(Received for publication, September 25, 1995; and in revised form, December 21, 1995)
From the
Differential association of regulatory B subunits with a core
heterodimer, composed of a catalytic (C) and a structural (A) subunit,
is an important mechanism that regulates protein phosphatase 2A (PP2A).
We have isolated and characterized three novel cDNAs related to the B`
subunit of bovine cardiac PP2A. Two human (B`1 and B`
2) and a
mouse (B`
3) cDNA encode for alternatively spliced variants of the
B` subunit. The deduced primary sequences of these clones contain 12 of
15 peptides derived from the purified bovine B` subunit. Differences
between the deduced sequences of the B`
splice variants and the
cardiac peptide sequences suggest the existence of multiple isoforms of
the B` subunit. Comparison of the protein and nucleotide sequences of
the cloned cDNAs show that all three forms of B`
diverge at a
common splice site near the 3`-end of the coding regions. Northern blot
and reverse transcription-polymerase chain reaction analyses revealed
that the B`
transcripts (4.3-4.4 kb) are widely expressed
and very abundant in heart and skeletal muscle. The expressed human and
mouse B`
proteins readily associated with the PP2A core enzyme in
both in vitro and in vivo complex formation assays.
Immunofluorescence microscopy revealed that epitope-tagged B`
was
localized in both the cytosol and nuclei of transiently transfected
cells. The efficiency of binding of all three expressed proteins to a
glutathione S-transferase-A subunit fusion protein was greatly
enhanced by the addition of the C subunit. Expression of the B`
subunits in insect Sf9 cells resulted in formation of AC
B`
heterotrimers with the endogenous insect A and C subunits. These
results show that the B` subunit, which is the predominant regulatory
subunit in cardiac PP2A, is a novel protein whose sequence is unrelated
to other PP2A regulatory subunits. The nuclear localization of
expressed B`
suggests that some variants of the B` subunit are
involved in the nuclear functions of PP2A.
Protein phosphorylation is an essential mechanism regulating a
wide variety of cellular processes. The coordinate activity of protein
kinases and phosphatases is required for normal signal transduction.
Protein phosphatase 2A (PP2A) ()is a serine/threonine
phosphatase that has been implicated in the control of the cell
cycle(1) , growth and proliferation(2) , DNA
replication(3) , viral transformation(4) , and
morphogenetic events(5) . The PP2A holoenzyme is a heterotrimer
composed of a 38-kDa catalytic (C) subunit, a 63-kDa structural (A)
subunit, and a third subunit termed B or phosphatase regulatory (PR)
subunit(6, 7) . There are at least five distinct
families of proteins that interact with and regulate the PP2A core
enzyme. These include the B, B`, PR72 (B"), and
families of
regulatory subunits and the small and middle tumor antigens of DNA
tumor viruses(8) .
The diversity of PP2A regulatory subunits
suggests specific physiological roles for individual holoenzymes.
Genetic studies have shown that disruption of the yeast homolog of the
B subunit, CDC55, results in defects in budding and
cytokinesis(9) . Decreased expression of the Drosophila B
subunit causes defects in mitosis, duplication of wing
imaginal discs and is lethal in the late larval/early pupal stage of
development.(5, 10) . Biochemical studies have
demonstrated that the B subunits and tumor antigens affect the
substrate specificity and activity of
PP2A(11, 12, 13) . Sensitivity to polyamines,
polycations, and ceramide is also dependent on the type of B subunit
associated with the AC core
complex(13, 14, 15) . These data support an
emerging hypothesis that differential association of B subunits
regulates PP2A function in vivo.
Molecular cloning has
revealed diversity within the PP2A regulatory subunit families.
Multiple isoforms (,
,
) of the B subunit and a splice
variant of B
have been isolated from mammalian sources (9, 16, 17) . These proteins are 81-87%
identical and diverge primarily at the amino termini. Two cDNAs
encoding 72- and 130-kDa forms of the PR72 (B") subunit were obtained
from human heart and brain libraries(18) . PR130 has an
extension at the amino terminus that is generated either by alternative
splicing or the use of an alternative promoter site. In this report, we
describe the isolation and expression of novel cDNAs encoding human
(B`
1 and B`
2) and mouse (B`
3) members of the B` familiy
of PP2A regulatory subunits. The recombinant subunits readily form
heterotrimeric complexes with the AC core enzyme in vitro and in vivo. Transient expression of human B`
in mammalian
cells revealed the presence of both cytoplasmic and nuclear populations
of the regulatory subunits. The B`
subunits may be important for
the localization/translocation of PP2A into the nucleus.
