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(Received for publication, June 14, 1995; and in revised form, August 11, 1995) From the
Protein phosphatase 2A (PP2A) is a major intracellular protein
phosphatase that regulates multiple aspects of cell growth and
metabolism. The ability of this widely distributed heterotrimeric
enzyme to act on a diverse array of substrates is largely controlled by
the nature of its regulatory B subunit. Only two gene families encoding
endogenous B subunits have been cloned to date, although the existence
of several additional regulatory subunits is likely. We have identified
by two-hybrid interaction a new human gene family encoding PP2A B
subunits. This family, denoted B56, contains three distinct genes, one
of which is differentially spliced. B56 polypeptides
co-immunoprecipitate with PP2A A and C subunits and with an okadaic
acid-inhibitable, heparin-stimulated phosphatase activity. The three
B56 family members are 70% identical to each other but share no obvious
homology with previously identified B subunits. These phosphatase
regulators are differentially expressed, with B56
Protein phosphatase 2A (PP2A) (
Figure 1:
Application of the two-hybrid method to
identify PP2A B subunits. A, subunit interactions in the PP2A
heterotrimer and a schematic of the fusion proteins used. The PP2A B
and C subunits are thought to bind to the amino and carboxyl regions of
the A subunit as shown(4) . The 65-kDa A subunit was expressed
as a fusion with the LexA protein (LexA-65A). It was used to screen a
HeLa cell cDNA library in pGAD GH (see ``Experimental
Procedures'') for interacting proteins. Additional LexA constructs
(LexA-315, LexA-397, LexA-SUB, and LexA-lamin) were used to
characterize the interaction between the PP2A A subunit and putative B
subunits. Interaction between the LexA constructs and the GAL4 fusion
proteins activate transcription of HIS3 and lacZ genes in S. cerevisiae L40 and AMR70 cells. B,
B56 clones interact specifically and distinctively with the PP2A A
subunit. L40 cells expressing the indicated B subunit fusion proteins
were mated to AMR70 cells expressing LexA-65A, LexA-modified A
subunits, or the nonspecific bait LexA-lamin. The resulting diploids
were tested for ability to grow on His
Heterotrimeric PP2A enzymes with different B subunits have distinct
substrate specificities(7, 8, 21) , a mode of
phosphatase regulation that has important functional effects. For
example, PP2A can turn SV40 DNA replication on or off, depending on the
type of B subunit in the holoenzyme(6, 22) . Viral
replication in vitro is controlled by the activity of viral
initiator phosphoprotein, SV40 large T antigen. The heterotrimeric form
of PP2A containing B55 removes a phosphoryl group from threonine 124 (a
cyclin-dependent kinase site) (23) and inactivates T
antigen's ability to initiate SV40 DNA replication, while the
PP2A heterotrimer containing B72 removes inhibitory phosphoryl groups
from serines 120 and 123 (casein kinase I sites) (24, 25) and activates SV40 DNA
replication(6) . An additional role of B subunits may be to
act as targeting subunits that direct PP2A to specific subcellular
locations. This method of regulation has been most clearly demonstrated
for protein phosphatase 1, where the catalytic subunit is localized to
its substrates, e.g. phosphorylase, phosphorylase kinase, and
glycogen synthase, by association with a specific glycogen-binding
subunit(2) . Similarly, PP2A-B55 This current study was designed to identify novel PP2A B
subunits. Using the yeast two-hybrid method (27) with the PP2A
A subunit as bait, we identified a novel gene family encoding three
polypeptides with a predicted size of approximately 56 kDa that were
70% identical to each other but with no significant similarity to
either B55 or B72. Full-length polypeptides expressed in 293 cells
bound to PP2A A and C subunits and co-immunoprecipitated with a
heparin-stimulated, okadaic acid-inhibited phosphorylase phosphatase
activity. Northern blots of human tissue showed that these genes have
tissue-specific expression patterns, with two isoforms highly expressed
in heart and skeletal muscle and one highly expressed in the brain.
These results are further evidence that protein serine/threonine
phosphatase diversity is generated in large part by association of a
common catalytic subunit with an increasing array of regulatory or
targeting subunits.
The bait plasmid was based on pBTM116 (originally constructed by P.
