Two conserved domains in regulatory B subunits mediatebinding to the A subunit of protein phosphatase 2A Xinghai Li1and David M.. In vitro expression of a series of B56a fragments identi
Trang 1Two conserved domains in regulatory B subunits mediate
binding to the A subunit of protein phosphatase 2A
Xinghai Li1and David M Virshup1,2
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 gene 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 about how these B subunits interact with the PP2A
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 binding 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 that these B subunits share a common core structure and mode of interaction with the PP2A holoenzyme 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 regulatory B subunits These diverse B subunits in
the PP2A heterotrimer allow the phosphatase to localize to
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 of B subunits in cellular regulation 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 leads to imaginal disc duplication and defects in
mitosis [6,7], transposon insertions in B56c enhance the
metastatic ability of mouse melanoma cell lines [8],
muta-tions in the A subunit that alter B subunit binding are found
in lung, breast, colorectal and skin cancers [9,10], and
decreases in A subunit expression are seen in neuronal
tumors [11] Despite the signi®cant role the 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 PP2A A subunit serves as a scaffold for assembly of
the B and C subunits It is composed of 15 imperfect HEAT
repeats, each of 39 amino acids, which form a hook-shaped molecule [12] The repeats consist of two a helices connected
by an intrarepeat loop, and mutations in distinct loops 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 subunit binds to repeats 11±15 Interactions between the B and C subunits are also important for heterotrimer formation, as loss of C subunit binding sites prevents B subunit binding [14,15], and modi®cation of the C-terminus of the C subunit regulates B subunit binding [16±18]
To date, at least three families of PP2A B subunits have been identi®ed in eukaryotes They are designated B (PR55, B55, CDC55), B¢ (PR61, B56, RTS1), and B¢¢ (PR72/130) Each B subunit family is encoded by multiple genes, with multiple splice variants, generating an extraordinary diver-sity of these regulatory subunits [1,2] Although the three families of B subunits do not share apparent 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 region
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 has been proposed to interact with the AC heterodimer, while the nonconserved N- and C-ends may perform different functions, such as regulation of substrate 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 core of PP2A However, unlike the B subunits, T antigens and striatin 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 Institute,
University of Utah, Salt Lake City, UT 84112 Fax: + 801 587 9415,
Tel.: + 801 585 3408, david.virshup@hci.utah.edu
Abbreviations: PP2A, protein phosphatase 2A; ASBD, A subunit
binding domain; GST-A, glutathione S-transferase A subunit; NP-40,
nonidet p40; CMV, cytomegalovirus.
(Received 19 September 2001, revised 8 November 2001, accepted 16
November 2001)
Trang 2A subunit binding domains In this study, we used the B56a
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 families The predicted A subunit
binding domains in B/B55 and B¢¢/PR72 were also able to
interact with the PP2A A subunit The presence of a
conserved motif in the highly divergent B subunits suggests
a common ancestry, structure, and mode of A subunit
interaction for these important regulatory proteins
E X P E R I M E N T A L P R O C E D U R E S
Synthesis of [35S]protein
[35S]Methionine-labeled B subunits and their fragments, and
SV40 small t antigen and its mutant were generated by
coupled in vitro transcription and translation in rabbit
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 lL of
reticulocyte lysate in the presence of [35S]methionine The
reaction was incubated at 30 °C for 2 h In several cases,
additional lower molecular mass bands were seen which are
likely to be due to either premature termination or partial
proteolysis of the [35S]methionine-labeled proteins
Preparation of glutathioneS-transferase (GST)
and GST-A fusion proteins
The GST-A subunit of 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 performed as described previously
[24] The puri®ed proteins were thoroughly dialyzed against
buffer A (50 mM Tris/HCl pH 7.5, 20 mM NaCl, 2 mM
EDTA, 1 mMdithiothreitol, containing 3 lgámL)1
pepsta-tin and leupeppepsta-tin, 2 mM benzamidine, and 1 mM
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 lL of [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 incubation for 2 h at ambient temperature (or
