Báo cáo khoa học: Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A pdf

Two conserved domains in regulatory B subunits mediate
binding to the A subunit of protein phosphatase 2A
Xinghai Li
1
and David M. Virshup
1,2
1
Department of Oncological Sciences, Center for Children, Huntsman Cancer Institute, and
2
Department of Pediatrics,
University of Utah, Salt Lake City, UT, USA
Protein phosphatase 2A (PP2A) is an abundant heterotri-
meric serine/threonine phosphatase containing highly con-
served structural (A) and catalytic (C) subunits. Its diverse
functions in the cell are determined by its association with a
highly variable regulatory and targeting B subunit. At least
three distinct g ene families encoding B subunits are known:
B/B55/CDC55, B¢/B56/RTS1 and B¢¢/PR72/130. No
homology has been identi®ed among the B families, and little
is known a bout how these B subunits interact with the P P2A
A and C subunits. In vitro expression of a series of B56a
fragments identi®ed two distinct domains that bound inde-
pendently to the A subunit. Sequence alignment of these A
subunit b inding domains (ASBD) identi®ed conserved resi-
dues in B/B55 and PR72 family members. The alignment
successfully predicted domains in B55 and PR72 subunits
that similarly bound to the PP2A A subunit. These results
suggest t hat these B s ubunits share a common core structure
and mode of interaction with the PP2A ho loenzyme.
Keywords: phosphoprotein phosphatase; PP2A; subunit
interactions; phosphorylation.

Protein phosphatase 2A (PP2A) is an abundant cellular
serine/threonine-speci®c phosphatase that regulates a sig-
ni®cant array of cellular events. The PP2A holoenzyme is a
heterotrimer, containing a 65-kDa regulatory A subunit
(A/PR65), a 36-kDa catalytic C subunit, and one of a
variety of r egulatory B subunits. These diverse B subunits in
the P P2A heterotrimer allow the phosphatase to localize t o
distinct regions of the cell and to dephosphorylate speci®c
substrates, thereby allowing PP2A to regulate diverse
processes in the cell such as DNA replication, Wnt
signaling, apoptosis, and cytoskeletal function (reviewed in
[1,2]). The importance o f B subunits in cellular r egulation is
illustrated by the effect of mutations that alter B subunit
function. Over-expression of B56 blocks Wnt signaling in
Xenopus embryos [3±5], mutations in a Drosophilia B/B55
subunit l eads to imaginal disc duplication and defects in
mitosis [6,7], t ransposon insertions in B56c enhance the
metastatic ability of mouse melanoma cell lines [8], muta-
tions in the A s ubunit that a lter B subunit binding are found
in lung, breast, colorectal and s kin cancers [9,10], and
decreases in A subunit e xpression are seen in neuronal
tumors [11]. Despite the signi®cant role t he B subunits play
in cellular homeostasis, little is known about how they
physically interact with the PP2A holoenzyme to target the
phosphatase to its substrates.
The P P2A A subunit serves as a scaffold for assembly of
the B and C subunits. I t i s c omposed of 15 imperfect HEAT
repeats, each of 39 amino acids, which form a hook-shaped
molecule [12]. T he repeats consist of t wo a helices connected
by an intrarepeat loop, and m utations in distinct lo ops alter

the binding of the B and C subunits [13]. The B subunits
bind to repeats 1±10 of the A subunit, whereas the C s ubunit
binds to repeats 11±15. Interactions between the B and C
subunits are a lso important for heterotrimer formation, as
loss of C subunit binding sites prevents B subunit binding
[14,15], and modi®cation of the C-term inus of the C s ubunit
regulates B subunit binding [16±18].
To date, at least three families of PP2A B subunits have
been identi®ed in e ukaryotes. They are designated B (PR55,
B55, CDC55), B¢ (PR61, B56, RTS1), and B¢¢ (PR72/130).
Each B subunit family is encoded by m ultiple genes, with
multiple splice variants, generating an extraordinary diver-
sity of these regulatory subunits [1,2]. Although the three
families o f B subunits do not share a pparent sequence
similarities between the families, they do have signi®cant
sequence homology within each family. For example, within
the B56 family, each isoform shares a common core r egion
of 241 amino acids with 71±88% identity by protein
sequence, while both the N- and C -termini are signi®cantly
more divergent [19±21]. The conserved core region h as been
proposed to interact with the AC h eterodimer, while the
nonconserved N- and C-ends may perform different
functions, such as r egulation of s ubstrate speci®city and
subcellular targeting [20,22]. Two additional classes of
polypeptides also interact with the AC core of PP2A. Both
the small and middle T antigens encoded by polyomavirus
and SV40, and the calmodulin-binding proteins striatin and
SG2NA [ 23], bind to the AC c ore of P P2A. However, unlike
the B subunits, T antigens and s triatin do not require
interaction with, nor methylation, of the PP2A C subunit