GAL4 DNA binding domain fusions of human
B`, p53, and the A subunit were tested for binding specificity
with GAL4 activation domain fusions of the A and C subunits of PP2A.
The A and C subunits of PP2A were inserted into the EcoRI/XhoI and SmaI/SalI sites of
pACTII, respectively. NcoI/BamHI fragments from the
pRcCMV-B`
plasmids (see below) were subcloned into the appropriate
sites in the GAL4 DNA binding domain vector, pAS1-CYH2. Expression of
-galactosidase activity in the two-hybrid assay was determined as
described above(22) .
Poly(A) RNA was isolated from mouse tissues, and
first strand cDNA was synthesized as described previously(23) .
The mouse cDNA was used in a PCR with oligonucleotide primers
corresponding to nucleotides 837-857 (sense
5`-CTGAGTGTCTACCATCCCCAG-3`) and 1413-1436 (antisense
5`-CTCGGACTTGCGGCGTGCAAGAGG-3`) of B`
2. The thermal profile (94
°C, 1 min; 55 °C, 1 min; 72 °C, 1 min) was carried out for
30 cycles.
Figure 1:
Alignment of the deduced amino acid
sequences of human B`1, B`
2, mouse B`
3, and residues
127-561 of yeast RTS1. Peptides derived from purified bovine
cardiac B` are shown below the aligned sequences. The asterisks represent identities, and the dashed lines indicate gaps. The bipartite nuclear localization signal in
B`
1 is underlined. The nucleotide sequences of human
B`
1 (accession number U37352), and mouse B`
3 (accession
number U37353) have been deposited in the GenBank data base. The
accession number for human B`
2 (KG1 ORFY) is D26445 and for yeast
RTS1 is U06630.
Oligonucleotide primers
were designed from the human sequence in the data base, and a 854-bp
PCR product was amplified from a HUVEC ZAP cDNA library. The PCR
product was sequenced and found to be identical to nucleotides 775-1629
of the KG1 cDNA. Five positive clones were isolated from the HUVEC cDNA
library with the 854-bp probe using stringent hybridization and wash
conditions. The largest cDNA (HB`-7) was 4064 bp long and nearly
identical in sequence to the KG1 cDNA. Two base changes were present in
the 3`-untranslated region of the HUVEC cDNA; nucleotide 2082 was G
instead of A, and nucleotide 3847 was A instead of G. The HUVEC cDNA
extends the 5`- and 3`-untranslated regions of the KG1 sequence by 62
and 182 nucleotides, respectively. The sequence around the putative
initiator codon, AGCAGGATGGTGG, conforms to the consensus motif for
translation initiation in vertebrates(28) . Four potential
polyadenylation sites (AATAAA) are present at nucleotides 2480, 3677,
3851, and 4027. There is also an insertion of 117 nucleotides in the
HB`-7 cDNA that is absent in the KG1 cDNA. The insertion occurs after
nucleotide 1385 and encodes for an additional 39 amino acids
(433-472). We have designated this cDNA B`
1. The deduced
amino acid sequence encodes for a protein of 514 residues with a
predicted molecular weight of 59,995 and an isoelectric point of 6.1 (Fig. 1).
The remaining cDNAs were found to be partial
clones, three of which corresponded to B`1. The fourth cDNA
(HB`-5) lacked 617 nucleotides of the 5` sequence, terminated at
nucleotide 3138 of B`
1, and did not contain the 117-nucleotide
insertion. This cDNA corresponded to the KG1 sequence in the data base,
and we have designated it B`
2. The deduced amino acid sequence
encodes for a protein of 475 residues with a predicted molecular weight
of 55,958 and an isoelectric point of 6.5 (Fig. 1).
An
additional B` cDNA was isolated in an independent screen of a
mouse T-lymphocyte library for proteins that interact with the A
subunit of PP2A using the yeast two-hybrid assay. One of the cDNAs that
interacted with the GAL4 DNA binding domain-A subunit fusion protein
was a mouse homolog of human B`
. This cDNA (B`
3) was 1.35 kb,
lacked a portion of the 5` sequence, and contained 37 nucleotides of
the 3`-untranslated sequence. The partial open reading frame encodes
for a protein of 435 residues, with a predicted molecular weight of
51,158. An alignment of the human, mouse, and a portion of the yeast
RTS1 primary sequences is shown in Fig. 1. There are three
conserved substitutions in the mouse sequence compared with human
B`
1 (88% identity) and B`
2 (92% identity). The mouse sequence
terminates 10 residues after the B`
1 insertion begins.
