A new effector pathway links ATM kinase with the DNA damage response

A RT I C L E S
968 NATURE CELL BIOLOGY VOLUME 6 | NUMBER 10 | OCTOBER 2004
A new effector pathway links ATM kinase with the
DNA damage response
Constantinos Demonacos1,2, Marija Krstic-Demonacos1,2, Linda Smith1, Danmei Xu1, Darran P. O’Connor1,
Martin Jansson1 and Nicholas B. La Thangue1,4
The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a
limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase
signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue,
stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53
acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia
telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by
ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results
indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can
be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.
The DNA damage signalling pathway is a highly conserved response to
genotoxic stress1.In mammalian cells,the pathway functions to protect
cells from agents that induce cellular death or transformation, where it
participates in DNA repair and checkpoint control leading to survival
or apoptosis1. The related phosphatidylinositol-3-OH-kinase-like
kinases (PI(3)KK) ATM and ATR, which become activated in response
to DNA damage, transduce signals to downstream targets, including
p53 and the checkpoint kinases Chk1 and Chk2 (refs 2–9). In turn,
checkpoint kinases phosphorylate key substrates, such as p53, E2F-1,
cyclin dependent kinase 25A (Cdc25A) and Cdc25C10–14, to facilitate
the DNA damage response.
The tumour suppressor protein p53 has an important function in the
cellular response to DNA damage15,and consequently,TP53 is one of the
most frequently altered genes in human cancer16. Under normal condi-
tions, p53 is held in a latent inactive state but undergoes a significant
increase in protein stability after DNA damage15. p53 stability control is
believed to involve both families of DNA damage signalling kinases that
activate p53 function: the ATM/ATR and Chk1/Chk2 families (refs 6, 7,
17, 18). In cells derived from ataxia telangiectasia individuals, in which
ATM is missing or inactivated19, the DNA damage pathway and p53
response is abnormal2,4. Similarly, in some Li-Fraumeni patients with a
defective Chk2,the p53 response is also compromised20.
p53 functions as a stress-responsive transcription factor, inducing
genes that facilitate cell-cycle arrest and apoptosis. The phosphoryla-
tion of p53 seems to be tightly coordinated with p53 acetylation, medi-
ated by the histone acetyltransferase (HAT) in co-activators such as
p300/CBP and PCAF (p300/CBP-associated factor)21–23. During the
DNA damage response, two cofactors JMY and Strap bind to p300
(refs 24, 25), resulting in a co-activator complex which enhances p53
transcriptional activity. Interestingly, Strap contains a tandem series of
TPR repeats (tetratricopeptide repeats), consistent with its role in pro-
tein assembly25. Thus, different domains of Strap interact with p300
and JMY, which augments interaction between p300 and JMY.
Here, we have explored the possibility that Strap is regulated by the
DNA damage signalling pathway, and report a new pathway through
which ATM kinase signals to Strap and activates the DNA damage
response. ATM phosphorylates Strap at a single serine residue, and
phosphorylated Strap accumulates in the nucleus, thereby allowing
Strap to assemble into a co-activator complex with p300. Strap activ-
ity enhances p53 acetylation and augments the DNA damage
response. In ataxia telangiectasia cells, Strap remains localized in the
cytoplasm under DNA damage conditions, and a mutant derivative of
Strap that cannot be phosphosphorylated by ATM phosphorylation
localizes to the cytoplasm in cells with normal ATM activity.
Moreover, targeting Strap to the nucleus is sufficient to stabilize Strap
protein and activate the DNA damage response. Our results define in
mechanistic detail a new checkpoint effector pathway that links ATM
kinase with the DNA damage response through the phosphorylation
and altered intracellular distribution of Strap.
RESULTS
Strap is phosphorylated by ATM kinase
To investigate the regulation of Strap by the DNA damage signalling
pathway we assessed whether Strap was induced by different geno-
toxic stresses. In cells treated with etoposide, hydroxyurea or strep-
tonigrin, there was an increase in the level of Strap (Fig. 1a; bottom).
1Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK. 2Current address: School of Pharmacy and
Pharmaceutical Sciences (C.D.), School of Biological Sciences (M.K-D.), University of Manchester, Manchester M13 9PT, UK. 4Correspondence should be addressed
to N.B.L.T. (e-mail: n.lathangue@bio.gla.ac.uk).
Published online: 19 September 2004; DOI:10.1038/ncb1170
print ncb1170 14/9/04 2:58 PM Page 968
©2004 NaturePublishing Group

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NATURE CELL BIOLOGY VOLUME 6 | NUMBER 10 | OCTOBER 2004 969
A substrate-specific antibody that recognizes phospho-serine/threo-
nine (S/T) ATM/ATR was used. This antibody recognised Strap in
DNA damaged cells, but not in untreated cells (Fig. 1a; top), suggest-
ing that Strap is phosphorylated by ATM or ATR kinase during the
DNA damage response.
