Published Ahead of Print 29 October 2007.
2008, 28(1):448. DOI: 10.1128/MCB.00983-07.
Mol. Cell. Biol.
Geoffrey P. Shouse, Xin Cai and Xuan Liu
Protein Phosphatase 2A
Suppressor Activity of B56
and the Tumor
Its Interaction with B56
Serine 15 Phosphorylation of p53 Directs
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MOLECULAR AND CELLULAR BIOLOGY, Jan. 2008, p. 448–456
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 1
Serine 15 Phosphorylation of p53 Directs Its Interaction with B56? and
the Tumor Suppressor Activity of B56?-Specific Protein
Geoffrey P. Shouse, Xin Cai, and Xuan Liu*
Department of Biochemistry, University of California, Riverside, California 92521
Received 4 June 2007/Returned for modification 20 July 2007/Accepted 19 October 2007
Earlier studies have demonstrated a functional link between B56?-specific protein phosphatase 2A (B56?-
PP2A) and p53 tumor suppressor activity. Upon DNA damage, a complex including B56?-PP2A and p53 is
formed which leads to Thr55 dephosphorylation of p53, induction of the p53 transcriptional target p21, and the
inhibition of cell proliferation. Although an enhanced interaction between p53 and B56? is observed after DNA
damage, the underlying mechanism and its significance in PP2A tumor-suppressive function remain unclear.
In this study, we show that the increased interaction between B56? and p53 after DNA damage requires
ATM-dependent phosphorylation of p53 at Ser15. In addition, we demonstrate that the B56?3-induced inhi-
bition of cell proliferation, induction of cell cycle arrest in G1, and blockage of anchorage-independent growth
are also dependent on Ser15 phosphorylation of p53 and p53-B56? interaction. Taken together, our results
provide a mechanistic link between Ser15 phosphorylation-mediated p53-B56? interaction and the modulation
of p53 tumor suppressor activity by PP2A. We also show an important link between ATM activity and the
tumor-suppressive function of B56?-PP2A.
Protein phosphatase 2A (PP2A) is a very important family of
holoenzyme complexes that functions within a diversity of sig-
naling pathways inside the cell (9, 21, 26, 27). PP2A consists of
either a core complex containing a catalytic (C) subunit and
scaffolding (A) subunit (29) or a trimer containing the AC core
with one of many possible regulatory (B) subunits bound to it
(30). The known B subunits have been divided into four gene
families based on sequence homology: the B (B55 or PR55), B?
(B56 or PR61), B? (PR48/59/72/130), and B? (PR93/110) fam-
ilies (25). Each of these many B subunits can combine with the
PP2A core to form complexes with distinct activities and sub-
strate specificities. As such, PP2A is able to perform various
functions in multiple regulatory pathways, depending on which
B subunit is bound.
In the past, PP2A was thought to have primarily dull house-
that PP2A may have more-active regulatory roles and may actu-
ally function as a tumor suppressor under certain conditions. It is
believed that a small subset of B subunits is most likely respon-
sible for promoting this function of PP2A. In support of this view,
at least two B56 subunit family members have been implicated in
conferring tumor-suppressive functions on the holoenzyme. The
B56 family consists of five different genes, ? (PPP2R5A), ?
(PPP2R5B), ? (PPP2R5C), ? (PPP2R5D), and ε (PPP2R5E) (5,
16). In addition, the B56? gene encodes four differentially spliced
was shown to function in a mitotic checkpoint in Xenopus laevis
(15) and B56?3-specific PP2A in blocking the proliferation of
lung cancer cell lines (3). Importantly, evidence from our labora-
tory indicates that B56?-PP2A participates in the activation of the
tumor suppressor protein p53 after DNA damage (13).
p53 is a highly regulated tumor suppressor protein that is
very important in cancer suppression. In response to genotoxic
stress, p53 is activated through a series of posttranslational
modifications (2). Once activated, it acts as a transcription
factor, eliciting the transcription of genes that induce cell cycle
arrest or programmed cell death (23, 28). Our studies have
shown that, under cell growth conditions, p53 is phosphory-
lated at Thr55 by TAF1, which helps to keep the protein
inactive, and upon genotoxic stress, B56?-PP2A complexes
dephosphorylate p53 at this residue, leading to p53 activation,
p21 expression, and G1cell cycle arrest (12, 13). Interestingly,
in the course of our studies, we observed an enhanced inter-
action between B56? and p53 upon DNA damage; however, its
significance in p53 activation and in PP2A tumor suppressor
function remains unknown.