Based on
the nucleotide sequence identity, the two human forms are likely to
arise by alternative splicing of a single gene. A putative splice
acceptor site boundary, CCCAGG (nucleotides 764-769 in B`2,
1319-1324 in the KG1 cDNA, and 1379-1384 in B`
1), is
present at the point of divergence between the B`
1 and B`
2
sequences. Consistent with these observations is the conservation of
the same splice site junction in the mouse B`
3 cDNA (nucleotides
1231-1236). The presence of the 39-amino acid insertion in
B`
1 produces a bipartite nuclear targeting signal (29) that is absent in both B`
2 and B`
3. The yeast
homolog of B`
diverges from its mammalian counterparts at the
amino and carboxyl termini, which are not shown in Fig. 1. The
overall identity between the yeast protein and B`
1, B`
2, and
mouse B`
3 is 39, 40, and 43%, respectively. However, if the
divergent termini are omitted and the comparison done is on residues
127-561 of the yeast protein, the identity increases to 68%.
Figure 2:
Expression and tissue distribution of
B` mRNA. Panel A, Poly(A
) RNA (2 µg)
from the indicated human tissues was hybridized with a B`
specific
probe as described under ``Materials and Methods.'' Panel
B, first strand cDNA was prepared from poly(A
)
RNA isolated from mouse tissues and used in a PCR with B`
specific
primers as described under under ``Materials and Methods.''
An aliquot of each reaction was resolved on a 1% agarose gel and
stained with ethidium bromide (0.5 µg/ml). The migration of
molecular size standards (kb) are indicated to the left of
each panel.
The probe used in the Northern analysis does not distinguish
between the alternatively spliced forms of human B`. Therefore,
expression of the alternatively spliced forms of B`
mRNA was
examined in mouse tissues by reverse transcription PCR (Fig. 2B). Two major bands were amplified from each
tissue that had mobilities identical to the B`
1 (717 bp) and
B`
2 (600 bp) controls. Similar amounts of cDNA were present in
each PCR reaction, since the ubiquitously expressed mRNA for
cyclophilin was amplified to similar levels with all of the cDNAs (data
not shown). Approximately the same ratios of B`
1 and B`
2 were
observed in mouse tissues except liver and especially brain, where
B`
2 was more prevalent. In contrast to the human Northern blot,
mouse skeletal muscle contained significantly lower levels of the
B`
transcripts. Interestingly, bands of
650 and
750 bp
were also amplified with the heart, brain, and skeletal muscle cDNAs.
Although we have not identified these products, they may be due to the
presence of additional splice variants of B`
or other isoforms of
the B` regulatory subunit.
In
vitro transcription/translation of B` cDNAs in reticulocyte
lysates directed the synthesis of
[
S]methionine-labeled human and mouse B`
proteins. The expressed proteins had apparent molecular masses of 60
kDa (Fig. 3A, lane 1, B`
1), 56-kDa (Fig. 3A, lane 2, B`
2), and 52-kDa (Fig. 3A, lane 3, B`
3), which were nearly
identical to the predicted molecular weights of each splice variant.
The interaction of the expressed proteins with the A subunit was
assayed using a GST-A subunit fusion protein. A low level of binding to
GST-A was observed with B`
1 (Fig. 3B, lane
1), B`
2 (Fig. 3C, lane 1), and
B`
3 (Fig. 3D, lane 1). However,
preincubation of the GST-A fusion protein with the C subunit of PP2A
significantly enhanced the binding of all three forms of B`
(lane 2). The interaction of the B`
proteins with the A
subunit was specific, as no binding was observed with GST alone (lane 3) or when GST was preincubated with the C subunit (lane 4). The minor bands present in the GST-A lanes may be
due to proteolysis of B`
1 and B`
2 or initiation from an
internal methionine.
Figure 3:
Binding of B` proteins to a GST-A
subunit fusion protein. Panel A, SDS-PAGE of in vitro translation products of human B`
1 (lane 1), human
B`
2 (lane 2), and mouse B`
3 (lane 3) cDNAs.
A control sample containing no cDNA template was applied to lane
4. An aliquot of the translation mixture containing B`
1 (panel B), B`
2 (panel C), and B`
3 (panel D) were assayed for binding to a GST-A subunit fusion
protein as described under under ``Materials and Methods.'' Lane 1, GST-A subunit; lane 2, GST-A subunit +
purified bovine C subunit (2.5 µg); lane 3, GST fusion
protein; lane 4, GST fusion protein + purified bovine C
subunit (2.5 µg). The migration of molecular mass standards
including bovine serum albumin (66-kDa), glutamate dehydrogenase
(55-kDa), ovalbumin (44-kDa), and aldolase (40-kDa) are shown to the left of each panel.