[17].
Little is known about the molecular basis for the
interaction of the B subunits with the AC heterodimer.
None of the B subunits have been mapped to de®ne the
Correspondence to D. M. Virshup, Huntsman Cancer Institu te,
University of Utah, Salt Lake City, UT 84112. Fax: + 801 587 9415,
Tel.: + 801 585 3408,
Abbreviations: PP2A, protein phosphatase 2A; ASBD, A subunit
binding dom a in; GST-A, glu t athione S-transferase A s ubunit; NP-40,
nonidet p40; CMV, cyto megalovirus.
(Received 19 S eptember 2001, revised 8 November 2001, accepted 16
November 2001)
Eur. J. Biochem. 269, 546±552 (2002) Ó FEBS 2002
A subunit binding domains. I n t his s tudy, w e u sed t he B56 a
isoform as a model regulatory protein to identify structural
elements involved in the interaction with PP2A. We
identi®ed two distinct domains within the B56a core region
that are each suf®cient for interaction with the A subunit.
Sequence alignment analyses demonstrated that these two
distinct regions are signi®cantly conserved among the three
eukaryotic B subunit f amilies. The predicted A subunit
binding domains in B/B55 and B¢¢/PR7 2 were also able to
interact with the PP2A A subunit. The p resence of a
conserved motif in the highly divergent B subunits suggests
a common ancestry, structure, and mode of A subunit
interaction for these i mportant regulatory proteins.
EXPERIMENTAL PROCEDURES
Synthesis of [
35
S]protein

[
35
S]Methionine-labeled B subunits and t heir fragments, and
SV40 small t antigen a nd its mutant were g enerated by
coupled in vitro transcription and translation in r abbit
reticulocyte lysates ( TNT, Promega) using PCR-generated
templates. All N-terminal PCR primers incorporated a T3
or T7 promoter sequence. Ampli®ed PCR products were
puri®ed using a PCR puri®cation kit (Qiagen) and
200±400 ng of puri®ed DNA was added to 50 lLof
reticulocyte lysate in the presence of [
35
S]methionine. The
reaction was incubated at 30 °C for 2 h. In several cases,
additional lower molecular mass bands were seen which a re
likely to b e due to either premature termination o r partial
proteolysis of the [
35
S]methionine-labeled proteins.
Preparation of glutathione
S
-transferase (GST)
and GST-A fusion proteins
The GST-A subunit o f PP2A ( GST-A) construct was a
generous gift from M. Mumby (UT Southwestern, Dallas,
TX, USA) [24]. Puri®cation of GST-A and GST proteins
from Escherichia coli was p erformed as described previously
[24]. The puri®ed proteins were thoroughly dialyzed against
buffer A (50 m
M

Tris/HCl pH 7.5, 20 m
M
NaCl, 2 m
M
EDTA, 1 m
M
dithiothreitol, containing 3 lgámL
)1
pepsta-
tin and leupeptin, 2 m
M
benzamidine, and 1 m
M
phen-
ylmethanesulfonyl ¯uoride). The resultant protein
preparation was stored at )70 °C in buffer A containing
50% glycerol until use.
GST precipitation assay
The binding reactions contained 10 lLof[
35
S]methionine-
labeled polypeptides from programmed reticulocyte lysates,
2 lg of GST or GST-A and buffer A in a ®nal volume of
50 lL. After i ncubation for 2 h at ambient temperature (or
4hat30°C, where indicated), the reaction was diluted to
500 lL with buffer B [buffer A containing 0.1% nonidet
p40 (NP-40) and 0.25% BSA] and 20 lLofapre-
washed 1 : 1 slurry of glutathione±Sepharose (Amersham
Pharmacia) was added. Incubation continued f or 2 h at
4 °C . The beads we re then washed four times w ith 1 mL of

