, a high degree of homology (55-66%) was
found with a yeast gene, RTS1, encoding a multicopy suppressor
of a rox3 mutant. Our data indicate that at least seven B`
subunit isoforms may participate in the generation of a large number of
PP2A
holoenzymes that may be spatially and/or functionally
targeted to different cellular processes.
Protein phosphatase 2A (PP2A) ()is one of the major
serine/threonine protein phosphatases present in the cell and is
involved in the control of many cellular functions and metabolic
pathways (reviewed by Cohen(1989), Mumby and Walter(1993),
DePaoli-Roach et al.(1994), and Mayer-Jaekel and Hemmings
(1994)). The Ser/Thr protein phosphatases, with the exception of PP2C,
consist of multimeric structures. Their catalytic subunit associates
with specific proteins, which serve as targeting/regulatory subunits
and play substantial roles in the control of phosphatase activity.
PP2A is a family of holoenzymes containing a common core of a 36-kDa
catalytic (C2) subunit and a 63-kDa A subunit associated with a variety
of regulatory B-subunits (B, B`, and B") to form the trimeric
PP2A, PP2A
, and polycation-stimulated protein
phosphatase M, respectively (Tung et al., 1985; Waelkens et al., 1987; Mumby et al., 1987; Zolnierowicz et
al., 1994). Takeda and co-workers (Usui et al., 1988)
also isolated from human erythrocytes a PP2A form that contained a
polypeptide of 74 kDa associated with the A
C2 core. Molecular
cloning has identified in mammals two isoforms each of the C2 (da Cruz
e Silva and Cohen, 1987; Green et al., 1987; Stone et
al., 1987) and the A (Walter et al., 1989, 1990; Hemmings et al., 1990) subunits, which are evolutionarily highly
conserved.
The B-subunit is the most diverse and comes in three
variants, B, B`, and B", of 52, 53, and 74-130 kDa,
respectively. The B subunit, associated with PP2A
, and the
B` subunit, associated with PP2A
, appear to be structurally
unrelated based on peptide mapping (Tung et al., 1985) and
immunoreactivity (Zolnierowicz et al., 1994). Molecular
cloning of the B subunit has identified three closely related isoforms,
,
and
(Healy et al., 1991; Mayer et
al., 1991; Zolnierowicz et al., 1994), that are more than
80% identical. Drosophila (Mayer-Jaekel et al., 1993;
Uemura et al., 1993) and Saccharomyces cerevisiae (Healy et al., 1991) homologs have also been isolated.
Cloning of the cDNA for the B" subunit, constituent of the
polycation-stimulated protein phosphatase M, predicts the existence of
two, 72- and 130-kDa, alternatively spliced forms (Hendrix et
al., 1993) that show no homology to the B subunit isoforms. The
dimeric A
C2 phosphatase has also been found to be associated with
polyomavirus middle and small tumor antigens and with SV40 small tumor
antigen (Pallas et al., 1990; Walter et al., 1990).
The control of PP2A activity is not fully understood. Recent reports suggest that post-translational modifications of the C2 subunit, such as phosphorylation and carboxymethylation, might have regulatory function (Chen et al., 1992; Lee and Stock, 1993; Xie and Clarke, 1993; Turowski et al., 1995). Several findings also point to a key role of the A and B-subunits in the regulation of phosphatase activity. In vitro studies indicate that the substrate specificity is affected by the subunit composition (Imaoka et al., 1983; Cohen, 1989; Agostinis et al., 1992; Kamibayashi et al., 1992, 1994). Association of the regulatory subunits with C2 has been demonstrated to cause either increased or decreased activity toward different substrates (Agostinis et al., 1992; Ferrigno et al., 1993; Mayer-Jaekel et al., 1994).
Support for the involvement of the regulatory B-subunits in the control of specific cellular functions comes from several studies. Association of the SV40 and polyomavirus antigens with the dimeric PP2A by displacement of the B-subunits might subvert the function of the enzyme and contribute to cell transformation (for a review see Mumby and Walter(1993)). Transient expression of SV40 small t antigen in CV-1 cells stimulated the mitogen-activated protein kinase pathway and cell growth, most likely through inhibition of PP2A (Sontag et al., 1993). Mutations of the B subunit were found to result in defective cytokinesis in S. cerevisiae (Healy et al., 1991) and in abnormal anaphase progression in Drosophila (Mayer-Jaekel et al., 1993). Although the mechanisms responsible for the defects are not completely understood, it appears that the B subunit targets the phosphatase to distinct cellular structures (Sontag et al., 1995) and/or confers substrate specificity (Mayer-Jaekel et al., 1994).