Recombinant B` proteins were also
expressed in the baculovirus-insect cell system. Homogenates from
infected cells were partially purified by heparin-Sepharose and size
exclusion chromatography to determine if the expressed B`
proteins
interacted with the endogenous AC core enzyme. The B`
proteins in
infected Sf9 cell extracts were eluted from heparin-Sepharose with both
0.3 and 0.5 M NaCl (data not shown). When B`
1 in the 0.3 M NaCl fraction was applied to a gel filtration column, the
peak of immunoreactivity co-eluted with the C subunit of PP2A (Fig. 4A) in fractions 24-26, which correspond to the
elution position of purified cardiac AC
B` (M
= 156,000). The 0.5 M NaCl heparin-Sepharose
fraction, which did not contain any detectable C subunit (data not
shown), eluted in fractions 27-29, corresponding to the predicted
molecular weight of monomeric B`
1 (Fig. 4C; left half of panel). Incubation of the 0.5 M NaCl heparin-Sepharose fraction with purified bovine cardiac AC
dimer shifted the peak of B`
1 to fractions 24-26,
corresponding to heterotrimeric PP2A (Fig. 4C; right half of panel). Similar results were obtained
with recombinant B`
2 (Fig. 4, B and D).
Formation of the AC
B`
complexes was not disrupted by
chromatography in 0.5 M NaCl. These properties of the
expressed B`
1 and B`
2 proteins are identical to those
described previously for the B` subunit purified from cardiac
tissue(20) . These data demonstrate that the cloned B`
proteins bind directly to the A subunit of PP2A and readily form
heterotrimeric complexes with the AC core enzyme in vitro and in vivo.
Figure 4:
In vivo and in vitro reconstitution of expressed B` proteins with the AC core
enzyme of PP2A. Homogenates of insect cells infected with baculoviruses
encoding B`
1 (panels A and C) and B`
2 (panels B and D) were partially purified on a
heparin-Sepharose column. Aliquots of the fraction eluted with 0.3 M NaCl (panels A and B) and 0.5 M NaCl (left half of panels C and D) were
separately chromatographed on a Superdex 200 10/30 column. A second
aliquot of the 0.5 M NaCl fractions (right half of panels C and D) were incubated with purified bovine
AC (20 µg) for 30 min (4 °C) prior to chromatography. The
fractions from the Superdex column were analyzed by immunoblotting for
B`
1 (panels A and C), B`
2 (panels B and D), and the C subunit of PP2A (panels A and B). The homogenates were used as positive controls (+)
for the expression of B`
1 and B`
2. The migration of molecular
mass standards including bovine serum albumin (66-kDa), glutamate
dehydrogenase (55-kDa), and aldolase (40-kDa) are shown to the left of each panel.
Figure 5:
Localization of transiently expressed
human B` in NIH3T3 cells. NIH3T3 cells were transfected with
pFLAG-CMV2-B`
1 (panels A and B),
pFLAG-CMV2-B`
2 (panels C and D), or
pFLAG-CMV2-B
(panels E and F). The cells were
fixed and stained with an anti-FLAG antibody and analyzed by indirect
immunofluorescence microscopy (panels A, C, and E) as described under ``Materials and Methods.'' Panels B, D, and F are phase contrast images
of the same field of cells shown in panels A, C, and E.
We have isolated three novel cDNAs related to the bovine
cardiac B` regulatory subunit of PP2A. All three forms appear to be
generated from a single gene by alternative splicing. Transcripts of
B` are widely expressed in human and mouse tissues and are
especially abundant in muscle. The high level of B`
transcripts
detected in heart and skeletal muscle is consistent with the
biochemical composition of PP2A purified from these
tissues(17, 24) . These B` subunits have no apparent
homology to the B or PR72/130 (B") regulatory subunits or other
proteins that interact with and regulate PP2A, including viral tumor
antigens and the phosphotyrosyl phosphatase activator
protein(31) . Interaction of these proteins with PP2A is
apparently not due to regions of conserved sequence but may involve
domains of similar higher order structure.
Heterotrimeric complexes
of PP2A were formed when recombinant human and mouse B` proteins
were reconstituted with the AC core enzyme in vivo and in
vitro. All three forms of B`
interacted with a GST-A subunit
fusion protein and with a GAL4 activation domain-A subunit fusion
protein in the yeast two-hybrid assay. A role for the C subunit in
stabilizing oligomeric complexes was originally suggested by
reconstitution experiments with porcine cardiac PP2A(32) . This
idea is supported by chemical cross-linking studies (13, 20) , and analysis of A subunit
mutants(33, 34) . The increased binding of recombinant
B`
proteins to GST-A in the presence of C is also consistent with
a role in stabilizing the AC
B` heterotrimer. We do not know if
the endogenous yeast A subunit participates in the two-hybrid
interaction. However, no hybrids were formed between GAD-C and
GDB-B`
, suggesting that B`
has very weak affinity for C and
that the yeast A subunit homolog does not participate in hybrid
formation. Another possibility is that the fusion with the DNA binding
domain to the amino terminus of B`
hinders interaction with GAD-C.