buffer B , o r R IPA buffer (50 m
M
Tris, pH 7 .5, 150 m
M
NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) where
indicated, for 10 min each wash. Bound proteins were then
eluted by incubating the beads with 20 lLof10m
M
reduced glutathione in buffer A on ice for 30 min The
eluted polypeptides w ere analyzed by either conventional
SDS/PAGE or on tricine/glycine gels for small molecular
mass peptides [25] and i maged u sing a Molecular D ynamics
PhosphorImager.
RESULTS AND DISCUSSION
Identi®cation of two A subunit-binding domains in B56a
To determine the minimal r egion of B56 that interacted with
the PP2A subunit, we utilized an in vitro binding assay using
GST-A subunit and reticulocyte lysate-synthesized B f rag-
ments [10,21]. To optimize c onditions for the assay, full-
length B56a was ®rst tested for binding to GST-A. B56a
full-length protein bound well to GST-A, but not to GST
alone (Fig. 1A). To f urther con® rm the speci®c binding,
SV40 small t antigen and a truncation mutant w e re used a s a
binding control. Consistent with p revious reports, GST-A
speci®cally bound to wild-type small t, but not to a mutant
small t antigen l acking the A subunit binding site (m#3,
Fig. 1. Binding of B56a to the A subunit of PP2A. [
35
S]Methionine-
labeled proteins generated in vitro w ere incu ba ted with GST or GST-A

for 2 h at ambient temperature, and precipitated w ith glutathione±
Sepharose beads. The bound proteins were eluted with the reduced
glutathione and analyzed b y SDS /PAGE followed b y Pho sphorIm ager
analysis. (A) Added C subunit does not enhance the GST-A:B56a
interaction. Binding of B56a wild type pr otein to PP2A A was assessed
in the presence or a bsence of 1 lg of puri®e d PP2A C and/or 10 lLof
35
S-labeled PP2A C synthesized in vitro. ( B) GS T-A bound spe ci®cally
to the full-length SV40 small t, but not to the m#3 mutant small
t (1±110 fragment).
Ó FEBS 2002 Conserved PP2A A subunit binding domains (Eur. J. Biochem. 269) 547
small t 1±11 0 f ragment, Fig. 1B) [ 24,26]. A lso c onsistent
with previous reports, we saw no enhancement of B56a
binding when the reactions were supplemented w ith puri®ed
C subunit or [
35
S]methionine-labeled C subunit synthesized
in the reticu locyte lysate (Fig. 1A), suggesting the C subun it
present in the reticulocyte lysate may contribute t o the
formation of heterotrimers [27].
To map the region(s) of B56a responsible for binding to
the A subunit, multiple B56 fragments were generated by
PCR followed by in vitro transcription and translation. The
ability of t he fragments to bind to GST-A was assessed as
described above and the results shown in Fig. 2. Two
distinct domains that interacted with GST-A but not the
GST control w ere identi®ed. Generally less than 10% of
input B56a was recovered from the glutathione±Sepharose
beads w hen GST-A s ub unit was included. This low r ecovery
may be due to a high level of nonspeci®c adsorption of the

B56a polypeptides to the beads, and suboptimal binding in
the absence of cotranslation of the A and C subunits. The
smallest N-terminal fragmen t of B56a tha t interacted with
GST-A encompasses r esidues 200±303 (Fig. 2). A second
domain e xtending from amino acids 325 ±383 wa s capable of
independently binding to GST-A ( Fig. 2). These regions
were named A subunit b inding domains (ASBD) 1 and 2.
Given that the two distinct regions can bind to the
structural A subunit, an effort was undertaken to express
these domains in vivo. We reasoned that over-expression o f
an A subunit binding domain at high levels might displace
endogenous B subunits, thereby blocking speci®c interac-
tions with substrates and leading to alterations in speci®c
signaling pathways. A series of epitope-tagged B56a frag-
ments (amino acids 1±142, 142±303, 200±383, 303±383, and
383±486) were expressed in human embryonic kidney
(HEK293) cells using a cytomegalovirus (CMV)-promoter
driven construct. Unfortunately, only the 1±142 fragment
was highly expressed by immunoblot analysis, w hile the
142±303 fragment was barely e xpressed in comparison with
expression of the full-length protein (1±486). Expression of
other B56a fragments was not detectable (data not shown).
Similar r esults were obtained with t wo additional expression
vectors. In addition, fusion of green ¯uorescent protein to
either end of a polypeptide containing B56a amino acids
180±383 did not result in detectable protein. Considering
that these fragments can be well expressed in reticulocyte
lysates, it seems likely that t he failure to detect the e xpressed
fragments in c ultured cells is due to enhanced degradation
by intracellular proteases. One possibility is that these B56a