Bartel and S. Fields; with the selectable marker TRP1 and
modified by the insertion of the ADE2 gene (31) in the PvuII site) and expresses LexA fused to the full-length human
PP2A A
Full-length clones were
isolated from a human fetal brain cDNA library in bacteriophage lambda
by standard methods (35) and sequenced on both strands using
Sequenase 2.0 according to the manufacturer's instructions.
Sequence
analysis was performed with the Wisconsin package (38) and
GenBank searches using the BLAST algorithm(39) .
A HeLa cell cDNA library in pGAD-GH was then
screened for proteins that interact with the LexA-65A bait (Fig. 1). 17 To identify regions of the PP2A A subunit bait required for
interaction with these putative B subunits, yeast strain AMR70 carrying
mutant bait constructs (as described under ``Experimental
Procedures'') were mated to L40 yeast carrying only the prey
plasmid; interaction was assessed by growth on His
Figure 5:
B56 family members have tissue-specific
mRNA expression. A multiple tissue Northern blot (Clontech) was probed
using fragments of each of the B56 family member cDNAs as described
under ``Experimental Procedures.''
Figure 2:
The B56 family contains closely related
proteins. B56 is encoded by one yeast and three human cDNAs with 70%
amino acid identity. Predicted amino acid sequences were aligned using
the Genetics Computer Group PILEUP program (38) and displayed
using SeqVu(45) . Only residues 219-758 of RTS1 are shown. Amino acid identities are shaded, and
similarities are boxed; gaps introduced to optimize the
alignments are indicated by a dash.
Figure 3:
The A and C subunits of PP2A
co-immunoprecipitate with HA-tagged B56. A, 293 cells were
transiently transfected with a CMV expression plasmid encoding
HA-tagged B56, B55, or ERK1. Soluble extracts from transfected cells
were separated by SDS-PAGE on a 10% gel and then transferred to a
nitrocellulose membrane. HA-tagged proteins were detected by
immunoblotting with the 12CA5 mAb. Asterisk indicates a 12CA5
mAb cross-reacting band present in untransfected cells. B and C, soluble extracts from transfected cells were subjected to
immunoprecipitation with 12CA5 mAb and protein A-agarose.
Immunoprecipitates were eluted in 1
Having established that the constructs indeed produced
soluble proteins of the expected size, extracts from transfected cells
were subjected to immunoprecipitation with 12CA5 mAb.
Immunoprecipitated proteins were separated by SDS-PAGE and
immunoblotted with antibodies that recognize PP2A A and C subunits. As Fig. 3, B and C, demonstrates, HA-tagged
B56
Figure 4:
PP2A activity binds to HA-tagged B56. 293
cells were transiently transfected with the CMV expression plasmids.
The cytosolic fractions were incubated with 12CA5 mAb and protein
A-agarose. Phosphatase activity in immunoprecipitates was assayed using
Finally, to evaluate whether the B56 polypeptides bound to PP2A
heterotrimers (forming heterotetramers) or formed novel heterotrimers,
soluble extracts from transfected cells were analyzed by glycerol
gradient velocity sedimentation followed by immunoblot analysis with
12CA5 mAb. The majority (>75%) of HA-B56
Reversible protein phosphorylation is one of the most widely
utilized mechanisms to regulate cellular processes. The activity of
many cellular enzymes is determined by the net protein phosphorylation
level, which is determined by the balance of specific protein kinase
and protein phosphatase activities. The substrate specificity of the
regulatory protein kinases is determined largely by the diversity of
their catalytic subunits, as demonstrated by the estimate that over
2000 distinct protein kinase catalytic subunits are encoded by the
human genome(43) . However, there appears to be a more limited
number of protein serine/threonine phosphatase catalytic subunits.