In the present study, we show that the p53-B56? interaction
is required for p53 and B56?-PP2A cooperative tumor sup-
pression. Mechanistically, we show that the kinase activity of
ATM is required for Thr55 dephosphorylation in response to
DNA damage. ATM is an important kinase involved in cellular
responses to DNA double-strand breaks. Once activated, ATM
directly phosphorylates p53 at Ser15 and promotes Ser20 phos-
phorylation indirectly by activating Chk2 kinase. We show that
Ser15, but not Ser20, mutant p53 is unable to interact with
B56? and significantly reduces the ability of B56?3 to inhibit
cell proliferation and transformation, suggesting that Ser15
phosphorylation primes p53 for the p53-B56? interaction and
Thr55 dephosphorylation by PP2A. Taken together, our results
demonstrate the importance of the Ser15-mediated p53-B56?
interaction in the activation of p53 by B56?-PP2A and in PP2A
tumor suppressor function. In addition, our results also pro-
* Corresponding author. Mailing address: Department of Biochem-
istry, University of California, Riverside, CA 92521. Phone: (951) 827-
4350. Fax: (951) 827-4434. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 29 October 2007.
on June 10, 2014 by guest
vide a functional link between ATM and PP2A tumor suppres-
sor activity in response to DNA damage.
MATERIALS AND METHODS
Cell culture and plasmids. U2OS cells were cultured in McCoy’s 5A medium
supplemented with 10% fetal calf serum. H1299 and H1437 cells were cultured
in RPMI 1640 medium supplemented with 10% fetal calf serum. Normal
GM02254 and ATM-deficient GM01526 lymphoblasts were cultured in RPMI
1640 supplemented with 15% fetal calf serum. To induce DNA damage, the cells
were subjected to UV radiation (10 J/m2for U2OS cells) or gamma radiation (6
Gy for U2OS, 8 Gy for GM, and 10 Gy for H1299). The ATM inhibitor KU55933
was a gift from Kudos Pharmaceuticals. In ATM inhibition experiments, cells
were treated with 10 ?M KU55933, 7 mM caffeine, 30 ?M wortmannin, or
dimethyl sulfoxide control as indicated.
The Flag-ATM and Flag-ATM-KD plasmids were gifts from M. Kastan. The
p53 mutants S15A, S15D, and S20A were generated by using a QuikChange
site-directed mutagenesis kit (Stratagene). All plasmids were verified by se-
Western blot and immunoprecipitation. Whole-cell extract was prepared by
lysing the cells in a buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl,
0.5% NP-40, 1 mM dithiothreitol, 2 ?g/ml aprotinin, and 2 ?g/ml leupeptin. Cell
lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis followed by immunoblotting analysis with anti-p53 (DO1; Santa Cruz
Biotechnology), anti-phospho-Ser15 (Cell Signaling Technology), anti-phospho-
Ser20 (Cell Signaling Technology), anti-phospho-Ser37 (Cell Signaling Technol-
ogy), anti-acetyl-Lys373 (Upstate), anti-p21 (C-19; Santa Cruz), anti-PP2A A
subunit (Upstate), anti-PP2A C subunit (1D6; Upstate), anti-PP2A B56?3
(against full-length B56?3), anti-FLAG (M2; Sigma), or antivinculin (VIN-11-5;
Sigma) antibodies. For Thr55 dephosphorylation, the cell lysate was immuno-
precipitated with phosphospecific antibody for Thr55 (Ab202 ) and immuno-
blotted with anti-p53 antibody. For the interaction of transfected p53 with en-
dogenous B56?3, H1299 cells were transfected with various p53 plasmids by
using Lipofectamine (Invitrogen) and lysed 28 h after transfection. Immunopre-
cipitation was performed using either anti-p53 polyclonal antibody (FL393;
Santa Cruz) or anti-B56? polyclonal antibody. The amounts of coprecipitated
proteins were determined by immunoblotting.
Cell cycle profile analysis. H1299 cells were transfected with empty vector or
B56?3 together with wild-type p53, S15A, or S20A, as well as a green fluorescent
protein expression vector. Cells were harvested 60 h after transfection, fixed in
paraformaldehyde, and stained with propidium iodide. Cell cycle phase distri-
butions of green fluorescent protein-positive cells were determined by FACScan
Cell proliferation and anchorage-independent growth assays. To generate
proliferation curves for H1299 cells, cells were cotransfected with either B56?3
or a control cytomegalovirus empty vector and either wild-type p53, the S15A
mutant, or the S20A mutant by using Fugene (Roche); seeded in triplicate; and
counted at 0, 24, 48, 72, 96, and 120 h postseeding.
For anchorage-independent growth assays of H1299 and H1437 cells, the cells
were cotransfected with either B56?3 or a control cytomegalovirus empty vector
and either wild-type p53, the S15A mutant, or the S20A mutant; seeded in
triplicate in 0.35% Noble agar (Fisher); and counted at 4 weeks postseeding.