Several consensus phosphorylation sites (SQ or TQ) for ATM and
ATR kinase are present in Strap. To identify functionally important ser-
ine/threonine residues, each of the three theoretical phosphorylation
sites was altered (from serine/threonine to alanine) and the phospho-
rylation status and stabilization of the mutant derivative assessed in
cells treated with etoposide. One derivative, with a serine to alanine
change at residue 203 (referred to as StrapS203A) in the third TPR motif
(Fig. 1b) was of particular interest. This was because, in contrast to
wild-type Strap,StrapS203A was poorly phosphorylated by immunopre-
cipitated ATM kinase (Fig. 1c). Moreover, the protein level of
StrapS203A was not increased in cells treated with etoposide but retained
the constitutive level of expression observed with wild-type Strap in
untreated cells (Fig. 1d); similar effects were not observed with any
other Strap mutant (data not shown). Thus, Ser 203 is phosphorylated
by ATM kinase and is a functionally important residue in regulating
Strap stability in response to DNA damage. It is consistent with these
results that Strap is an intrinsically unstable protein, the half life of
which is increased on etoposide treatment (30 min to 3 h, respectively;
see Supplementary Information, Fig. S1).
IP: Strap
IP: ATM kinase
− E Hu S
IB: anti-P-S/T
IB: Strap
P-Strap
Strap
P-Ser 203 Strap
p53
PCNA
Strapp53
Strap
1 2 3 4
1
0 1 3 6 16
1 2 3 4 5
: Bleomycin (h)
2 3 4
1 2 3 4 5 6
1 440l ll lll lV V Vl
N P K I S Q Q A L : WT
A : StrapS203A
a
c
e
d
b
Strap
p53 −
−
− + − + − +
WT S203A
WT S203A
Strap:
Etoposide:
Figure 1 DNA damage induces phosphorylation of Strap. (a) Lysates from
HeLa cells treated with etoposide (E, 10 µM for 12 h; lane 2),
hydroxyurea (Hu; 0.5 µM for 12 h; lane 3), streptonigrin (S; 5 ng µl−1 for
1 h; lane 4) or untreated (lane 1) were immunoprecipitated with the anti-
Strap peptide 510 antibody that recognizes endogenous Strap and then
immunoblotted with the anti-phospho-S/T ATM/ATR substrate-specific
antibody (anti-P-S/T, top). The bottom panel shows the level of input
Strap by immunoblotting the cell extracts with the anti-Strap 510
antibody. Equal amounts of protein (5 µg) were loaded. (b) Schematic
representation of Strap, showing the six TPR repeats (dark shading). The
sequence surrounding the serine (S) residue (at position 203) in wild-
type (WT) Strap is shown, together with the alanine (A) derivative in
StrapS203A. (c) An extract from HeLa cells (about 50 µg) was
immunoprecipitated with the anti-ATM Ab3 antibody and kinase reactions
performed in the presence of purified His-tagged p53, Strap or StrapS203A
(1 µg of each) as indicated, together with a control treatment lacking
recombinant protein (lane 2). (d) U20S cells transfected with expression
vectors encoding WT Strap (10 µg; lanes 3 and 4), StrapS203A (10 µg;
lanes 5 and 6) or empty vector (lanes 1 and 2) were treated with etoposide
(10 µM for 12 h) and immunoblotted with an anti-HA monoclonal
antibody recognizing exogenous Strap. pCMV-β-gal was co-transfected as
an internal control, and used to normalize the amount of protein resolved
for each treatment. (e) U2OS cells were treated with bleomycin (5 µg ml−
1) and harvested at the times indicated. Total cell extracts were
immunoblotted with the anti-phospho Ser 203 peptide antibody (top),
anti-p53 antibody (middle) or anti-PCNA antibody (lower).
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To gain evidence that Ser 203 is phosphorylated in cells by ATM or
ATR kinase, we monitored the level of phosphorylation of StrapS203A
with the anti-phospho-S/T ATM/ATR antibody in U2OS cells.Although
wild-type Strap exhibited an increased level of phosphorylation after
etoposide treatment, phosphorylation of StrapS203A was not evident (see
Supplementary Information, Fig. S1), even though similar levels of
StrapS203A and wild-type Strap were expressed (lower panel). Moreover,
we used a phospho-specific peptide antibody against phosphorylated Ser
203 (see Supplementary Information, Fig. S1) to confirm the phospho-
rylation of Ser 203 in cells. Using a tetracycline-inducible stable cell line
in which Strap levels could be regulated, Strap was shown to be specifi-
cally phosphorylated on Ser 203 (see Supplementary Information,
Fig. S2). The anti-phospho Ser 203 antibody recognised endogenous
Strap (see Supplementary Information, Fig. S2), and in cells treated with
the DNA damaging agent bleomycin or ionising radiation, Strap under-
went increased phosphorylation at Ser 203 (Fig. 1e; also see
Supplementary Information, Fig. S2). Under the conditions of
bleomycin and etoposide treatment, active ATM kinase26 coincided with
the presence of phosphorylated Ser 203 (see Supplementary
Information,Fig. S2).Thus,Ser 203 is a major site of phosphorylation by
ATM kinase and, combined with the earlier results (Fig. 1d), implies that
phosphorylation is required for Strap stabilization.