Their substrate specificity is determined instead by association with a
variety of regulatory and targeting subunits(2) . We now
report the identification, using the two-hybrid method, of a new gene
family encoding PP2A regulatory subunits. These genes encode authentic
PP2A B subunits based on the following evidence. First, all members of
the gene family B56 (- It is of note
that the B56 gene family has no obvious similarity to previously
identified gene families encoding polypeptides that bind to the
amino-terminal end of the PP2A A subunit (B72/130, B55, B56, and
polyoma and SV40 small t antigens). Thus, it has not been possible to
define an interaction domain that is required for binding to the A
subunit of PP2A. The B56 family has a very highly conserved (80%
identical) central region, while both the carboxyl terminus and the
amino terminus are significantly more divergent. This suggests that the
conserved region is required for interaction with the A and possibly
the C subunit, whereas the ends may perform different functions such as
regulation of substrate specificity or intracellular location of PP2A. The lack of sequence similarity between the different B subunit
families suggests that they each bind differently to the PP2A core
A A yeast gene 68% similar to
the B56 gene family has been independently identified by two groups (Fig. 2)(24, 25) . SCS1 (suppressor of
chaperonin sixty-1), was isolated as a high copy suppressor of several
temperature-sensitive alleles of hsp60 (a mitochondrial
chaperonin)(24) . SCS1 in budding yeast is a cytosolic protein
that when overexpressed appears to positively regulate transcription of
additional chaperonin genes. The identical gene, termed RTS1, was
cloned as a multicopy suppressor of ROX3, which encodes a
transcriptional regulator involved in the response to anaerobic
conditions. Thus, one function of the B56 homologues in yeast appears
to be as regulators of the transcriptional response to environmental
stress. The PP2A regulatory subunits we have identified are similar
in size and tissue distribution to a previously purified regulator of
PP2A with an M PP2A has been implicated in the control of the cell cycle
and the initiation of DNA replication. Since the PP2A A and C subunits
are distributed in multiple cellular compartments, one function of a B
subunit may be to target the heterotrimer to the nucleus, where it can
participate in the regulation of these processes. The expression of the
B55, B56, and B72 genes largely in terminally differentiated tissues
such as heart and brain suggests that the PP2A B subunit that has
direct involvement in cell cycle regulation or DNA replication remains
to be identified.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
L42373[GenBank],
L42374[GenBank], and
L42375[GenBank].
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26123-26128
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and B56
highly expressed in heart and skeletal muscle and B56
highly
expressed in brain. The identification of this novel phosphatase
regulator gene family will facilitate future studies on the control of
protein dephosphorylation and the role of PP2A in cellular function.
)is a major
intracellular phosphatase that regulates such diverse cellular
processes as DNA replication, transcription, signal transduction, and
intermediary metabolism(1, 2, 3) . PP2A is a
heterotrimer, containing A, B, and C subunits. The catalytic activity
of PP2A resides in the C subunit, a 36-kDa protein encoded by two 97%
identical genes. The C subunit binds stably to the carboxyl-terminal
region of the A subunit, a 65-kDa rod-shaped polypeptide consisting of
15 imperfect repeats. The B subunits bind to the amino-terminal region
of the A subunit (Fig. 1A) and determine the substrate
specificity of the complex (4, 5, 6, 7, 8) . Three
distinct B subunits have been biochemically isolated from a variety of
mammalian
tissues(9, 10, 11, 12, 13, 14) ,
and several studies have suggested the existence of additional B
subunits(15, 16) . Additionally, several DNA tumor
viruses encode polypeptides that can function as PP2A B
subunits(17, 18, 19) . The B subunits
purified to date migrate in SDS-PAGE with the apparent molecular masses
of 54 kDa (B54), 55 kDa (B55), and 72 kDa (B72). Three cDNAs encoding
55-kDa B subunits have been
identified(10, 11, 20) ; the B55 family
members are 80-90% identical, and their level of expression
varies by tissue type. A cDNA encoding the 72-kDa B subunit has also
been cloned, and a splice variant encoding a 130-kDa protein has been
identified (12) . The sequence of the 54-kDa B subunit cDNA has
not yet been reported. Interestingly, the amino acid sequences of the
B55 and B72 subunits and the viral PP2A binding proteins show little
homology to each other; thus, no common motif mediating the interaction
of the B subunit with the PP2A A and C subunits has been discovered.
plates. +
signifies growth was similar to growth on His
plates,
± indicates growth was less than half the rate of growth on
His
plates, and - indicates no growth. SV40
small t antigen (t ag) was not tested in the mating assay; the
data indicated here in italics are predicted from the results
of Ruediger et
al.(4, 5) .
has recently been shown
to associate with microtubules(26) . While PP2A activity has
also been found in membrane and nuclear fractions, the B subunits of
PP2A that direct the heterotrimer to these sites have not yet been
identified.