fragments h ave substantially lower af®nity for the PP2A AC
heterodimer than does full-length B56a. As B56 subunits
over-expressed in v ivo are detected only in PP2A he terotri-
mers [20], B subunits and their fragments unable to be
stabilized by PP2A b inding in vivo may be inherently
unstable and rapidly l ost.
Identi®cation of two conserved regions present
in all three families of B subunits
Although no apparent sequence homology has been
discovered among B s ubunits of the t hree families identi®ed
thus far, all B subunits do bind to overlapping N-terminal
regions of PP2A A (intraloop repeats 1±10) [13,27]. These
data sugge st t he possibility that B subunits contain common
structural elements that are responsible for t he PP2A A
binding. To test whether these two PP2A A binding
domains identi®ed in B56a are conserved among different
B subunits, the
CLUSTALW
multiple sequence alignment
program (available at ) was used
to align a diverse collection o f B subunits (either functionally
identi®ed or characterized by sequence homology from
various species) against these two domains. While full-
length B56 failed to produce a signi®cant alignment with
other B subunits, homology with B/B55 and P R72 family
members was found when only the B56 binding domains
were used in the a lignment ( Fig. 3). For ASBD 1, the region
of homology ( amino a cids 188±292 of hsB56a) substantially
overlaps the experimentally deduced A subunit b inding
domain (amino acids 200±303), while for A SBD 2, t he

overlap is even tighter (homology, 329±386; binding 325±
383). Conserved hydrophobic, charged, and po lar residues
are distributed along the length of the two domains. The
two domains are s eparated by a l ess-conserved region o f
between 20 and 41 amino acids. A conserved amino-acid
pro®le ( Fig. 3) was generated by visual inspection of the two
aligned s equences, a nd used to search the nonredundant
protein database at the Swiss I nstitute for Experimental
Cancer Research web site ().
Each pro®le identi®ed over 9 5% of the approximately 1 05
B/B55/CDC55, B¢/B56/RTS1, and B¢¢/PR72 related seq-
uences contained in the database. Neither pro®le identi®ed
any novel types o f B subunits, strongly suggesting no
additional conventional B subunit families exist, at least in
the n onredundant protein database. Neither pro®le identi-
®ed irrelevant proteins. The pro®les did not match SV40
and polyomavirus t antigens nor members of the striatin/
SG2NA families, implying the se PP2A-interacting proteins
have a distinct ancestry and mechanism of interaction.
Notably, the pro®les identi®ed B, B56, and PR72-type B
subunits in organisms as diverse as Neurospora crassa,
Candida tropicalis, Dictyostelium discoideum, Medicago
varia (alfalfa), Arabidopsis t haliana, Oryza sativa (rice),
Caenorhabditis elegans, Drosophila m elanogaster, Xenopus
laevis, a nd mammals. Combining the ASBD 1 a nd 2 pro®les
with a variable linker b etween them also identi®ed o ver 90%
of the B subunits in the database. Similar results were
obtained when a
PROSITE
pro®le, generated from the

multiple sequence alignment data using the
MOTIF
program
at was used to search t he
Swiss-Prot protein database. We conclude that these pro®les
accurately re¯ect con served amino acid s in the P P2A B
subunit families.
Fig. 2. Binding of full-length and truncated B56a to the A subunit of
PP2A. [
35
S]Methionine-labeled reticulocyte lysate-synthesized B56a
and f ragments were mixed with GST-A or GST for 2 h at the ambient
temperature as described, and the resultant complexes were precipi-
tated with glutathione±Sepharose b eads. After washing, bound com-
plexes were elute d with re duced glu tathion e and analyzed by
SDS/PAGE and PhosphorImager. (A) Schematic summary of the
binding pro perties of the B56a fragmen ts. Th e emp ty bar represents
full-length B56a or its fragments, and the gray boxes represent the
deduced A s ubunit-bin ding d omains. (B) Representative autoradio-
graphs from the binding assays. The left panel shows 5 lL of input
reticulocyte lysate, and the r ight panel d emonstrates which B 56a
fragments precipitated with GST-A and GST beads. Each experiment
was repeated at least thre e times with sim ilar results.
548 X. Li and D. M. Virshup (Eur. J. Biochem. 269) Ó FEBS 2002
Ó FEBS 2002 Conserved PP2A A subunit binding domains (Eur. J. Biochem. 269) 549
The binding of the two conserved regions
from B and PR72 to GST-A
To test whether these two conserved regions of B subunits
found in B/B55/CDC55 and PR72/B¢¢ family members
indeed form domains capable of interaction with the