Thus, strong
evidence from different experimental approaches and systems points to a
key role of the B-subunits in the control of PP2A holoenzymes. The
elucidation of their primary structure is a critical step toward
understanding their function. In this paper we report the isolation and
characterization of cDNA clones encoding at least seven isoforms of the
B` subunit of PP2A that are not related to other
B-subunits. The large number of isoforms identified and the relative
tissue-specific distribution of their mRNAs suggest that distinct
holoenzyme forms may be generated that either spatially and/or
functionally direct the enzyme to different cellular targets. The high
homology of the B` subunit to sequences derived from various organisms
also indicate that the PP2A
enzyme is highly conserved
through evolution.
Reverse transcriptase polymerase chain reactions (PCR) were
performed using degenerate oligonucleotide primers and rabbit skeletal
muscle mRNA. Total RNA from rabbit skeletal muscle was purified
according to Chomczynski and Sacchi(1987). Poly(A) RNA
was isolated by affinity chromatography on oligo(dT)-cellulose (5 Prime
3 Prime, Inc.). First strand cDNA was synthesized from 0.2
µg of mRNA with Moloney murine leukemia virus reverse transcriptase
(200 units) and reverse oligonucleotide primers 1 or 2 (100 pmol) as
described previously (Healy et al., 1991). The single stranded
cDNA was then amplified with 100 pmol each of oligonucleotide 1, 2, 3,
or 4 as forward primer. The amplified products were separated on 1%
agarose gel and analyzed by Southern hybridization (Southern, 1975)
with 5` endlabeled oligonucleotides that had not been used in the
amplification reaction. A 641-bp DNA fragment, M PCR (Fig. 1),
obtained with oligonucleotides sense 1 and antisense 2, hybridized with
the 5` end-labeled oligonucleotide 3. The M PCR fragment was subcloned
into pCR
II vector (TA Cloning System, Invitrogen) and
sequenced by the dideoxynucleotide chain termination method (Sanger et al., 1977) with vector- and cDNA-specific oligonucleotide
primers.
Figure 1:
cDNA clones encoding protein
phosphatase 2A B` subunit. Clones were isolated from rabbit
skeletal muscle (M) and rabbit brain (BR) cDNA
libraries. Thick solid lines indicate regions that were
sequenced in both direction. Thin lines indicate regions
sequenced in one direction. Dashed lines indicate regions not
sequenced. The angled line in clone M 7-1B`
indicate a
deletion. Diverging sequences of the different
forms are marked
with different dashed lines. The sizes of the individual
clones are indicated in parentheses. Partial restriction maps
of cDNAs for different isoforms are also
shown.
The initial
screening of the rabbit skeletal muscle cDNA library with the 641-bp M
PCR fragment identified 38 positive clones. Fifteen clones were
rescreened, and nine held positive after plaque purification. Four were
fully characterized (M 2-1, 1338 bp; M 5-1, 1125 bp; M 1-1, 877 bp; and
M 6-2, 1157 bp; Fig. 1), and five were found to overlap with the
other four. Nucleotide sequence analysis of the cDNA clones showed
70% identity to the M PCR sequence. The four clones fell into two
groups, one comprising M 2-1 and M 5-1 and the other comprising, M 1-1
and M 6-2 clones, indicating the existence of at least three isoforms.
The isoforms encoded by M 2-1/M 5-1, termed
, and by M 1-1/M 6-2,
termed
, shared 78 and 92% identity, respectively, with that
encoded by M PCR, termed
. The combined
cDNAs contained an
open reading frame with a stop codon close to the 3` end but did not
have a translation start ATG codon. The
cDNAs had an ATG start
codon but no stop codon.
In order to obtain the complete coding
sequences, the 5` end 367-bp EcoRI-BglII and
the 3` end 329-bp NaeI-EcoRI fragments of the
isotype M 2-1 cDNA were used to rescreen the original filters.
