(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 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.
Protein phosphatase 2A (PP2A) ()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.
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 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) .
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 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.
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 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).
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 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 B56
1.
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 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.
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.'' -Actin cDNA was used
as a positive control.
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 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.
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 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.
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 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.
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 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.
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 (-, -
, -
) 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.
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
AC 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.
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 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
.
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].