Yeast Two-hybrid Screen Strains and
Constructs
A two-hybrid screen based on the method of
Fields and Song (28) (Fig. 1) was performed using the Saccharomyces cerevisiae strain L40(MATa, his3
200, trp1-901, leu2-3, 112, ade2,
LYS::(lexAop)
-HIS,
URA3::(lexAop)
-lacZ (constructed by S.
Hollenberg). AMR70 (MAT, his3, lys2, trp1, leu2,
URA::(lexAop)-lacZ) (constructed by R.
Sternglanz, the gift of S. Hollenberg) was used in mating assays to
test for specific interactions. Untransformed yeast were grown in YPD,
and transformed yeast were grown in minimal media with necessary
supplements(29) . The HeLa cDNA library in pGAD-GH (with the
selectable marker LEU2) contains inserts averaging 1500 bp in
length and was the gift of Greg Hannon. pGAD-GH expresses inserts as
fusion proteins with the transcriptional activation domain of GAL4 (Fig. 1A)(30) . All the baits and positive
controls used in the two-hybrid method were cloned by use of polymerase
chain reaction using Taq polymerase. High levels of substrate
(100 ng) and low numbers of cycles(10, 11, 12, 13, 14, 15) were
used to minimize inadvertent mutagenesis. Polymerase chain reaction
primers (Table 1) added appropriate restriction sites for
in-frame insertion into the polycloning sites of pBTM116 or pGAD-GH.
subunit(5, 18) . Expression of this
construct, LexA-65A, was confirmed by immunoprecipitation of the fusion
protein from [S]methionine-labeled yeast
extracts with an antibody to the PP2A A subunit. Constructs encoding
mutant A subunits were also prepared to facilitate classification of
isolated clones by the mating assay. These included COOH-terminal
truncations A397 (LexA-A397) and A315 (LexA-A315) and the 12/5 loop
substitution mutant (LexA-SUB), all described by Ruediger et
al.(4, 5) (Fig. 1A). The 12/5
(LexA-SUB) loop mutant was generated by site-directed mutagenesis; this
mutation has been previously shown to eliminate B55 and SV40 small t
antigen binding to the A subunit. Human B72 and C subunit genes were
cloned into pGAD-GH in frame with the activation domain of GAL4 for use
in control experiments (GAL4-B72, GAL4-C). Rat B55
cDNA (20) was cloned into the two-hybrid expression vector pVP16 (32) for use in control experiments (pVP16-B55).Two-hybrid Screen Methods
500 µg of
HeLa cDNA plasmid was used for the large scale transformation of the
L40 strain carrying the LexA-65A bait plasmid(33) , and
interacting cDNA clones were selected by rapid growth on
His plates. Positive colonies were further screened
for lacZ expression using the colony lift method (29) . lacZ
yeast were then allowed
to lose the bait plasmid by removal of selection (Trp
Leu
media) and were identified by accumulation of a
red metabolite due to loss of the ADE2 gene also carried on
the bait plasmid. Yeast containing only the library plasmids encoding
putative interacting proteins were then tested for specific interaction
by mating to AMR70 yeast-carrying plasmids encoding LexA-65A, LexA-SUB,
LexA-A315, LexA-397, or LexA-lamin (Fig. 1)(32, 34) .
Expression of Hemagglutinin (HA)-tagged B
Subunits
The protein coding sequences of
B55(20) , B56, and B56 were cloned using the
indicated polymerase chain reaction primers into pCEP-4/Lerner, a CMV
promoter-driven expression vector based on pCEP4 (Invitrogen) that
encodes a hemagglutinin epitope tag (36) at the amino terminus
of the expressed polypeptide. 293 cells (human embryonic kidney cells
transformed with adenovirus) grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) plus 10% supplemented
calf serum were transfected using lipofectamine (Life Sciences)
following the manufacturer's instructions. Using a plasmid that
expresses lacZ from the CMV promoter, the transfection
efficiency was determined to be about 40%. Cells were lysed 24-36
h after transfection on ice in 20 mM Tris-HCl, pH 7.5, 0.2%
Nonidet P-40, 10% glycerol, 200 mM NaCl, 1 mM EDTA, 1
mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2
µg/ml leupeptin, 1 mM benzamidine, and 2 µg/ml
pepstatin. Lysates were then centrifuged at 14,000 
g for 3 min, and complexes were immunoprecipitated from the
supernatant by the addition of 12CA5 mAb (a mouse monoclonal antibody
that recognizes the hemagglutinin epitope) (36) and protein
A-agarose. For immunoblotting, washed immunoprecipitates were
solubilized in 1 SDS-PAGE loading buffer, separated by SDS-PAGE
on 10% gels, transferred to nitrocellulose, then probed with rabbit
anti-PP2A-A and PP2A-C antibodies and visualized by enhanced
chemiluminescence (Amersham). Phosphatase activity in the
immunoprecipitate was determined by quantitating the release of
trichloroacetic acid-soluble P from
[
P]phosphorylase(37) . All phosphatase
assays were performed in duplicate, and the amount of PP2A activity was
calculated by subtracting radioactivity released from
immunoprecipitates of untransfected cells or cells transfected with a
HA-ERK1 construct.