Out of fifteen positive signals, eight were new clones. Seven were
plaque purified, and four of these (M 1-7B`
, 1013 bp; M
1-8B`
, 421 bp; M 5-4B`
, 537 bp; and M 7-1B`
, 819 bp)
were sequenced (Fig. 1).
Because only partial clones were
isolated for the B` isoform, the original filters of the rabbit
skeletal muscle library were rescreened with the labeled 5` end 343-bp EcoRI-PstI and the 3` end 395-bp XbaI-EcoRI fragments of M 6-2B`
cDNA.
Fifteen positive clones were identified, only one of which, M
8-6B`
1 (1514 bp), extended the 5` and 3` ends (Fig. 1).
This cDNA had an open reading frame with an ATG at position 24,
immediately preceded by a stop codon, but no stop codon was present at
the 3` end. Therefore the 5` 428-bp EcoRI-PstI,
the 3` 304-bp XmnI-EcoRI, and the 3` 426-bp RcaI-EcoRI fragments of M 8-6B`
1 were used for
screening additional 320,000 recombinants of the rabbit skeletal muscle
cDNA library. Sequence analysis revealed that the 3` end regions of
five out of the eight clones characterized (M 19-1B`
2, 316 bp; M
10-1B`
3, 620 bp; M 17-2B`
3, 922 bp; M 13-1B`
4, 515 bp;
and M5-1B`
4, 1502 bp) diverged from the M 8-6B`
1 at different
positions ( Fig. 1and 3). The subtypes of the
isoform of
the B` subunit were termed
1,
2,
3, and
4.
320,000 additional recombinant phages were also screened with
the radiolabeled 641-bp M PCR fragment in order to isolate
isotype cDNAs. Two partial overlapping clones were identified, M
20-1B`
(996 bp) and M 23-1B`
(956 bp) (Fig. 1).
Although there are 11 differences (3 G/A and 8 T/C) between the
nucleotide sequences of the 918 bp overlapping portion of the two
clones, their deduced amino acid sequences are identical, suggesting
that they represent allelic forms. The M 23-1B`
clone is identical
to the M PCR in the overlapping region, except for some differences in
the primer regions. These cDNA clones did not contain the complete
coding sequence, and rescreening of the rabbit skeletal library failed
to provide additional sequences. Because Northern analysis (see below)
had indicated that the
and
isoforms were most abundant in
brain, 220,000 recombinants from a
gt10 rabbit brain
oligo(dT)-primed cDNA library (Clontech Laboratories Inc.) were
screened with the 641-bp M PCR fragment, the 536-bp M 5-4B`
, the
421-bp M 1-8B`
, the 773-bp SspI-EcoRI
fragment of M 2-1B`
, the 505-bp EcoRI-XbaI
fragment, and the 665-bp XbaI-EcoRI fragment of
M 8-6B`
1. Several positive clones were isolated, eight of which
encoded B`
and two of which coded for a distinct isoform, termed
(Fig. 1).
Plasmids B`pET-8c and B`
pET-15b were
transformed into E. coli strain BL21(DE3). The cells were
grown at 37 °C to an A
of 0.8 units and then
induced for 2 h with 0.4 mM (B`
pET-8c) or 1.0
mM (B`
pET-15b)
isopropyl-1-thio-
-D-galactopyranoside. The cells were
harvested by centrifugation at 5,000
g for 15 min and
lysed by 2
20 s sonication in 10 volumes of 50 mM Tris-HCl (pH 7.5), 0.2%
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1
mM N
-p-tosyl-L-lysine-chloromethyl
ketone, and 10 µg of leupeptin/ml. After centrifugation at 10,000
g for 20 min, the soluble fraction was removed. The
pellet fraction was washed twice with the same buffer containing 1%
Triton X-100 and then resuspended in the same volume of buffer without
Triton X-100. The samples were analyzed by SDS-PAGE and Western
immunoblotting using antibodies raised against the bovine B` subunit.