Modifications of p53 may promote interaction with B56?
after DNA damage. After genotoxic stress, B56? and p53 pro-
tein levels are induced and B56?-PP2A associates with p53,
promoting p53 activation through dephosphorylation of Thr55
(13). To better understand the mechanism of the enhanced
interaction between B56?-PP2A and p53 after DNA damage
and its functional significance in tumor suppression, we re-
cently generated polyclonal antibody against full-length B56?
protein (see Fig. S1 in the supplemental material). U2OS cells
were either mock treated or treated with ionizing radiation
(IR). B56?-containing immunocomplexes were then precipi-
tated using this antibody, and interacting proteins were assayed
by Western blotting (Fig. 1A). A dramatic increase in B56?-
p53 interaction is observed after IR (9.8-fold). Although both
p53 and B56? protein levels increase after IR (2.1- and 2.6-
FIG. 1. Modifications of p53 may be important for DNA damage-induced p53-B56? interaction. (A) U2OS cells were subjected to IR and the
association of endogenous B56?3/B56?2 with p53 and with the PP2A core after DNA damage was detected by immunoprecipitation with anti-B56?
antibody and immunoblotting with anti-PP2A A, anti-PP2A C, and anti-p53 antibodies. Five percent of the input was loaded on the gel. (B) U2OS
cells were pretreated with MG132 and subjected to IR, and the association of p53 and the PP2A core with GST-B56?3 was assayed in GST
pull-down assays. Five percent of the input was loaded on the gel. (C and D) Cell lysates were prepared from U2OS cells treated either with IR
and (C) or UV(D) for the indicated times and then subjected to immunoblotting to detect Thr55, Ser15, and Ser20 phosphorylation, as well as
Lys373 acetylation, with the corresponding antibody. p53 protein levels were normalized by treating cells with MG132. IgG, immunoglobulin G;
vinc, vinculin; P, phosphorylation; DMSO, dimethyl sulfoxide.
VOL. 28, 2008Ser15 PHOSPHORYLATION MEDIATES p53 -B56? INTERACTION 449
on June 10, 2014 by guest
fold, respectively), they do not appear to increase significantly
enough to completely explain the enhanced interaction. Like-
wise, an increased interaction is also observed between B56?
and both PP2A A and PP2A C after IR (1.8-fold); however,
these increases seem to be consistent with the increased B56?
protein levels. Thus, in addition to the increased B56? and p53
protein levels, other mechanisms may also promote B56?-p53
interaction after IR. Since p53 and B56? are both posttransla-
tionally modified proteins, it is possible that modifications of one
or both proteins after IR may modulate their ability to interact.
In an attempt to clarify the role of p53 modification in its
enhanced interaction with B56? after DNA damage, we per-
formed a glutathione S-transferase (GST)–B56?3 pull-down
experiment using bacterially expressed GST-B56?3 that is pre-
sumably unmodified. U2OS cells, pretreated with MG132 to
normalize p53 protein levels, were either mock treated or
treated with IR. The cell lysates were then incubated with
GST-B56?3 fusion protein or a GST-negative control, and the
proteins interacting with the B56?3 fusion protein were as-
sayed by Western blotting (Fig. 1B). With normalized p53
protein levels, p53 interaction with B56?3 increased signifi-
cantly after IR, suggesting that some modifications of p53 may
have occurred, causing the increased interaction. Consistent
with this result, we observed increased levels of p53 Ser15
phosphorylation after IR, as well as in the GST-B56?3 pull-
down assay, demonstrating that modified p53 can be brought
down by the GST-B56?3 fusion protein. Both endogenous
subunits PP2A A and PP2A C bound to the GST-B56?3 fusion
protein, suggesting that the bacterially expressed fusion pro-
tein was in its native conformation.
In order to gain insight into potential p53 modifications that
may be priming the molecule for interaction with B56? after
DNA damage, we assayed the timing of several p53 modifica-
tions relative to Thr55 dephosphorylation. U2OS cells were
pretreated with MG132 to normalize p53 protein levels or with
dimethyl sulfoxide to check p53 protein induction, followed by
either IR or UV treatment. As shown in Fig. 1C and D, Ser15
and Ser20 phosphorylation increased 10 min after IR and 30
min after UV treatment and continued to increase over time,
while Thr55 phosphorylation levels only began to decrease 30
min after IR and 2 h after UV treatment. K373 acetylation, on
the other hand, occurred at around 40 min after IR and 2 h
after UV treatment. The acetylation of K382, another major
acetylation site on p53 after DNA damage, showed the same
timing as the acetylation of K373 (data not shown). The finding
that Ser15 and Ser20 phosphorylation precede Thr55 dephos-
phorylation after DNA damage suggests that both of these
modifications could potentially be involved in promoting p53’s
association with PP2A, while K373 and K382 acetylation,
which occur later, are probably not involved.