Northern Blot Analysis
A multiple tissue
human Northern blot (Clontech) was probed sequentially with P-labeled cDNAs encoding B56
(800-bp NcoI-EcoRI fragment), B56 (605-bp EcoRI
fragment), and B56 (entire prey plasmid isolated by the two-hybrid
method) and human
-actin (supplied by Clontech).
Identification of HeLa Cell cDNA Library Clones
That Interact with the PP2A A Subunit in the Yeast Two-hybrid
Screen
The A subunit of PP2A was expressed in S.
cerevisiae strain L40 as a fusion protein with the DNA binding
protein LexA (LexA-65A) and used as the bait for a two-hybrid screen (Fig. 1). Two-hybrid interactions in this strain activate
transcription of the HIS3 and lacZ genes. In pilot
experiments, plasmids expressing the fusion proteins VP16-B55,
GAL4-B72, and GAL4-C subunit were constructed and tested for
interaction with LexA-65A. The GAL4-B72 fusion protein interacted
strongly and specifically with LexA-65A (Fig. 1B),
demonstrating the feasibility of identifying PP2A B subunits with the
two-hybrid method. 10
transformants were plated on
His plates, and rapidly growing colonies were further
screened by blue/white assay for lacZ expression and for
specific interactions in a mating assay. 212 clones were isolated that
interacted strongly and specifically with the A subunit. These 212
clones were separated into 16 groups based on dot blot hybridization
and restriction digests of polymerase chain reaction-amplified inserts.
The 5`- and 3`-ends of representative clones from each of the 16 groups
were then sequenced. The three groups that are the subject of this
report encoded polypeptides that are 70% identical to each other and
are referred to as B56
, -, and -1. Five clones encoded
B56
, two clones encoded B56, and one clone encoded B561.
media (Fig. 1). The B56 family of B subunits isolated in
this screen did not interact with either the carboxyl-terminal
truncated A subunits or the 12/5 loop mutant. This is similar to the
result reported when B55 binding to PP2A AC complex was assessed in an in vitro assay(4, 5) . In contrast, GAL4-B72
interacted with a carboxyl-truncated A subunit, suggesting a different
mode of interaction with the A subunit. None of the clones isolated in
this two-hybrid screen encoded the PP2A C subunit, B55, or B72. This is
consistent with the inability of the GAL4-C and VP16-B55 fusions to
interact strongly with LexA-65A in control experiments and the reported
absence of B72 mRNA in HeLa cells(12) . Ruediger and co-workers (4, 5) have demonstrated in in vitro studies
that B55 binding to PP2A A subunit requires a C subunit binding site,
suggesting that B55
C contacts are essential. Thus, the absence of
human C subunit may have prevented formation of a stable two-hybrid
complex between B55 and LexA-65A.Full-length cDNA Clones Were Identified for the
Partial Clones Found by the Yeast Two-hybrid
Screen
Full-length cDNA clones were obtained for B56
and B56 by screening a human fetal brain cDNA library with P-labeled probes from the 5`-end of clones from the HeLa
two-hybrid library. Two independent phage clones containing similar
amounts of additional 5`-sequence were obtained for B56
, and 4
phage clones with additional 5`-sequence were obtained for B56.
Since the reading frame of each isolate was known from the two-hybrid
screen, in-frame stop and start sites could be used to deduce putative
protein coding regions. The sequence of B56 was determined using
the phage clones for bases 1-456 and the HeLa two-hybrid library
clone for bases 456-3120. B56 sequence was entirely determined
from the phage cDNA clone. For B56, only the 3`- and 5`-ends (350
bases) of the HeLa library clone were sequenced, and no attempt was
made to find full-length clones. The full-length B56
cDNA is 3120
bp, with an open reading frame encoding a polypeptide of 56.1 kDa. The
translational start was determined to be at nucleotide 572, since this
was the first in-frame ATG and gives a start site offset only four
amino acids from that of B56. Additional ATG codons in the
B56 sequence were either far downstream (252 bp) or out of frame.