Phosphorylation of Strap at Ser 203 governs intracellular location
We investigated the mechanism through which the phosphorylation of
Ser 203 may influence Strap activity, and to this end considered a role
in regulated intracellular localization.In normal cells,Strap localized to
the nucleus, with an increase in staining intensity occurring after
etoposide treatment (Fig. 2a, c), correlating with the increased level of
Strap after etoposide treatment (Fig. 1a). A similar effect was observed
with exogenous Strap, again exhibiting enhanced nuclear staining in
cells treated with etoposide (Fig. 2e, g). Remarkably, the StrapS203A
mutant was excluded from nuclei,being localized to the cytoplasm,and
treating cells with etoposide failed to alter its localization (Fig. 2i, k;
also see Supplementary Information, Fig. S4). Residue Ser 203 there-
fore regulates the intracellular location of Strap.
Strap localizes to the cytoplasm in AT cells
Ataxia telangiectasia is an autosomal recessive human disease, charac-
terized by abnormal chromosome breakage and haematological malig-
nancy, that results from mutation in the ATM gene and loss of ATM
kinase activity27,28. Given the implied role for ATM kinase in the phos-
phorylation and regulation of Strap, we were interested to evaluate
Strap in ataxia telangiectasia cells. In two different ataxia telangiectasia
cell lines (1BR and GM02530) endogenous Strap localized to the cyto-
plasm and was excluded from the nucleus, and etoposide treatment
had little effect on this distribution (Fig. 3a, c and e). Exogenous wild-
type Strap possessed similar properties to endogenous Strap in that it
localized to the cytoplasm, and similarly was not affected by etoposide
(data not shown). Furthermore, Strap did not undergo protein stabi-
lization in ataxia telangiectasia cells, and phosphorylated Strap was not
apparent in etoposide-treated ataxia telangiectasia cells (see
Supplementary Information, Fig. S1). Thus, the properties of wild-
type Strap in ataxia telangiectasia cells recapitulate the properties of the
StrapS203A mutant derivative in cells with normal ATM activity (Fig. 2i,
k). Because in ataxia telangiectasia cells ATM is missing or inactivated,
these results further strengthen the idea that ATM kinase phosphory-
lates Ser 203 to regulate the intracellular location of Strap.
a b c d
e f g h
i j k l
Etoposide: − − + +
Endogenous Strap
Exogenous Strap
StrapS203A
Figure 2 Intracellular localization of Strap and StrapS203A. (a–d) U20S cells
were immunostained with the anti-Strap 510 antibody recognizing endogenous
Strap pre (a and b) or post (c and d) treatment with etoposide (10 µM for 12 h).
(e–l) U20S cells were transfected with expression vectors (5 µg) encoding wild-
type Strap (e–h) or StrapS203A (i–l) and immunostained with the anti-HA
monoclonal antibody pre (e, f, i, j) or post (g, h, k, l) etoposide treatment (10
µM for 12 h). DAPI staining shown in b, d, f, h, j, and i. Arrows in k indicate the
reduced level of nuclear staining. Original magnification ×630.
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Additional experiments were performed to further support the idea
that ATM regulates Strap activity. In the first approach, we introduced
wild-type ATM into ataxia telangiectasia cells to assess whether rein-
stating wild-type ATM altered the distribution of endogenous Strap.
Under these conditions, Strap in different ataxia telangiectasia cell lines
predominantly localized to the nucleus (Fig. 3g, k), an effect that relied
on ATM activity because a kinase-dead mutant6 failed to cause nuclear
accumulation (Fig. 3i); both wild-type and kinase-dead ATM proteins
were expressed at similar levels in nuclei (Fig. 3l; and data not shown).
In the second approach, Strap distribution was investigated in cells
with normal ATM that were treated with wortmannin, which inhibits
the protein kinase activity of many PI(3)K family members, includ-
ing ATM29,30. In U2OS cells, wortmannin caused wild-type Strap to
localize to the cytoplasm (Fig. 3m), rather than being present in
nuclei as observed in untreated cells (Fig. 2a). The low level of
nuclear Strap in non-stressed cells may reflect constitutively active
ATM31, or constitutive activity from a related kinase. In this respect,
however, ATR did not seem to have a significant role in Ser 203 phos-
phorylation (data not shown). Collectively, these results support the
regulation of Strap activity by ATM kinase.
Phosphorylation of Strap augments p53 acetylation
We compared the ability of wild-type Strap and StrapS203A to interact
with p300 in normal and DNA-damaged cells by immunoprecipitating
the p300 complex and measuring the amount of Strap.Wild-type Strap
co-immunoprecipitated with p300, and there was a significant increase
in the Strap/p300 complex in damaged cells (Fig. 4a). In contrast, a
greatly reduced level of StrapS203A was observed in the p300 complex in
DNA-damaged cells (Fig. 4a), arguing that Strap phosphorylation by
ATM kinase augments the interaction between Strap and p300.