The kinase activity of ATM is required for Thr55 dephos-
phorylation. It has been demonstrated that ATM is one of the
major kinases that phosphorylate p53 at Ser15 after IR and
that ATM also phosphorylates and activates Chk2 after IR,
which then phosphorylates p53 at Ser20 (18). We therefore
investigated the importance of ATM activity in Thr55 dephos-
phorylation after DNA damage by using the phosphatidylino-
sitol 3-kinase-like kinase inhibitors caffeine and wortmannin
and the more-specific ATM inhibitor KU55933 (8). U2OS cells
were pretreated with MG132 and one of the three inhibitors or
a control treatment and then subjected to IR. Interestingly,
Thr55 dephosphorylation after IR was completely blocked in
the presence of each inhibitor (Fig. 2A, B, and C). As previ-
ously described, Ser15 and Ser20 phosphorylation were also
FIG. 2. Inhibition of ATM abolishes DNA damage-induced Thr55 dephosphorylation. (A, B, and C) U2OS cells were pretreated with ATM
inhibitor caffeine (A), wortmannin (B), or KU55933 (C) and then subjected to IR. Cells were harvested at the indicated time points (’, min) and
subjected to immunoblotting to detect Thr55, Ser15, and Ser20 phosphorylation (P) levels with the corresponding antibody. p53 protein levels were
normalized by treating the cells with MG132. (D) A control empty vector (EV), Flag-tagged wild-type ATM (ATM), or KD mutant was transfected
into U2OS cells. The presence of overexpressed ATM was verified by immunoblotting using anti-Flag antibody. The p53 protein and the Thr55,
Ser15, Ser20, and Ser37 phosphorylation levels were detected by immunoblotting with the corresponding antibody. (E) U2OS cells were transfected
with a control empty vector (control), Flag-tagged wild-type ATM (ATM), or KD mutant and then treated with IR and harvested at the indicated
time points after treatment. ATM and p53, as well as the Ser15, Thr55, Ser20, and Ser37 phosphorylation levels, were detected by immunoblotting
with the corresponding antibody. (F) Normal and ATM-deficient (A?T) human lymphoblasts were subjected to IR and harvested 1 h after
treatment. The B56?-p53 association was analyzed by immunoprecipitation with anti-B56? antibody and immunoblotting (IB) with anti-p53
antibody. The p53 and B56? protein levels, as well as the Thr55, Ser15, and Ser20 phosphorylation levels, were assayed with the corresponding
antibody. IgG, immunoglobulin G; vinc, vinculin.
450 SHOUSE ET AL.MOL. CELL. BIOL.
on June 10, 2014 by guest
blocked, providing additional evidence of their potential role
in mediating p53-B56? interaction after DNA damage. These
data suggest that ATM kinase activity is involved in promoting
Thr55 dephosphorylation after DNA damage.
In order to directly demonstrate the role of ATM in Thr55
dephosphorylation, we overexpressed the kinase and tested its
effect on Thr55 phosphorylation. As shown in Fig. 2D, the
expression of wild-type ATM, but not a kinase-dead (KD)
dominant-negative mutant (14, 22), led to a modest decrease in
Thr55 phosphorylation. Ser15 phosphorylation levels also in-
creased modestly when the wild-type kinase was overex-
pressed, as expected. These results indicate that ATM-medi-
ated phosphorylation, particularly at Ser15, may promote
Thr55 dephosphorylation. The modest effects observed with
ATM overexpression may be due to the tendency of ATM to
form inactive multimer complexes under normal growth con-
ditions (1). To overcome this problem, we assayed the effect of
ATM on Thr55 dephosphorylation under DNA damage con-
ditions. Compared to the results with the control, the overex-
pression of wild-type ATM led to higher Ser15 and Ser20
phosphorylation levels and earlier Thr55 dephosphorylation,
while the overexpression of the KD mutant completely blocked
Thr55 dephosphorylation, as well as Ser15 phosphorylation,
after IR (Fig. 2E). The p53 protein levels were normalized by
MG132 pretreatment. The phosphorylation, as a control, of
Ser37, a previously described PP2A dephosphorylation site,
was unaffected by ATM. To provide further evidence, we show
that the IR-induced p53-B56? interaction and Thr55 dephos-
phorylation were completely abolished in ATM-deficient
(GM01526), but not in normal (GM02254), human lympho-
blasts (Fig. 2F). Overall, these findings provide evidence that
ATM kinase activity is required for Thr55 dephosphorylation
after IR. In addition, the results suggest that Ser15 and Ser20
phosphorylation play a role in modulating p53-B56? interac-
Ser15 phosphorylation is required for PP2A interaction and
Thr55 dephosphorylation after DNA damage. To further in-
vestigate the role of ATM-mediated phosphorylation of p53 in
Thr55 dephosphorylation, we generated Ser15 to Ala (S15A),
Ser15 to Asp (S15D), and Ser20 to Ala (S20A) p53 mutants to
either abolish or mimic ATM-induced phosphorylation. We
overexpressed these mutants in H1299 cells that lack endoge-
nous p53 protein and assayed for their ability to interact with
B56?-PP2A (Fig. 3A). The assay shows that the wild-type p53
and S15D and S20A mutant proteins were able to interact with
B56? in vivo, while the S15A mutant could not. These results
support our finding of the importance of ATM activity in
Thr55 dephosphorylation. The phosphomimic S15D mutant
showed levels of interaction similar to those of the wild-type
p53, suggesting that perhaps this mutant does not perfectly
mimic Ser15-phosphorylated p53 under our assay conditions.