The interaction assays suggested that the B` subunits have
different apparent affinities for PP2A. Of the three proteins, B`
1
displayed the strongest interaction in both the yeast two-hybrid and
GST-A binding assays. It is not known whether the absence of 8 amino
acids from the amino terminus of mouse B`
3 affects binding to
PP2A. However, there is evidence that a portion of the amino terminus
of the B
and B
subunits is involved in binding to the AC core
enzyme (13) . The fact that B`
1 and B`
2 have
different apparent affinities for the AC complex suggests that regions
near the carboxyl terminus are also important for subunit interactions.
The purified bovine cardiac B` subunit has a very high affinity
(IC = 0.58 nM) for the AC form of
PP2A(20) . Although cardiac B` and B`
2 have the same
mobility in SDS-PAGE, functional data and other lines of evidence
suggest that human B`
2 and bovine B` are not the same protein.
Three of the peptides derived from the bovine cardiac protein were not
found in the human or mouse B`
sequences. Although these peptides
may have been generated from a contaminant(s), they were not similar to
any other sequences in the data bases. Anti-peptide antibodies raised
against the human B`
proteins cross-reacted with the bovine
cardiac subunit; however, no cross-reactivity with the human proteins
was observed with an antiserum (E005) raised against purified bovine
cardiac B` (data not shown). Taken together, these data indicate that
the human B`
and bovine cardiac B` subunits are closely related
isoforms of a larger B` family. (
)
PP2A has generally been
regarded as a soluble cytoplasmic enzyme, but significant amounts of
PP2A are also present in the nucleus(35, 36) . Partial
purification of rat liver nuclear PP2A suggests that the nuclear enzyme
is a heterotrimer; however, the type of B subunit in nuclear PP2A has
not been identified(37) . A significant feature of all three
B` variants is the presence of a cluster of basic residues near
the carboxyl terminus and a consensus bipartite nuclear localization
signal in B`
1 (residues 462-478). The bipartite motif has
been found in 60% of all known nuclear proteins and less than 4% of
nonnuclear proteins(29) . Although the bipartite nuclear
localization signal is absent in B`
2, localization of
FLAG-B`
2 was similar to that of B`
1 in our transient assays.
While some nuclear proteins contain large T antigen-type nuclear
targeting signals or the bipartite motifs, there are many nuclear
proteins that do not contain a concensus sequence for nuclear
localization(29) . The only significant feature that has been
recognized in these proteins is the presence of clusters of basic
residues within the signaling domain. The basic residues present in the
carboxyl termini of the B`
proteins may serve as a signal for
nuclear import. Mutational analysis of this region will be required to
determine the critical residues for nuclear localization.
Exclusion
of transiently expressed FLAG-B from the nucleus is consistent
with previous immunofluorescence data showing that AC
B
is
largely cytoplasmic and that a subpopulation is associated with the
microtubule cytoskeletal network(30) . We have not quantitated
the amount of FLAG-B
that is associated with endogenous AC in the
transient assays. However, other studies have shown that expressed
small t antigen interacts with endogneous AC in mammalian
cells(2, 38) . In addition, we have shown here that
expressed B`
subunits form heterotrimeric complexes with
endogenous AC in Sf9 cells. Presumably, some FLAG-B
associates
with AC and is bound to microtubules, preventing nuclear import. We
have no evidence that AC
B`
binds to microtubules, and
failure of the AC
B`
complex to interact with the
cytoskeleton may allow nuclear uptake. Regardless of the mechanism, it
is clear that expressed B`
subunits are highly localized within
the nucleus, while the B
subunit is not. This result suggests that
some members of the B` family may be present in nuclear PP2A and that
B` may be important in nuclear functions of PP2A. PP2A has been
implicated in the dephosphorylation of nuclear cAMP-response element
binding protein(37, 38) . The AC
B`
heterotrimer may be involved in the control of cAMP-mediated changes in
gene transcription. Yeast RTS1 is a suppressor of a mutation in the
ROX3 transcription factor. The similarity of RTS1 and B`
also
suggests a role for the mammalian homolog in regulating the activity of
transcription factor(s).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37352 [GenBank]and U37353[GenBank].