We reasoned that the regulation of the interaction between Strap and
p300 by ATM may augment the acetylation of target proteins, as p300
possesses an intrinsic HAT activity32. In cells treated with etoposide,
Strap enhanced the levels of p53 acetylation at Lys 382 (Fig. 4b; refs 22,
33). In comparison, the significant increase in p53 acetylation apparent
in the presence of wild-type Strap was less in the presence of StrapS203A
(Fig. 4b). Therefore the phosphorylation of Ser 203 seems to aid the
interaction between Strap and p300 and the acetylation of p53.
In support of this idea, we measured HAT activity in Strap immuno-
complexes. For this experiment, either Strap or StrapS203A was
immunoprecipitated from normal or DNA-damaged cells, and the
a b c d
e f g h
i j
m n
k l
Endogenous Strap Endogenous Strap
Figure 3 Strap localization in ataxia telangiectasia cells. (a–d) Ataxia
telangiectasia 1BR (AT 1BR) cells immunostained for endogenous Strap
with the anti-Strap peptide 510 antibody pre (a and b) or post (c and d)
treatment with etoposide (10 µM for 12 h). (e, f) Ataxia telangiectasia
GM02530 (AT GM02530) cells immunostained for endogenous Strap as
described above in the absence of etoposide. (g–j) AT 1BR cells
transfected with expression vectors encoding wild-type ATM (g and h;
4 µg) or kinase-dead ATM (i and j) were immunostained for endogenous
Strap with the anti-Strap peptide 510 antibody (g and i). (k, l) AT
GM02530 cells transfected with expression vectors encoding wild-type
ATM (4 µg) were immunostained with either anti-Strap 510 antibody for
endogenous Strap in k or anti-ATM antibody to detect exogenous ATM in l.
(m, n) U2OS cells were immunostained for endogenous Strap using the
anti-Strap peptide 510 antibody after treatment with wortmannin (50 µM)
30 min before the addition of etoposide (10 µM for 12 h). DAPI staining
shown in b, d, f, h, j and n.
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acetylation of recombinant p53 at Lys 382 was assessed. The acetylase
activity in the Strap immunocomplex was significantly greater in DNA-
damaged cells than normal cells (Fig. 4c). In contrast, the StrapS203A
immunocomplex lacked p53 acetylase activity (Fig. 4d).
Phosphorylation of Ser 203 is required for apoptosis in response
to DNA damage
It was important to assess the function of Ser 203 in regulating the
DNA damage response. Thus, we compared the ability of wild-type
Strap and StrapS203A to enhance p53-dependent apoptosis in
response to DNA damage. In SAOS2 cells (which lack p53 activity),
the introduction of exogenous p53 followed by treatment with etopo-
side caused significant cell-cycle delay, increasing the G1 population
and the level of cells undergoing apoptosis (sub-G1) (Fig. 5c). The
presence of wild-type Strap significantly increased the level of apop-
totic cells (Fig. 5f; 21% compared with 35%), whereas in the absence
of p53 Strap had minimal effect (Fig. 5d). In sharp contrast,
StrapS203A failed to augment apoptosis (Fig. 5e, g). These results sup-
port the idea that Ser 203 phosphorylation by ATM kinase is a func-
tionally important event in the DNA damage response.
Targeting Strap to the nucleus activates the DNA damage response
To determine whether the nuclear accumulation of Strap is sufficient to
induce the DNA damage response, we targeted Strap to nuclei by attach-
ing a nuclear localization signal (NLS) to both wild-type Strap and
StrapS203A (Fig. 6i–l). In ataxia telangiectasia cells, NLS–Strap accumu-
lated in nuclei in contrast to the behaviour of wild-type Strap (Fig.6a–d).
Also, in U2OS cells NLS–StrapS203A localized to the nucleus, in contrast
with StrapS203A which remained in the cytoplasm (Fig.6e–h).
As expected from the earlier results, StrapS203A failed to stabilize
under DNA damage conditions (Fig. 6m, n). However, targeting
StrapS203A to nuclei with NLS–StrapS203A re-instated its stabilization in
a
c
b
+ −
− WT S203A − WT S203A− WT S203A
+ −
+ − + − + −
+ − + − + − + − +− +− +−
Strap: Strap:
Etoposide: Etoposide:
Input IP: anti-p300 / IB: anti-Strap (HA)
1 2 3 4 5 6
1 2 3 4 5 6
1 2 3 4 5 6
7 8 9 10 11 12
p300
Strap
IP: anti-p53
IB: anti-Ac Lys 382
IB: anti-Ac Lys 382
IB: anti-p53
IB: anti-p300
Actin
p53 BSA p53
anti-Strap anti-GAL4
WT Strap
Ac Lys 382-p53
StrapS203A
d
Figure 4 Strap Ser 203 regulates the interaction between Strap and p300
and the acetylation of p53. (a) Lysates prepared from U2OS cells
transfected with expression vectors encoding wild-type Strap (15 µg; lanes
3, 4, 9 and 10) or StrapS203A (15 µg; lanes 5, 6, 11 and 12) and p300 (10
µg; lanes 1–12) and treated with or without etoposide (10 µM for 12 h)
were immunoprecipitated with anti-p300 monoclonal antibody, and either
immunoblotted with the same antibody (top) or anti-HA polyclonal Y-11
antibody for exogenous Strap (bottom). Lanes 1 to 6 show the input level of
p300 (top) and Strap (bottom), and Lanes 7 to 12 show the level of the
immunoprecipitated p300–Strap complex. (b) Lysates prepared from U2OS
cells transfected with expression vectors encoding wild-type Strap (10 µg;
lanes 3 and 4), StrapS203A (10 µg; lanes 5 and 6) or empty vector (10 µg;
lanes 1 and 2) were treated with etoposide as above, immunoprecipitated
with anti-p53 DO-1 antibody, and then immunoblotted with anti-acetylated
p53 Lys 382 (top). Input extracts immunoblotted with anti-Ac Lys 382,
DO-1, anti-p300 monoclonal antibody or actin are shown. (c, d) Lysates
prepared from SAOS2 (p53−/−) cells transfected with expression vectors
encoding wild-type Strap (10 µg, c) or StrapS203A (10 µg, d) either pre or
post etoposide treatment (10 µM for 12 h) were immunoprecipitated with
the anti-HA monoclonal antibody (lanes 1, 2, 3 and 4) or a control anti-
Gal4 antibody (lanes 5 and 6). The level of HAT activity in the
immunocomplex was measured in vitro using His-tagged wild-type p53 (1
µg) as the substrate. The acetylation of p53 was assessed by
immunoblotting with an antibody recognizing acetylated Lys 382, a site on
p53 which is known to be acetylated in damaged cells by p300 (ref. 22).