The insoluble cell fraction, containing the His-tagged B`,
prepared from 100 ml of cultured E. coli, was solubilized in
50 mM Tris-HCl (pH 7.5) and 6 M guanidine-HCl at room
temperature for 1 h. Renaturation was carried out by a rapid 100-fold
dilution of the solubilized proteins in 50 mM Tris-HCl (pH
7.5), 0.8 M NaCl, 0.4%
-mercaptoethanol, and 0.02% Tween
20 (Berndt and Cohen, 1990). After 2 h at room temperature,
1.2 ml
of Ni
-NTA-agarose (QIAGEN)/liter of solution was
added and batch-absorption was allowed for 2 h at 4 °C. The resin
was then packed into a column and washed with 25 mM Tris-HCl
(pH 7.5), 5 mM imidazole, and 0.8 M NaCl, followed by
25 mM Tris-HCl (pH 7.5), 20 mM imidazole, and 0.2 or
0.8 M NaCl. B`
was eluted with 100 or 200 mM imidazole in 25 mM Tris-HCl (pH 7.5) and 0.2 or 0.8 M NaCl. Fractions were collected and analyzed by SDS-PAGE and
Western immunoblotting with B` antibody.
Figure 2:
Nucleotide and deduced amino acid
sequences of the rabbit skeletal muscle B` isoform of
PP2A
. The sequence presented is the combination of the
overlapping B`
clones from Fig. 1: M 1-7B`
, M
5-4B`
, M 15-1B`
, M 9-1B`
, M 2-1B`
, M 5-1B`
,
and M 1-8B`
. The amino acid sequence is shown below the
nucleotide sequence; both sequences are numbered on the right
side. The dot represents the stop
codon.
Figure 3:
Nucleotide and deduced amino acid
sequences of the rabbit skeletal muscle B` isoforms of
PP2A
. Combined sequences of all the B`
cDNA clones in Fig. 1are shown. Single underlines indicate the
potential splice sites where the
1, 2, 3, and 4 isoforms diverge.
The double underline shows the polyadenylation signal, and the dots represent the stop codons.
The 2 and
3 clones start to diverge from
1 and from each other at
nucleotide 1467 (Fig. 3). The
2 cDNA has a stop codon 16 bp
downstream, and the
3 cDNA has a stop codon 130 bp downstream.
Combination of the common region of the B
cDNAs with the
2 or
3 isotypes yields sequences of 1458 or 1572 nucleotides, encoding
proteins with predicted molecular weights of 56,500 or 60,900,
respectively. Interestingly, the divergent region of
3 contains a
putative bipartite nuclear localization signal RKTVSDEARQAQKDPKK
(Dingwall and Laskey, 1991; Robbins et al., 1991).
Figure 4:
Nucleotide and deduced amino acid
sequences of the rabbit skeletal muscle and brain B` isoforms of
PP2A
. A combined sequence of the muscle and brain cDNA
clones in Fig. 1is shown. The dot indicates the stop
codon.
Figure 5:
Alignment of amino acid sequences of
different B` subunit isoforms. Deduced amino acid sequences of the
rabbit skeletal muscle (RSM) and brain (RB) B`
subunit isoforms, human myeloid cell cDNA (HUMORFY)), and S.
cerevisiae rox3 suppressor gene product, Rts1p, were aligned with
the sequence of the B` isoform. Identical amino acids are
designated by small dots; dashed lines indicate gaps,
and solid diamonds designate stop codons. The numbers in the Rts1p sequence indicate residues that were not included in
the alignment. The peptide sequences obtained from the purified rabbit
skeletal muscle (RSM B` Peptide) B` subunit are also shown. The underlined sequences indicate the putative nuclear
localization signals.
Screening of the rabbit brain library
identified two additional clones, BR 6-1B` and BR 8-1B`
, that
are identical at the 3` end but differ at the 5` end (Fig. 1).
This region might be the result of the presence of concatamerization of
brain cDNAs in the library (Zolnierowicz et al., 1994). A
putative translation start codon is present in a reasonable
(AGTAGGGATATGT) consensus sequence for initiation (Kozak, 1989).
The 495-bp partial coding region encodes 165 amino acids that share 64,
55, and 56% identity with the corresponding regions of the
,
, and
isoforms, respectively ( Fig. 5and Table 1).
Figure 6:
Northern analysis of the B`,
,
, and
mRNA. Total RNA (20 µg/lane) from different rabbit
and rat tissues and cell lines was electrophoresed through an agarose
gel under denaturing conditions and transferred to a nitrocellulose
membrane. The bound RNA was hybridized with:
P-labeled
B`
(1338 bp, M 2-1B`
cDNA) (A),
P-labeled B`
(1157 bp, M 6-2B`
, cDNA) (B),
P-labeled B`
(641 bp, M PCR cDNA) (C), and
P-labeled B`
(594 bp, 3` end SmaI-EcoRI fragment of BR 6-1B`
cDNA) (D). The ribosomal RNA stained with ethidium bromide is shown below the autoradiograms. The numbers on the right sides indicate the sizes of the molecular markers in
kilobases. The numbers and arrows on the left
sides designate the sizes of the B` subunit transcripts. The
migration of the 28 S and 18 S ribosomal RNAs is indicated in B. Sk. Muscle, skeletal muscle; Bovine
Ao.Sm.M.C., bovine aorta smooth muscle
cells.