Total p53 protein was normalized in the immunoprecipitation.
Taken together, these results suggest that Ser15 phosphoryla-
tion is required for Thr55 dephosphorylation. Interestingly, the
interaction between p53 and PP2A A and C showed a similar
trend in that the AC core interacted with wild-type p53, the
S15D mutant, and the S20A mutant, but not the S15A mutant.
This finding underscores the importance of the B56? subunit in
bridging the interaction between p53 and the PP2A core and
demonstrates the requirement of Ser15 for this interaction.
FIG. 3. Ser15 phosphorylation is required for the B56?-p53 interaction and Thr55 dephosphorylation. (A and B) Whole-cell extracts of H1299
cells transfected with either wild-type (WT) p53 or p53 mutants S15A, S15D, and S20A were immunoprecipitated with either p53 (A) or B56?
(B) antibody. The precipitated proteins were then analyzed for the presence of p53, endogenous B56?3/B56?2, PP2A A, PP2A C, or vinculin (vinc)
by immunoblotting (IB) as indicated. (C) H1299 cells were transfected with wild-type p53, the S15A mutant, or the S20A mutant and then treated
with IR. The B56?-p53 association was analyzed by immunoprecipitation with anti-B56? antibody and immunoblotting with anti-p53 antibody. The
p53 and B56? protein levels, as well as the Thr55, Ser15, Ser20, and Ser37 phosphorylation (P), were assayed with the corresponding antibody.
(D) Double modifications on p53 were assayed by immunoprecipitation with either Lys373 acetyl-specific (KAc) or Thr55 phosphospecific antibody
at 0 (Mock), 20, or 80 min after IR, followed by immunoblotting with anti-p53 antibody, as well as Ser15 or Ser20 phosphospecific antibodies, of
cell lysates from U2OS cells. ?, anti; IgG, immunoglobulin G.
VOL. 28, 2008 Ser15 PHOSPHORYLATION MEDIATES p53 -B56? INTERACTION451
on June 10, 2014 by guest
To confirm the results, we performed reciprocal immuno-
precipitation experiments using anti-B56? antibody (Fig. 3B).
As expected, the interaction between p53 and B56? was similar
when wild-type p53, the S15D mutant, or the S20A mutant was
expressed, while no interaction was detected in the presence of
the S15A mutant. As a control, the expression of p53 con-
structs had no effect on B56? interaction with PP2A C. These
results provide further evidence for the importance of Ser15 in
the interaction between PP2A and p53.
Since p53 is dephosphorylated at Thr55 by B56?-PP2A after
IR, we investigated the role of Ser15 phosphorylation in this
process. H1299 cells were transfected with wild-type p53, the
S15A mutant, or the S20A mutant and either mock treated or
treated with IR. Cell lysates were then subjected either to
immunoprecipitation/immunoblotting for the p53-B56? inter-
action or to immunoblotting for p53 modifications (Fig. 3C).
The assay shows that S15A mutant p53 was unable to interact
with B56? after IR treatment, which is consistent with the lack
of Thr55 dephosphorylation after IR. As we would expect,
wild-type p53 and the S20A mutant both had significantly en-
hanced interactions with B56? after IR, which correlated well
with their ability to be phosphorylated at Ser15 and dephos-
phorylated at Thr55. Ser20 phosphorylation of wild-type p53
and the S15A mutant, as well as Ser37 phosphorylation of all
four constructs, was used as a control for the cellular response
to IR. Under the experimental conditions, transfected p53
protein levels were not affected by IR. Taken together, these
results clearly demonstrate the importance of Ser15 in p53
Thr55 dephosphorylation through increased association with
B56?-PP2A after IR.