BSA (1 µg) served as a control substrate.
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A RT I C L E S
response to DNA damage (Fig. 6n), arguing that a nuclear location is
necessary to mediate Strap stabilization in response to DNA damage.
Subsequently, we examined the importance of Strap for p53 activity by
reducing the level of Strap through expressing short interfering RNA.
A reduced level of Strap caused lower levels of p53 and p53 target
genes, such as p21 (see Supplementary Information, Fig. S3).
Moreover, p53 targets were induced in a stable cell line in which Strap
levels could be upregulated (see Supplementary Information, Fig. S3),
providing evidence that together with previous results25 supports a
role for Strap in control of the p53 response.
We compared the ability of StrapS203A and NLS–StrapS203A to induce
the DNA damage response by studying p53 in U2OS cells. Although
StrapS203A had little effect on p53, a significant increase in p53 levels
occurred with the expression of NLS–StrapS203A (Fig. 7a). Given the
regulation of p53 by NLS–StrapS203A, we reasoned that NLS–StrapS203A
may affect the level of apoptosis in U2OS cells. StrapS203A exhibited
negligible apoptotic activity relative to the control treatment, whereas
NLS–StrapS203A was able to cause a significant increase in apoptosis
(Fig. 7b). Nevertheless, it remains to be determined whether other tar-
gets in addition to p53 are regulated by Strap.
DISCUSSION
The induction of Strap after DNA damage suggested that the DNA
damage signalling pathway controls Strap activity. Our results have
identified a residue in Strap, Ser 203, as a major site of phosphorylation
and show that ATM is one of the kinases responsible for this phospho-
rylation. At a biochemical level, StrapS203A failed to undergo a DNA
damage-dependent response and, in contrast with wild-type Strap,
accumulated in the cytoplasm. The cytoplasmic location of endoge-
nous Strap in ataxia telangiectasia cells, combined with the similar
location of StrapS203A in cells with normal ATM kinase activity, argues
that ATM functions in regulating the intracellular location of Strap. It
is consistent with the idea that re-instating ATM activity in ataxia
telangiectasia cells allowed Strap to attain a nuclear location, and in
normal cells a nuclear location was necessary to acquire protein stabi-
lization. Collectively, these results argue that ATM kinase is an effector
enzyme, following DNA damage, that controls Strap activity although
they do not rule out that other PI(3)K family members are also
involved in the control of Strap.
The DNA damage response is abnormal in ataxia telangiectasia cells,
and these cells are hypersensitive to ionizing radiation and
radiomimetic drugs2,4,27. This abnormality has been attributed to
defective ATM and the failure to execute an efficient DNA damage
response4,5. The results described here imply that the aberrant regula-
tion of Strap in these cells is likely to contribute to the defective DNA
damage response, in part because of its role in regulating p53 activity.
In support of this idea, NLS–StrapS203A activates p53 and initiates
apoptosis more efficiently than StrapS203A. As the activation of p53 and
induction of apoptosis are key facets of the DNA damage response,
these results argue that the regulation of Strap activity by ATM is an
important point of control in the survival response to DNA damage.