The
NH-terminal His-tag allowed a rapid purification of the
recombinant B`
by metal chelate affinity chromatography. The
insoluble B`
was solubilized in 6 M guanidine HCl and
renatured by 100-fold dilution, as described under ``Experimental
Procedures.'' The solubilized B`
was then bound to
Ni
-NTA-agarose and eluted with 200 mM imidazole. SDS-PAGE and Western analyses indicated that the
purified protein was nearly homogeneous (Fig. 7, A and B). Moreover, after purification the protein was soluble. From
100 ml of E. coli culture, 3.8 mg of purified recombinant
B`
was obtained.
Figure 7:
Purification of His-tagged B`
expressed in E. coli. The B`
polypeptide expressed from
the pET-15b vector was solubilized, renatured, and purified on a
Ni
-NTA-agarose resin as described under
``Experimental Procedures.'' A, Coomassie Blue
staining of samples separated on 9% SDS-PAGE. B, Western blot
using antibody raised against B` subunit of bovine heart
PP2A
. Fl.t., flow through fraction; Wash
1, fractions eluted with 5 mM imidazole in 25 mM Tris-HCl (pH 7.5) and 0.8 M NaCl; Wash 2,
fractions eluted with 20 mM imidazole in 25 mM Tris-HCl (pH 7.5) and 0.8 M NaCl; lanes
1-7, fractions eluted with 200 mM imidazole in 25
mM Tris-HCl (pH 7.5) and 0.8 M NaCl. Samples of 15
µl from each fraction were loaded onto the gel. The numbers at the sides of the panels indicate molecular mass markers:
94 kDa, phosphorylase b; 67 kDa, bovine serum albumin; 53 kDa,
glutamic dehydrogenase; 43 kDa, ovalbumin; and 30 kDa, carbonic
anhydrase.
We took advantage of the His-tag in a
reconstitution experiment to determine whether the recombinant protein
could associate with the AC2 core of PP2A. Incubation of the
purified His-tagged B`
with dimeric PP2A (A
C2) and
chromatography of the mixture on Ni
-NTA-agarose
generated a complex with approximately equimolar amounts of the three
subunits as judged by SDS-PAGE and immunoblotting analyses utilizing
antibodies to the individual A
, B`, and C2 subunits (Fig. 8, A, upper and lower panels).
The dimeric form of the phosphatase did not bind to the column (Fig. 8, B, upper and lower panels).
These results strongly indicate that the B` cDNAs encode regulatory
subunits of PP2A that are able to interact with the dimeric form to
generate trimeric PP2A
holoenzymes.
Figure 8:
Reconstitution of the
AB`
C2 trimeric holoenzyme form of protein phosphatase
2A
. A
C2 dimeric PP2A was incubated with (A)
and without (B) recombinant B`
as described under
``Experimental Procedures.'' The mixture was then subjected
to chromatography on Ni
-NTA resin. The upper
panels show Coomassie Blue-stained samples separated by SDS-PAGE,
and the lower panels show Western blotting of the same
samples. The Western blot was carried out with antibodies against human
A
, bovine heart B`, and C2. Panel A, B`
,
0.4 µg of purified recombinant B`
; PP2A
,
1.6 µg of purified rabbit skeletal muscle A
C2 dimer; lane
1, flow through fraction; lanes 2-5, fractions
eluted with 20 mM imidazole in 25 mM Tris-HCl (pH
7.5) and 0.2 M NaCl; lanes 6-9, fractions
eluted with 200 mM imidazole in 25 mM Tris-HCl (pH
7.5) and 0.2 M NaCl. Panel B, lane 1, flow
through fraction; lanes 2-4, fractions eluted with 20
mM imidazole in 25 mM Tris-HCl (pH 7.5) and 0.2 M NaCl; lanes 5-7, fractions eluted with 200 mM imidazole in 25 mM Tris-HCl (pH 7.5) and 0.2 M NaCl. Samples of 10 µl from each fraction were loaded onto the
gel. The numbers at the left sides of the panels
indicate molecular mass markers. A, B`, and C designate the position of the
subunits.