Based on our results, we reasoned that if Ser15 phosphory-
lation promoted Thr55 dephosphorylation after IR, we should
not be able to detect phosphorylation at both residues on the
same p53 molecule. To test this, p53 protein was immunopre-
cipitated with the Thr55-phosphospecific antibody at time
points when Thr55 is phosphorylated, specifically mock and 20
min after IR. Ser15 and Ser20 phosphorylation, as well as p53
levels, in the immunocomplexes was then assayed by immuno-
blotting (Fig. 3D). No Ser15 phosphorylation was detected in
the immunoprecipitation using Thr55-phosphospecific anti-
body, whereas Ser20 phosphorylation was. It has been shown
that at later time points after IR, Thr55 phosphorylation recovers
to levels similar to those seen with mock treatment (12). We
therefore investigated whether Ser15 and Thr55 phosphorylation
occurred on the same p53 molecule at one of these later time
points by performing a Thr55 phosphoimmunoprecipitation of
lysate from cells harvested 80 min after IR treatment. Even at this
later time point, no Ser15 phosphorylation was detected in the
immunoprecipitation using Thr55-phosphospecific antibody,
while Ser20 phosphorylation was. As a control for the assay, when
Lys373-acetylated p53 was immunoprecipitated with an acetyl-
Lys373-specific antibody, Ser15- and Ser20-phosphorylated p53
were brought down in the immunocomplexes. These findings
clearly show that Ser15 phosphorylation and Thr55 phosphoryla-
tion do not occur on the same p53 molecule and provide further
evidence that the presence of Ser15 phosphorylation promotes
Thr55 dephosphorylation. Collectively, our findings delineate the
requirement of Ser15 phosphorylation for Thr55 dephosphoryla-
tion through enhanced association of p53 with B56? after DNA
Ser15 phosphorylation is required for the p53-dependent
tumor suppressor activity of B56?-PP2A. We previously dem-
onstrated both p53-dependent and -independent tumor sup-
pressor activities of B56?-PP2A (13). To determine the impor-
tance of Ser15 phosphorylation in the p53-dependent function
of B56?-PP2A, we investigated the ability of B56?3 to inhibit
cell proliferation in the presence of different p53 constructs
(Fig. 4A). B56?3 overexpression in the presence of wild-type
p53 significantly inhibited cell growth in H1299 cells, demon-
strating that the two proteins function together synergistically
to block cell proliferation. Further, we show that the overex-
pression of B56? inhibits endogenous p53-MDM2 interaction
(see Fig. S2 in the supplemental material), which is consistent
with our previous observation that Thr55 phosphorylation pro-
motes the p53-MDM2 interaction (12). In contrast, the over-
expression of B56?3 in the presence of the S15A mutant in-
hibited H1299 cell proliferation only modestly. The presence
of B56?3, p53, and p21 during the analysis was verified by
immunoblotting (Fig. 4A). As controls, the overexpression of
either p53 or B56?3 individually had only a small effect on cell
proliferation. In a manner similar to that of wild-type p53, the
S20A mutant was able to decrease cell proliferation slightly on
its own and much more dramatically when combined with
B56?3 overexpression. The cell-doubling times from three par-
allel cell growth experiments are shown in Fig. 4C. Because of
the presence of endogenous B56? in H1299 cells (Fig. 4A) the
S15A mutant shows slightly reduced activity compared to that
of wild-type p53 without B56?3 overexpression. Consistent
with this result, the S15A mutant shows a slightly higher level
of Thr55 phosphorylation than wild-type p53 in H1299 cells
(Fig. 3C). Interestingly, compared to the results with wild-type
p53, cells cotransfected with the S15A mutant and B56?3
showed reduced levels of endogenous p21 expression (Fig.
4A), while cells cotransfected with the S20A mutant and B56?3
showed similar levels of p21 expression (data not shown).
These results provide evidence for the functional importance
of Ser15 phosphorylation in p53-dependent B56?-PP2A tu-
The observation that Ser15 phosphorylation is required for
p21 induction prompted us to investigate its role in cell cycle
G1arrest. The combination of wild-type p53 with B56?3 over-
expression led to a significant increase in G1arrest (Fig. 4B
and D), while the combination of wild-type p53 with B56?3
knockdown led to a decrease in G1arrest in H1299 cells (see
Fig. S3 in the supplemental material). Similarly, the presence
of the S20A mutant along with B56?3 overexpression caused
significant increases in G1arrest. The S15A mutant on the
other hand, led to only a marginal increase in G1arrest when
coupled with B56?3 overexpression. These results correlate
with the conditions of maximal p21 induction. B56?3 overex-
pression by itself caused a low level of G1arrest, as did the
overexpression of each of the p53 constructs individually.