The B56 cDNA is 2450 bp, and encodes a polypeptide of 57.3 kDa.
The putative initiator methionine for B56 at nucleotide 326 is the
first in-frame ATG upstream of the fusion site seen in HeLa prey
library clones and has an optimum context for translational
initiation(40) . The only other in-frame ATG is at bp 230 but
is followed by an in-frame stop codon at bp 242. We have not isolated a
full-length cDNA encoding B56; the 1447-bp clone obtained in the
two-hybrid screen codes for a predicted protein of 51.5 kDa. The first
1298 bp of B56
1 are identical to a 3700-bp expressed sequence,
HumORFY (GenBank accession number D26445)(41) . However,
HumORFY, encoding a 56.5-kDa polypeptide, appears to be an
alternatively spliced message, as it completely diverges from B56
1
7 codons upstream of the
1 stop site and encodes an additional 43
amino acids. This would predict that there are at least two splice
variant forms of B56
, a conclusion also supported by Northern blot
analysis (see Fig. 5and below). The three B56 cDNAs encode
polypeptides with 70% identity to each other, with divergence limited
to the extreme amino- and carboxyl-terminal regions. The B56 genes are
also homologous to the S. cerevisiae gene RTS1/SCS1 (see ``Discussion''). However,
the predicted B56 amino acid sequences show no obvious similarity to
previously cloned B subunits (Fig. 2).
-Actin cDNA was used
as a positive control.
B56 Associates with the A and C Subunits of PP2A in
Human Cells
By definition, PP2A B subunits form stable
heterotrimers with the A and C subunits of PP2A. To evaluate the
ability of these putative B subunits to form stable complexes with PP2A
A and C subunits in vivo, B56 and B56 polypeptide
coding sequences were cloned with a 5`-extension encoding the
hemagglutinin epitope into a CMV expression vector. To verify that
these constructs indeed encoded proteins of the predicted molecular
weight, cytosolic extracts from transfected cells were subject to
SDS-PAGE and immunoblotting with the 12CA5 mAb (Fig. 3A). HA-B56 migrates slightly faster than
HA-B55, and HA-B56 migrates at about the same speed as
HA-B55.
SDS-PAGE loading buffer,
separated on a 10% polyacrylamide gel, and transferred to a
nitrocellulose membrane, where they were probed with affinity-purified
rabbit antibodies that recognize the PP2A A and C subunits. As a
positive control, whole lysate was run in lane one (EXTRACT). Asterisk indicates IgG heavy chain present in the
immunoprecipitates.
and B56 polypeptides co-immunoprecipitate with PP2A A and
C subunits in transfected human cells, indicating that B56 forms a
stable complex with PP2A in human cells.PP2A Phosphatase Activity Co-immunoprecipitates with
HA-tagged B56
PP2A heterotrimers contain
a catalytic (C) subunit whose phosphatase activity is (i) inhibited by
nanomolar concentrations of okadaic acid and (ii) stimulated toward
nonspecific substrates such as phosphorylase a by heparin
(presumably by facilitating dissociation of the catalytic subunit from
the A and B subunits(42) ). To determine whether the complex
containing HA-B56 indeed contained active PP2A-like phosphatase, 12CA5
immunoprecipitates were incubated with and B56P-labeled
phosphorylase a (Fig. 4). HA-B56
and HA-B56
both co-immunoprecipitate with a phosphorylase phosphatase activity
that is 50-80% inhibited by the inclusion of 1 nM okadaic acid (Fig. 4A). While both PP2A and PP1
can be inhibited by okadaic acid, PP1 is 10-100-fold less
sensitive than PP2A
, with an IC50 of 10-20
nM. Thus, inhibition by 1 nM okadaic acid strongly
suggests that the phosphatase activity present in these assays is PP2A.
Phosphorylase phosphatase activity was also increased 2-4-fold in
the presence of 15 µg/ml heparin (Fig. 4B), a
result expected for PP2A complexes. Thus, the data strongly suggest
that the B56 polypeptides are part of a stable and active PP2A complex.