4 h at 30 °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 lL of a
pre-washed 1 : 1 slurry of glutathione±Sepharose (Amersham
Pharmacia) was added Incubation continued for 2 h at
4 °C The beads were then washed four times with 1 mL of
buffer B, or RIPA buffer (50 mM Tris, pH 7.5, 150 mM
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 lL of 10 mM
reduced glutathione in buffer A on ice for 30 min The eluted polypeptides were analyzed by either conventional SDS/PAGE or on tricine/glycine gels for small molecular mass peptides [25] and imaged using a Molecular Dynamics PhosphorImager
R E S U L T S A N D D I S C U S S I O N
Identi®cation of two A subunit-binding domains in B56a
To determine the minimal region of B56 that interacted with the PP2A subunit, we utilized an in vitro binding assay using GST-A subunit and reticulocyte lysate-synthesized B frag-ments [10,21] To optimize conditions 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 further con®rm the speci®c binding, SV40 small t antigen and a truncation mutant were used as a binding control Consistent with previous reports, GST-A speci®cally bound to wild-type small t, but not to a mutant small t antigen lacking 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 were incubated with GST or GST-A for 2 h at ambient temperature, and precipitated with glutathione± Sepharose beads The bound proteins were eluted with the reduced glutathione and analyzed by SDS/PAGE followed by PhosphorImager analysis (A) Added C subunit does not enhance the GST-A:B56a interaction Binding of B56a wild type protein to PP2A A was assessed
in the presence or absence of 1 lg of puri®ed PP2A C and/or 10 lL of
35 S-labeled PP2A C synthesized in vitro (B) GST-A bound speci®cally
to the full-length SV40 small t, but not to the m#3 mutant small
t (1±110 fragment).
Trang 3small t 1±110 fragment, Fig 1B) [24,26] Also consistent
with previous reports, we saw no enhancement of B56a
binding when the reactions were supplemented with puri®ed
C subunit or [35S]methionine-labeled C subunit synthesized
in the reticulocyte lysate (Fig 1A), suggesting the C subunit
present in the reticulocyte lysate may contribute to 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 the 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 were identi®ed Generally less than 10% of
input B56a was recovered from the glutathione±Sepharose
beads when GST-A subunit was included This low recovery
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 fragment of B56a that interacted with
GST-A encompasses residues 200±303 (Fig 2) A second
domain extending from amino acids 325±383 was capable of
independently binding to GST-A (Fig 2) These regions
were named A subunit binding 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 of
an A subunit binding domain at high levels might displace
endogenous B subunits, thereby blocking speci®c
interac-tions with substrates and leading to alterainterac-tions 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, while the
142±303 fragment was barely expressed in comparison with
expression of the full-length protein (1±486) Expression of
other B56a fragments was not detectable (data not shown)
Similar results were obtained with two 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 the failure to detect the expressed
fragments in cultured cells is due to enhanced degradation
by intracellular proteases One possibility is that these B56a
fragments have substantially lower af®nity for the PP2A AC
heterodimer than does full-length B56a As B56 subunits
over-expressed in vivo are detected only in PP2A
heterotri-mers [20], B subunits and their fragments unable to be
stabilized by PP2A binding in vivo may be inherently
unstable and rapidly lost
Identi®cation of two conserved regions present
in all three families of B subunits
Although no apparent sequence homology has been
discovered among B subunits of the three 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 suggest the possibility that B subunits contain common
structural elements that are responsible for the 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 http://workbench.sdsc.edu) was used
to align a diverse collection of 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 PR72 family members was found when only the B56 binding domains were used in the alignment (Fig 3) For ASBD 1, the region