Overall, these data provide further support for the importance
of S15 phosphorylation in the modulation of p53 function by
In addition to blocking cell proliferation, B56?3 overexpres-
sion has also been shown to block anchorage-independent
growth (3, 13). We previously demonstrated a p53-dependent
inhibition of anchorage-independent growth by B56?-PP2A, so
we tested the importance of Ser15 in this process in H1299
452SHOUSE ET AL.MOL. CELL. BIOL.
on June 10, 2014 by guest
FIG. 4. The S15A mutant is unable to cooperate with PP2A in inhibition of cell proliferation. (A) The left four panels show results
representative of cell proliferation of the H1299 human lung cancer cell line transfected with either B56?3 (HA-B56?3) or a control empty vector
(Control) along with an empty vector (EV), wild-type p53, the S15A mutant, or the S20A mutant. Error bars show the averages ? standard
deviations of the results from triplicate plates in one representative experiment. The right panels show immunoblots of transfected HA-B56?3,
endogenous B56?3/B56?2, transfected wild-type (WT) or S15A mutant p53, and endogenous p21 from the cell proliferation assay. M, mock; vinc,
vinculin. (B) H1299 cells were transfected with B56?3 or empty vector (EV) control along with wild-type p53, the S15A mutant, or the S20A
mutant. The cell cycle profile was analyzed 60 h after transfection. (C) Cell-doubling times. The values are the averages ? standard deviations of
the results from three independent experiments. (D) Increases (positive values) and decreases (negative values) of percentages in each cell cycle
distribution compared to the percentages in the corresponding cell cycle stage of the EV control. ?, present; ?, absent.
VOL. 28, 2008 Ser15 PHOSPHORYLATION MEDIATES p53 -B56? INTERACTION453
on June 10, 2014 by guest
cells and another lung cancer cell line, H1437, which also lacks
functional p53. The results from the anchorage-independent
growth assays were similar for both cell lines tested, and the
trend was similar to that seen with the cell proliferation assays
(Fig. 5A and B). When wild-type p53 or the S20A mutant
construct was expressed along with B56?3, a significant de-
crease in colony number was observed. The decrease was more
than could be attributed to an additive effect of both p53 and
B56?3 expression individually and can only be attributed to a
cooperative function between the two proteins. This coopera-
tive function is abolished if the S15A mutant is expressed,
however, and an additive effect only is observed in this case. As
a control, the overexpression of B56?3 in the absence of p53
caused a small decrease in the number of colonies present on
the soft agar. Furthermore, the expression of any of the p53
constructs in the absence of B56?3 overexpression also caused
a small decrease in colony number. The presence of p53 and
hemagglutinin (HA)-tagged B56?3 (HA-B56?3) was verified
by immunoblotting (Fig. 5C). Interestingly, it was previously
reported that H1437 cells lack endogenous B56?3 protein (3),
but we were able to detect it with our antibody (Fig. 5C; see
Fig. S1 in the supplemental material). In addition, the overex-
pression of B56?3 promoted Thr55 dephosphorylation of wild-
type p53 and the S20A mutant, but not the S15A mutant. The
expression of p21 was also verified, which correlated well with
Thr55 dephosphorylation and with the ability of the p53 con-
structs to interact with B56?, as shown in Fig. 3C. The strong
correlation between the p53-B56?3 interaction, Thr55 dephos-
phorylation, and the induction of p21 expression suggests that
B56?3 promotes p53 transcriptional activation leading to inhi-
bition of tumor formation through interacting with p53. These
results also clearly demonstrate the importance of p53 Ser15
phosphorylation in the tumor suppressor activity of B56?-
PP2A, shedding light on the mechanism by which these two
proteins are cooperatively functioning to inhibit cell transfor-
In the present study, we demonstrate that the B56?-p53
interaction is mediated by Ser15 phosphorylation after DNA
damage and is required for p53 and PP2A synergistic tumor-
suppressive function. Specifically, we show that, without Ser15
phosphorylation, the B56?-mediated, p53-dependent inhibi-
tion of cell proliferation and transformation are lost. Impor-
tantly, we identify ATM as having a key role in promoting
Thr55 dephosphorylation of p53, providing insight into the
molecular mechanisms regulating PP2A tumor suppressor ac-
tivity in response to DNA damage. Overall, our results indicate
a stepwise procession of events beginning with the activation of
FIG. 5. The S15A mutant is unable to cooperate with PP2A in inhibition of cell transformation. (A) Anchorage-independent growth of H1299
or H1437 cells transfected with empty vector (EV), wild-type (WT) p53, the S15A mutant, or the S20A mutant in the absence (Control) or presence
of overexpressed B56?3 (HAB56?3). (B) Colony numbers. The values are the averages ? standard deviations of the results of three representative
experiments. (C) Immunoblots of the transfected HA-B56?3 and p53 and endogenous B56?3/B56?2 and p21 proteins, along with Thr55
phosphorylation (P) levels, in H1299 (left) and H1437 (right) cells. Ser37 phosphorylation in H1299 cells was also assayed with the corresponding
antibody. ?, present; ?, absent; vinc, vinculin.