The PP2A has been isolated in a variety of trimeric forms
that differ in the associated B subunits. The 52-kDa and the
72-130-kDa constituents of the PP2A and the
polycation-stimulated protein phosphatase M holoenzymes, respectively,
have been previously cloned. In this paper we report the molecular
cloning of cDNAs encoding the B` subunit of the previously
characterized PP2A
(Zolnierowicz et al., 1994).
Screening of rabbit skeletal muscle and brain cDNA libraries has led to
the isolation of at least seven cDNA isotypes that fall into four
subgroups, termed
,
,
, and
. The
subgroup
comprises four isotypes that appear to be generated by alternative
splicing at the 3` end. Complete coding sequences have been obtained
for five isoforms,
,
2,
3,
4, and
(Fig. 5)
Eight of nine peptides isolated from the rabbit
skeletal muscle protein were found to be identical or to share high
homology with the predicted amino acid sequences of the B` isoforms (Fig. 5). Three of the peptides have sequences identical to
those in the isoform. It is possible that this was the
predominant form in our PP2A
preparation and that sequences
identical to those of the other peptides would have been found if the
complete coding region had been isolated. The lack of complete identity
could also be explained either by errors in sequencing of the peptides
or by the presence of a mixture of different isoforms in the purified
phosphatase. Alternatively, not all the existing isoforms have been
isolated. Nevertheless, the reactivity of the recombinant B`
with
antibodies against the bovine heart B- subunit, which we have shown to
be of the B` form (Zolnierowicz et al., 1994), and the ability
of the recombinant protein to associate with the A
C2 dimer,
clearly indicate that the cloned cDNAs encode B` subunit isoforms.
The encoding cDNAs predict a protein of 57.6 kDa. The start
codon is preceded 30 bp upstream by an in frame stop codon. The
presence of a deletion that eliminates the initiator codon in one of
the cDNAs suggests that alternative splice variants of the
isoform may also exist. The
subgroup comprises four isotypes,
1,
2,
3, and
4, that most likely are generated by
alternative splicing at the 3` end. All four contain an identical 442
amino acid region and diverge at their COOH termini. The nucleotide
sequence around the points of divergence is in agreement with mammalian
splice junction boundaries, AG/GT for
2 and
4, AG/GC for
3, and AG/CA for
1 (Senapathy et al., 1990). The
detection of three B`
transcripts by Northern analysis further
supports the existence of multiple forms. The nucleotide sequence
surrounding the first ATG (GGAGTCTAGATGT) is in moderate
agreement with the consensus sequence for initiation (Kozak, 1989).
This putative start codon is immediately preceded by a stop codon. The
next downstream potential initiation codon is at position 54. The
nucleotide sequence (GGCAGCAGGATGG) around this second ATG
complies better with the consensus sequence for translational
initiation, but at this time it is not clear which one is actually
used.
Nucleotide and amino acid sequence analysis indicated that all
seven isoforms share a high degree of homology (Fig. 5, and Table 1). The and
1, 2, 3, or 4 isoforms show
58%
nucleotide and
62% amino acid sequence identity. The
isoform
shares higher amino acid homology with the
isoforms
(77-82%) than with the
(61%). Conversely, the
is more
similar to the
(64%) than the
(56%) and the
s (55%).
The homology is higher in the central regions and diverges most at the
NH
and COOH termini (Fig. 3). Thus, one could
speculate that the homologous region may be involved in interaction
with the A and/or C2 subunits, whereas the termini could confer
specific properties to the holoenzyme. In support of this hypothesis,
two of the isoforms,
and
3, contain the bipartite nuclear
localization motif (K/R)(K/R)XXXXXXXXXXKXXKK (Dingwall and Laskey, 1991; Robbins et al., 1991). This
sequence may be responsible for directing specific PP2A
holoenzyme forms to the nucleus. Indeed nuclear association of
PP2A activity has been described (Jakes et al., 1986; Turowski et al., 1995), although the exact type of the enzyme is not
known. The
isoform also contains at the NH
terminus
an 8-fold glutamine-proline (QP) repeat whose significance is not
clear. Interestingly the 74-kDa polypeptide present in the PP2A
holoenzyme isolated by Takeda and co-workers (Usui et al.,
1988) is an alternatively spliced variant of B`
. (
)Thus, the enzyme of Usui et al.(1988) belongs to
the PP2A
family.