of homology (amino acids 188±292 of hsB56a) substantially overlaps the experimentally deduced A subunit binding domain (amino acids 200±303), while for ASBD 2, the overlap is even tighter (homology, 329±386; binding 325± 383) Conserved hydrophobic, charged, and polar residues are distributed along the length of the two domains The two domains are separated by a less-conserved region of between 20 and 41 amino acids A conserved amino-acid pro®le (Fig 3) was generated by visual inspection of the two aligned sequences, and used to search the nonredundant protein database at the Swiss Institute for Experimental Cancer Research web site (http://www.isrec.isb-sib.ch) Each pro®le identi®ed over 95% of the approximately 105 B/B55/CDC55, B¢/B56/RTS1, and B¢¢/PR72 related seq-uences contained in the database Neither pro®le identi®ed any novel types of B subunits, strongly suggesting no additional conventional B subunit families exist, at least in the nonredundant 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 these 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 thaliana, Oryza sativa (rice), Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and mammals Combining the ASBD 1 and 2 pro®les with a variable linker between them also identi®ed over 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 theMOTIFprogram
at http://www.motif.genome.ad.jp was used to search the Swiss-Prot protein database We conclude that these pro®les accurately re¯ect conserved amino acids in the PP2A 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 fragments 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 beads After washing, bound com-plexes were eluted with reduced glutathione and analyzed by SDS/PAGE and PhosphorImager (A) Schematic summary of the binding properties of the B56a fragments The empty bar represents full-length B56a or its fragments, and the gray boxes represent the deduced A subunit-binding domains (B) Representative autoradio-graphs from the binding assays The left panel shows 5 lL of input reticulocyte lysate, and the right panel demonstrates which B56a fragments precipitated with GST-A and GST beads Each experiment was repeated at least three times with similar results.
Trang 5The 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 and [35
S]methionine-labeled in reticulocyte lysates, and tested in the GST
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 not bind to GST-A
(data not shown) SV40 small t antigen was used as
positive control for GST-A binding, while the C-terminal
truncated small t antigen (m#3) was used for 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 15 imperfect repeats, and the B subunits interact with repeats 1 through 10 Detailed mutagenesis and structural studies have shown that intrarepeat loops are binding sites for 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 subunits are likely to re¯ect a common conserved structure, while the variable residues and spacing may allow the B subunits to contact different residues on the A subunit intrarepeat loops One further implication of the sequence conservation is that the B subunits may have evolved from a single ancestral B subunit
In summary, in this study we have de®ned two separate PP2A binding domains in the regulatory and targeting B56a subunit, which are conserved in sequence and function in all three families of regulatory B subunits This ®nding may facilitate identi®cation of new B subunits and provide
Fig 3 PP2A B subunits have two conserved ASBDs Representative B subunits of the three families (B, B56, and PR72) from evolutionarily distant organisms were aligned against the two ASBD domains in human B56a (residues 200±303 and 325±383) The ®rst two characters on the left are the name of an organism (hs, homo sapiens; oc, Oryctolagus cuniculus; 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) Numbers in parentheses indicate the ®rst and last of 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 Residues marked with asterisks were included in the pro®les.
Trang 6information for further elucidating the structural basis of
interactions in the PP2A holoenzyme
A C K N O W L E D G E M E N T S
We thank Dr Marc Mumby, Estelle Sontag, and Matthew Movsesian
for plasmids and Joni Seeling and other members of the Virshup lab for
their assistance Oligonucleotide synthesis was supported by NIH grant
3P30 CA42014 This research was supported by NIH R01 CA80809
and the Huntsman Cancer Foundation
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Fig 4 Binding of the two conserved domains in Ba and PR72 to the A subunit of PP2A Fragments of rat Ba and human PR72 encompassing ASBD
1 and 2 were tested for binding to GST and GST-A by incubation for 4 h at 30 °C The precipitated proteins were washed with RIPA buer four times prior to elution from the glutathione±Sepharose beads (A) Binding of rat Ba ASBD 1 and 2; (B) binding of human PR72 ASBD 1 and 2 SV40 small t antigen and truncation m#3 were used for a speci®city control The data shown are representative of ®ve independent experiments (C) Diagrammatic representation of the two conserved A subunit-binding domains (ASBD 1 and ASBD 2) in human B56a, rat Ba, and human PR72, highlighted in gray.
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