P]phosphorylase a as a substrate. The
phosphatase activity was also assayed in the presence of 1 nmol of
okadaic acid (O.A.) (upper) or 15 µg/ml heparin (lower). Duplicate assays varied by less than 10%, and all
reactions converted less than 25% of phosphorylase a to
phosphorylase b. PP2A activity was calculated by subtracting
the background 12CA5-precipitatable activity present in non-transfected
cells. The specific activity was 4-10-fold higher than
background.
sedimented at the same
velocity as HA-B55 and as aldolase, a 158-kDa protein (data not
shown). These results are most consistent with the HA-B56 polypeptides
forming a heterotrimeric complex with the PP2A A and C subunits.Tissue-specific Expression of B56
Isoforms
The previously cloned PP2A regulatory subunits
have shown tissue-specific and developmentally regulated patterns of
gene expression, with specific isoforms preferentially expressed in
muscle and brain(10, 13) . To determine the expression
pattern of the B56 genes, a multiple human tissue Northern blot was
probed with sequences from the three B56 genes (Fig. 5).
B56 mRNA is approximately 3500 nucleotides, with expression in all
tissues examined but with the highest expression in heart and skeletal
muscle. B56 mRNA is 3000 nucleotides with highest expression in
brain. The apparent sizes of B56 and B56 mRNAs (3.5 and 3.0
kb) are 380-550 nucleotides longer than the cDNA clones we have
obtained. B56 probe hybridizes with three mRNA species of 1.8,
2.1, and 4.4 kb. The 4.4-kb transcript is most highly expressed in
heart and skeletal muscle, while the shorter transcripts are present
largely in heart alone. The 4.4-kb species most likely corresponds to
the 3700-bp HumORFY sequence, while the smaller more cardiac-specific
mRNAs most likely correspond to the B56
1 clone isolated in this
study.
, -, -) interacted with a LexA-A
subunit bait in the two-hybrid assay and failed to interact with
irrelevant baits such as LexA-lamin. Second, PP2A A and C subunits were
shown to associate with epitope-tagged B56
and polypeptides
in human cells in co-immunoprecipitation assays. Third, HA-B56 and
- co-immunoprecipitated with a phosphatase activity that was
inhibited by 1 nmol of okadaic acid and enhanced by heparin, the
results expected for a component of a PP2A complex.C complex and exert their effects on substrate specificity in
this manner. In support of this hypothesis, we note that test preys
encoding B55, B72, and B56 fusion proteins each interacted differently
in the two-hybrid screen when tested for association with the mutant
and wild type A subunits (Fig. 1B). Thus, B72, unlike
B55 and B56, interacted with carboxyl-truncated A subunit A397, and the
B56 preys interacted with full-length A subunit significantly more
strongly than did the B55 preys. B55 interaction with the A subunit is
reportedly stabilized by the presence of the C subunit(4) ;
this is supported by our finding that the B55 prey construct interacted
only weakly with the A bait in yeast, where no mammalian C subunit
exists. B72 and B56, like SV40 small t antigen, may bind more tightly
to the A subunit and thus have less of a requirement for a B-C
interaction. Additionally, within each B subunit family there are
non-conserved sequences that may contribute to unique interactions with
the PP2A A and C subunits or contribute to interactions with additional
cellular proteins. For example, PP2A heterotrimers containing either
B55 or B55 differ in their response to effector molecules
such as protamine and heparin (44) . Differential expression in
diverse tissues of B56 and B55 family members also implies that each
isoform performs a specific function.
of 54,000 as assessed by
SDS-PAGE(13) . If the B56 genes indeed encodes the 54-kDa
protein, we would predict based on Northern blot analysis that the
cardiac B subunit, also known as B`, is encoded by B56 or
B56.
), media lacking the amino acid histidine; CMV,
cytomegalovirus; mAb, monoclonal antibody; kb, kilobase(s).
We thank Rolf Rüediger, Gernot
Walter, David Pallas, Jozef Goris, and Brian Hemmings for plasmids
encoding wild type and mutant PP2A subunits; Stan Hollenberg and Graeme
Bolger for plasmids and yeast strains; Kimberly Fish for assistance
with methods; and John Phillips, David Stillman, and Andrew Thorburn
for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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