The tissue-specific expression of the
B` isoforms is also in keeping with the idea that different isoforms of
the B` subunit may direct the enzymes to different cellular functions.
The and
mRNAs are more abundant in brain. The 4.3-kb
transcript of
is highly expressed in testis, whereas the 1.7-kb
message is more abundant in heart and spleen. Testis, lung and brain
express the highest level of
mRNA.
Data base searches revealed
no homology between the B` subunit isoforms and the other known
B-subunits of PP2A, including the small and middle SV40 and polyoma
virus tumor antigens. However, matches with several other nucleotide
sequences present in GenBank were found. The protein sequence deduced
from a 382-bp cDNA, T09026 (Adams et al., 1993), is identical
to a region of B` if the T and the G at positions 339 and 373 of
the EST clone are deleted. A human open reading frame, HUMORFY (D26445,
3702 bp), shares more than 90% identity with the B`
group at the
level of amino acids (Table 1). Comparison of the amino acid
sequence of HUMORFY with the B`
3 revealed that the human clone
contains a deletion corresponding to amino acids 442-480 of the
B`
3, but the downstream sequences are almost identitical.
Interestingly, the B`
4 clone starts to diverge from the other
three
isotypes at the position where the deletion in the HUMORFY
starts, and
1,
2, and
3 diverge from each other at the
point where the deletion ends (Fig. 5). These positions
correspond to potential splice junctions. Therefore, it is possible
that a fifth alternative spliced form of B`
exists.
Two mouse
clones in the data base also share high homology with the B` isoforms.
The 332-bp sequence of MUSF354A (L26793) codes for a protein that is
97, 79, and 90% identical with B`, B`
, and B`
,
respectively, if minor changes are introduced. The other mouse clone,
MMTEG271G (X81059) is 86 and 90% identical to the B`
at the level
of nucleotides and amino acids, respectively. These clones appear to
represent the mouse
and
isotype of B`. In addition a rice
419-bp cDNA clone, RICC102651 (D22057), was found to have 64-65%
amino acid identity with the rabbit B` isoforms.
An especially
interesting match was found with a S. cerevisiae gene
(U06630), isolated as a high copy suppressor of rox3 mutants, RTS1. ()Rts1p showed high homology (53-56%
identity at the protein level) with the B` isoforms. The same gene has
also been isolated as a high copy suppressor, SCS1, of a yeast hsp60 mutant strain (Shu and Hallberg, 1995). Deletion of the
suppressor gene results in temperature-sensitive strains.
Overexpression of the mammalian cDNAs in yeast disrupted strains
rescues the temperature sensitivity. (
)In addition, PP2A
activity co-immunoprecipitates with Rts1 protein. These results
indicate that Rts1p and the B` subunit are functional homologs and
support the notion that the various B-subunits of PP2A confer
specificity to the enzyme. Defects in Cdc55p, the S. cerevisiae homolog of the B subunit, result in multiple elongated buds and
defective cytokinesis (Healy et al., 1991). This phenotype is
different from that elicited by disruption of RTS1, which
encodes the yeast B` homolog. Furthermore, overexpression of Rts1p does
not rescue the defect in cdc55 cells. Thus, the B and B`
subunits appear to target the phosphatase to distinct subsets of
substrates and consequently direct the enzyme to control different
cellular functions.
Although there are only two C2 catalytic subunit isoforms of PP2A, the number of the potential combinatorial associations of the different A and B/B`/B" regulatory subunit forms is very large. Counting two A, two C2, three B, two B", and possibly as many as ten B` subunit isoforms a total of some sixty different trimeric PP2A holoenzymes could be generated that may be able to cope with the large number of cellular functions in which this form of phosphatase has been implicated.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) U37769 [GenBank](B`), U37770 [GenBank](B`
1), U38190 [GenBank](B`
2), U38191 [GenBank](B`
3), U38192 [GenBank](B`
4), U38193 [GenBank]and U38195 [GenBank](B`
), and U38194 [GenBank](B`
).