454SHOUSE ET AL.MOL. CELL. BIOL.
on June 10, 2014 by guest
ATM by DNA damage (Fig. 6). Once activated, ATM phos-
phorylates p53 at Ser15, thereby promoting interaction with
B56?-PP2A and dephosphorylation of Thr55. These ordered
p53 modifications lead to maximal activation of the protein,
promoting cell cycle arrest and allowing for DNA repair to
The N terminus of p53 interacts with several molecules that
regulate p53 activity, including p300/CBP and MDM2. The
phosphorylation of serine and threonine residues within this
domain is involved in regulating p53 interaction with these
binding partners that are enzymes that further posttranslation-
ally modify p53 after binding. Specifically, Ser15 and Ser20
phosphorylation promote interaction with p300/CBP, which
leads to p53 C-terminal acetylation and enhanced transcrip-
tional activity (6, 10). In addition, the phosphorylation of either
Thr18 or Thr55 can affect the interaction with MDM2 by either
disrupting it or enhancing it, respectively, thereby regulating
p53 ubiquitination and stability (4, 12, 20). In this study, we
show that Ser15 phosphorylation promotes p53 interaction
with B56?-PP2A, thereby leading to Thr55 dephosphorylation.
Two possible mechanisms may contribute to this result. First,
Ser15 phosphorylation may directly affect p53 interaction with
B56?-PP2A. Second, Ser15 phosphorylation may affect the
tetramer formation of p53 and thereby change the stoichiom-
etry of the p53-B56? interaction. Nevertheless, our results pro-
vide additional evidence of p53 N-terminal phosphorylation
regulating its ability to bind to other enzymes that further
modify the protein. Importantly, our findings also suggest a
signal cascade leading to higher levels of p53 activation in
response to DNA damage, i.e., Ser15 phosphorylation pre-
cedes Thr55 dephosphorylation. Because Thr55 phosphoryla-
tion destabilizes p53, the link between these two modifications
provides an additional mechanism by which Ser15 phosphory-
lation can promote p53 stability. We demonstrate that this p53
signal transduction cascade is specifically necessary for the
cooperative tumor-suppressive function with B56?-PP2A.
PP2A substrate specificity is regulated through its B subunit
composition, and as such, specific B subunits bridge the inter-
actions between the PP2A core and target proteins. In fact,
PP2A was demonstrated to function as a tumor suppressor
through specific B-subunit–substrate interactions. B56?-PP2A
has been shown to function in a mitotic checkpoint by directing
the AC core to dephosphorylate and inactivate Cdc25 (15). In
addition, studies from our laboratory have shown that B56?-
PP2A functions to suppress cell proliferation and transforma-
tion through the interaction and regulation of p53. In this
paper, we have demonstrated the importance of both p53
Ser15 phosphorylation and p53-B56? interaction in mediating
this function. Specifically, in the presence of S15A mutant p53,
the cooperative tumor suppression between B56?3-PP2A and
p53 is abolished. Interestingly, the B56? subunit of PP2A is
also able to direct the AC core to another as-yet-unidentified
substrate, as B56?3-PP2A was shown to function by way of
suppressing tumor growth and formation independent of p53
protein (3, 13). Further investigation is necessary to identify
the additional substrates involved in p53-independent B56?-
PP2A tumor suppression.
Tumor suppressor functions typically fall into one of three
categories: DNA damage sensing, DNA repair, or cell cycle
regulation. Upon DNA damage, sensory proteins activate cell
cycle regulatory proteins and DNA repair proteins. In this
manner, deleterious mutations and tumor progression are
thought to be avoided. Although previous data has demon-
strated a role for B56?-PP2A as a tumor suppressor protein
functioning in the cell cycle regulatory category, its link to
DNA damage sensing has remained elusive. In this study, we
demonstrate a link between ATM, an important member of
the Mre11 complex which plays a critical role in recognizing
DNA double-strand breaks (11, 24), and B56?-PP2A function
through the phosphorylation of p53 at Ser15. However, be-
cause B56?-PP2A also functions in suppressing tumor growth
independent of p53, ATM may also regulate the B56?-PP2A
tumor suppressor function through other pathways. Thus, it
will be interesting to further investigate the role of ATM in the
regulation of the B56?-PP2A tumor suppressor function.
We are very grateful to M. Kastan for providing ATM plasmids, to
G. Smith and Kudos Pharmaceuticals for providing the ATM inhibitor
KU55933, and to H. Myllykangas for providing technical assistance.
We thank J. A. Traugh and all members of our laboratory, particularly
A. G. Li and P. Podlesny, for many helpful discussions and critical
reading of the manuscript.
This work was supported by NIH grant CA075180 from the National
Institute of Cancer.
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