Our results provide mechanistic insight into the role of Strap in the
DNA damage response (Fig. 7c). This is mediated in part through
interacting with p300 and promoting p53 acetylation, which thereby
increases p53 activity32,33. Strap facilitates the acetylation of p53 both
in vivo and in vitro, owing to the enhanced level of acetylase in the
Strap/p300 co-activator complex. Because StrapS203A failed to alter p53
acetylation, the interaction of Strap with p300 and acetylation of p53 is
a downstream effect of ATM-mediated phosphorylation. Taken
together, these results argue that Strap, and Strap regulation by ATM,
modulates p53 activity and thus defines a new pathway linking ATM
with downstream events in the DNA damage response.
a b c
d
g
e f
Control p53 p53 / etoposide
Strap / etoposide
p53 / StrapS203A / etoposide
StrapS203A / etoposide p53 Strap / etoposide
Sub G1 : 7
G1 : 63
S : 11
G2 : 15
Sub G1 : 11
G1 : 76
S : 4
G2 : 7
Sub G1 : 21
G1 : 60
S : 4
G2 : 12
Sub G1 : 8
G1 : 60
S : 9
G2 : 17
Sub G1 : 22
G1 : 61
S : 3
G2 : 11
Sub G1 : 10
G1 : 60
S : 9
G2 : 16
Sub G1 : 35
G1 : 52
S : 4
G2 : 8
Figure 5 Ser 203 is required for the p53 response. (a–g) Representative
examples of flow cytometry profiles from SAOS2 cells transfected with
expression vectors for p53 (4 µg), Strap (12 µg) or StrapS203A (12 µg) as
indicated followed by treatment with etoposide (10 µM for 12 h). The
percentage of cells containing a sub-G1 DNA content, together with cells in
either G1, S and G2/M phases is shown.
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A RT I C L E S
The regulation of intracellular location makes an important contri-
bution in the DNA damage response. For example, the Mdm2 onco-
protein functions to negatively control p53 activity through promoting
p53 export into the cytoplasm, which aids p53 degradation34. In this
respect, Mdm2 shuttles p53 out of the nucleus, perhaps involving ATM
which phosphorylates Mdm2 (refs 12, 13). It is interesting that the role
of ATM in regulating the nuclear location of Strap functions in the
opposite fashion to that of Mdm2. Perhaps one role of Strap is to
counter-balance the effects of Mdm2, possibly acting in competition
with the opposite action of Mdm2 in diminishing p53 activity25.
At the functional level, Strap provides an ideal target for regulat-
ing the DNA damage response, as the level of Strap activity seems to
directly relate to the DNA damage response. Indeed, the findings
that StrapS203A cannot augment apoptosis in response to DNA dam-
age, and that reducing Strap levels hampers the DNA damage
response, both emphasise the central importance of Strap.
a b
i
j
k
l
c d
e f
g
m
h
Strap
NLS−Strap
NLS−S203A
S203A
S203A
S203A
− + − +
Strap
− + − + − +
StrapS203A NLS−StrapS203A
NLS−Strap
Etoposide:
Etoposide:
Strap
Strap
1 2 3 4
1 2 3 4 5 6
NLS
NLS
n
Figure 6 Nuclear Strap undergoes DNA damage-dependent protein
stabilization. (a–d) Ataxia telangiectasia GM02530 cells were transfected
with either Strap (a and b; 8 µg) or NLS–Strap (c and d; 8 µg) and
immunostained with the anti-HA monoclonal antibody, which recognizes the
tagged Strap constructs. (e–h) U2OS cells were transfected with either
StrapS203A (e and f; 2 µg) or NLS–StrapS203A (g and h; 2 µg) and
immunostained with the anti-HA monoclonal antibody. (i–l) Diagrams
showing the position of the NLS in the Strap derivatives. (m, n) U2OS cells
were transfected with Strap, NLS–Strap, StrapS203A or NLS–StrapS203A (all
10 µg) as indicated in the absence or presence of etoposide (10 µm). The
level of exogenous Strap protein was assessed by immunoblotting with the
anti-HA monoclonal antibody. DAPI staining shown in b, d, f and h.
print ncb1170 14/9/04 2:58 PM Page 974

A RT I C L E S
METHODS
Plasmids and expression vectors. Site-directed mutagenesis was performed
using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla,
CA) to create StrapS203A. To generate wild-type and mutant Strap mam-
malian expression vectors, the same sized fragments containing the wild-
type and StrapS203A were sub-cloned into the BamH1/Xho1 cloning sites of
haemagglutinin (HA) tagged pcDNA3. NLS–Strap and NLS–StrapS203A were
prepared by cloning WT and StrapS203A cDNAs into the Xho1 site of pCMV-
HA-NLS containing the SV40 NLS. pCDNA-Flag-ATMwt and pCDNA-Flag-
ATMkd have been described previously6.
Cell culture and transfection. SAOS2, U2OS, HeLa and ataxia telangiectasia cells
(1BR and GM02530, Coriell Cell Repositories, Camden, NJ) were maintained in
Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine
serum. The calcium phosphate technique was used to transfect cells with DNA,
which were harvested at 36 h post-transfection as described previously25.
Inducible Strap stable cell lines were generated using the BD TET-ON gene
expression system (BD Biosciences, Franklin Lakes, NJ). A U2OS tet-on cell line
was transfected with the Tre2-hyg vector containing Flag–Strap and carrying the
hygromycin resistance gene. Positive clones were selected using double selection
media containing G418 (100 µg ml−1) and hygromycin (75 µg ml−1). Flag-tagged
Strap protein was induced by the addition of doxycyclin (1 µg ml−1) for 24 h and
inducible clones were identified by immunostaining and immunoblot analysis.