PP2A A subunit, the corresponding regions from rat Ba
and human PR72 were expressed a nd [
35
S]methionine-
labeled in reticulocyte lysates, and tested in the G ST
precipitation assay. As shown in Fig. 4, polypeptides
encompassing the two conserved regions from Ba and
PR72 bound well to GST-A, but not to GST alone.
Unrelated fragments of Ba and PR72 lying outside the
deduced A subunit binding domains did n ot bind to GST-A
(data not shown). S V40 small t antigen was used as
positive control for GST-A binding, while the C-terminal
truncated small t ant igen (m#3) was used f or a negative
control. The fact that the sequence alignment presented in
Fig. 3 correctly predicted domains in B/CDC55 and PR72
family members that interact with PP2A A subunit
strongly suggest that the sequence conservation is biolog-
ically relevant.
How do B subunits bind to the A subunit? The A subunit
is comprised of 1 5 imperfect repeats, and the B subunits
interact with repeats 1 through 10. Detailed mutagenesis
and structural s tudies have shown that intrarepeat loops are
binding s ites f or different types of B subunits [13,27].
Substitution of certain amino acids in the intrarepeat loops
abrogates the binding of some B subunits but not others
[13,28]. The results here de®ne two distinct PP2A binding
domains in the B subunits that are signi®cantly conserved
among all B subunits of the three known families. These
conserved residues in the B s ubunits are likely to r e¯ect a
common conserved s tructure, w hile the variable residues

and spacing may allow the B subunits to contact different
residues on th e A subunit i ntrarepeat loops. One further
implication of the sequence conservation is that th e B
subunits may h ave evolved from a single ancestral B
subunit.
In summary, in this study we have de®ned t wo separate
PP2A b inding domains in the regulatory a nd targeting B 56a
subunit, which a re conserved in sequence a nd function in all
three families of regulatory B subunits. This ®nding may
facilitate i denti®cation of new B subunits and p rovide
Fig. 3. PP2A B s ubunits have two conserved ASBDs. Representative B sub units of the three families (B, B56, and PR72) f rom evolutionarily distant
organisms were a ligned against the t wo ASBD domains i n human B56a (residues 2 00±303 and 325±383). The ®rst two characters on the left are the
name of an organism (hs, homo sapie ns;oc,Oryctolagus c unic ulus;dm,Drosophila melanogaster;xl,Xenopus laevis;ce,Caenorhabditis elegans;sc,
Saccharomyces cerevisiae;sp.,Schizosaccharomyces pombe;at,Arabidopsis thaliana;rn,Rattus norvegicus ;dd,Dictyostelium discoideum;os,Oryza
sativa;mm,Mus musculus). Nu mb ers in parentheses indicate the ®rst and last o f the aligned amino-acid residues in the individual protein sequence,
followed by the GenBank accession number. Amino acids that are invariant are highlighted in black. Identical residues conserved in more than 50%
of the aligned B subunits are highlighted in dark gray, while conserved similar residues are highlighted in light gray. The two deduced ASBD pro®les
are listed underneath the alignments. R esidues marked with asterisks were included in the pro®les.
550 X. Li and D. M. Virshup (Eur. J. Biochem. 269) Ó FEBS 2002
information for further elucidating the structural basis of
interactions in the PP2A holoenzyme.
ACKNOWLEDGEMENTS
We thank Dr Marc Mumby, Estelle Sontag, and Matthew Movsesian
for plasmids and Joni Seeling a nd other members of the V irshup lab for
their assistance. Oligonuc leotide synthesis was supported by NIH grant
3P30 CA42014. This research was supported by NIH R01 C A80809
and the Huntsman Cancer Foundation
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1 and 2 were tested f or b inding t o G ST and GST-A by in cubation for 4 h a t 3 0 °C. Th e precipitated proteins were washed with RIPA buer four
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SV40 small t antigen and trun cation m #3 w ere u sed f or a s peci® city c ontrol. T he data shown are representative of ®ve independent experiments.
(C) Diagrammatic representation of th e two conserved A su bunit-binding dom ains (ASB D 1 and ASBD 2 ) in human B 56 a,ratBa, and human
PR72, highlighted in gray.
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