Preparation of whole cell extracts and immunoblot analysis. Cells were washed
twice with PBS and lysed in 250 µl of TNN lysis buffer (50 mM Tris at pH 8,
120 mM NaCl, 0.5% NP-40, 1 mM dithiothreitol, 0.2 mM phenylmethylsulpho-
nyl fluoride and protease inhibitors) at 4 °C for 20 min. The extracts were cen-
trifuged at 16,000g for 10 min to remove cell debris. Immunoprecipitation and
immunoblotting were performed as described previously25. An equal amount of
protein was transferred to nitrocellulose and probed with the indicated antibody.
Enhanced chemi-luminescence (Pierce biotechnology, Rockford, IL) was used to
visualize antibody binding. The anti-acetylated Lys 382 p53 and anti-p300 Ab-1
antibodies were from Oncogene Research (Cambridge, MA), the anti-phospho-
S/T ATM/ATR substrate antibody from (Cell Signalling Technology, Beverly,
MA), the anti-Bax and anti-phospho-ATM (S1981) from Upstate (Lake Placid,
NJ), the Flag M2 antibody from Sigma (St Louis, MO), anti-p53 monoclonal
antibody DO1, anti-PUMA (N20), anti-p21 (F5) and anti-PCNA monoclonal
antibody PC10 from Santa Cruz (Santa Cruz, CA). The DNA damage agents:
etoposide, streptonigrin, hydroxyurea, bleomycin and wortmannin were from
Sigma. Irradiation of cells was performed as described25. The anti-Strap peptide
510 antibody was prepared by standard immunization protocol (Eurogentec,
Seraing, Belgium) against a peptide representing residues 211 to 234. The Strap
anti-P-Ser 203 antibody was generated by Eurogentec as described13.
Flag–Strap U2OS cells were seeded at 1.5 × 106 cells in a 150 cm3 dish and
grown in the presence of G418 and hygromycin. Flag–Strap protein expression
was induced for 24 h in the presence of doxycyclin (1 µg ml−1); control cells
were grown in the absence of doxycyclin. Cell extracts were prepared and Flag-
tagged protein was immunoprecipitated as described25. 1 mg of total cell extract
was used in each immunoprecipitation. Flag peptide eluted complexes were
resolved on a 10% SDS–polyacrylamide gel electrophoresis (PAGE) protein gel,
along with 20 µg of total cell extract and transferred to nitrocellulose.
−
−
−
StrapS203A
StrapS203A
NLS−StrapS203A
NLS−StrapS203A
+ − + − +Etoposide:
p53
Actin
1 2 3 4 5 6
a
c
b
20
10
DNA damage
ATM
Strap
P P
DNA damage response genes
Sub-G1(%)
Figure 7 Nuclear Strap activates the DNA damage response. (a) U2OS
cells were transfected with StrapS203A, NLS–StrapS203A or vector alone
(all 10 µg) and the level of p53 measured by immunoblotting with anti-
p53 antibody DO1 in the absence or presence of etoposide (10 µM). The
level of β-actin in the different treatments is shown. (b) U2OS cells were
transfected with StrapS203A, NLS–StrapS203A or vector alone (12 µg).
The percentage of cells containing sub-G1 DNA content, indicating that
they are apoptotic, is shown. (c) Model summarizing the role of Strap in
linking ATM kinase with the DNA damage response. It is envisaged that
DNA damage activates ATM kinase which then phosphorylates Strap at
Ser 203 (red circles). Phosphorylated Strap is stabilized and undergoes
nuclear accumulation where it assembles into a co-activator complex,
which includes p300 and cofactors such as JMY (blue and purple ovals).
One effect of Strap is to augment p53 activity through increased
acetylation and transcriptional activity, resulting in the activation of
DNA damage response genes.
print ncb1170 14/9/04 2:58 PM Page 975

A RT I C L E S
For the ATM kinase immunoprecipitation, HeLa cells were lysed in TNN
buffer and ATM immunoprecipitated with protein A agarose beads (25 µl) and
anti-ATM Ab-3 antibody (Oncogene Research Products, Cambridge, MA).
Kinase reactions were performed using precipitated ATM on beads (~15 µl)
with either His-tagged Strap, the His-tagged StapS203A mutant or His-tagged
p53 (ref. 35) purified from bacteria (~1 µg protein) in a kinase buffer (50 mM
Hepes at pH 7.4, 150 mM NaCl, 6 mM MgCl2, 4 mM MnCl2, 10% glycerol, 1
mM dithiothreitol, 0.1 mM NaOV and 15 µCi of [γ-32P] ATP] and incubated at
30 °C for 30 min. Proteins were separated by SDS–PAGE, transferred to nitro-
cellulose membranes and visualized by autoradiography.
Acetyl transferase assays. HA-tagged Strap was transfected into SAOS2 cells and
immunoprecipitated using anti-HA monoclonal antibody (Babco, Berkeley, CA).
Strap immunocomplexes were dissolved in HAT assay buffer (50 mM Tris at pH
8.0, 10% glycerol, 50 mM KCl, 0.1 mM EDTA, 10 mM butyric acid, 1 mM dithio-
threitol and 10 µM acetyl-CoA) and incubated with bacterially expressed recombi-
nant His-tagged p53 protein.The reaction mixtures were resolved on a SDS–PAGE
gel. Proteins were transferred onto nitrocellulose, which was then subjected to
immunoblot analysis and probed with the anti-acetylated Lys 382 p53 antibody.
Immunofluorescence microscopy. Cells were plated on to glass coverslips and
treated as described. Cells were fixed in 4% paraformaldehyde for 30 min, per-
meabilized with 0.1% Triton X-100 for 10 min, washed with PBS, blocked with
10% foetal calf serum in PBS for 10 min and then washed again with PBS. The
primary antibodies used were the anti-Strap peptide 510 antibody (detecting
endogenous protein, diluted 1 : 200) and the monoclonal anti-HA antibody (for
exogenous Strap and StrapS203A, diluted 1 : 1000). After washing with PBS, the
samples were incubated with FITC conjugated goat anti-mouse IgG (diluted 1 :
200) and stained with DAPI (0.1 µg m−1) for 30 min. The samples were exam-
ined using a fluorescent microscope (Olympus, Melville, NY).
siRNA expression. To generate the pSUPER-Strap knock down mammalian
expression vectors, four different short oligonucleotide fragments were
respectively subcloned into the HindIII/BglII cloning sites of pSUPER36.
pSUPER-Strap1 and 4 were found to be functional in reducing Strap protein
expression. The forward sequence of pSUPER-Strap1 was 5′-GATCCCCAA-
G AT G C A G G A C G G A A G C AT T C A A G AT G C T T C C G T C C T G -
CATCTTTTTTTGGAAA-3′ and the reverse 5′-AGCTTTTCCAAA
AAAAGATGCAGGACGGAAGCATCTCTTGAATGCTTCCGTCCTG-
CATCTTGGG-3′. The forward sequence of pSUPER-Strap4 was
5 ′ - G AT C C C C A A G A G AT G G A G A A G A C C C T T T C A A G A -
GAAGGGTCTTCTCCATCTCTTTTTTTGGAAA-3′ and the reverse
5 ′ - A G C T T T T C C A A A A A A A G A G A T G G A G A A G A C
CCTTCTCTTGAAAGGGTCTTCTCCATCTCTTGGG-3′.
Flow cytometry. U2OS cells were transfected with the indicated expression vec-
tors, together with pCMV CD20 (4 µg) to monitor transfection efficiency as pre-
viously described25. The profiles of the transfected population of cells are shown.
Note: Supplementary Information is available on the Nature Cell Biology website.
ACKNOWLEDGEMENTS
We thank M. Caldwell for assistance in preparing the manuscript. This work was
supported by the Medical Research Council, the Leukaemia Research Fund, Cancer
Research UK and the European Union. We thank M. B. Kastan for the wild-type
and kinase-dead ATM expression vectors.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
Received 18 February 2004; accepted 3 August 2004
Published online at http://www.nature.com/naturecellbiology.
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SUPPLEMENTARY INFORMATION
WWW.NATURE.COM/NATURECELLBIOLOGY 1
Figure S1 a) Strap is an intrinsically unstable protein: U2OS cells were
transfected with the expression vector encoding wild-type Strap (10µg) and
after 24h extracts harvested from either untreated or treated (cyclohexamide;
10mg/ml) cells at the indicated time points and immunoblotted with anti-HA
monoclonal antibody. b) Phosphorylation of Strap and S203A in U2OS and
AT cells: Lysates from U20S cells (tracks 1 to 6) transfected and treated as
described in (Fig. 1d) were immunoprecipitated with an anti-HA monoclonal
antibody followed by immunoblotting with anti-phospho S/T ATM/ATR
substrate-specific antibody (top) or the anti-Strap 510 antibody (bottom).
AT 1BR cells (tracks 7 to 10) transfected with the wild-type (WT) Strap
(10µg; track 3 and 4) or empty vector (10µg; track 1 and 2) were treated
with etoposide (+; 10µM for 12 h) and immunoprecipitated with an anti-HA
monoclonal antibody followed by immunoblotting with the anti-ATM/ATR
phospho S/T substrate specific antibody (top) or anti-Strap 510 antibody
(bottom). c) Strategy for preparing the Strap anti-P-S203 anti-peptide
antibody: Strap anti-P-S203 was prepared as described (13). Results from a
typical Elisa assay (bottom) representing activity against either the P-S203
or S203 peptide is shown.

2 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S2 Phosphorylation of Strap at S203. a) Flag-Strap U2OS cells were
either induced (tracks 1, 3, 4, 5 and 6) or not induced (tracks 2, 7, 8, 9
and 10) by doxycyclin and extracts prepared as described. Flag antibody
immunoprecipitates were eluted with Flag peptide and immunocomplexes
immunoblotted with either anti-Flag (tracks 6 and 10) or the anti-phospho
S203 peptide antibody (tracks 3, 4, 5, 7, 8 and 9) in the presence of the
phospho S203 peptide ((P); tracks 4 and 8) or non-phosphorylated peptide
(+; tracks 3 and 7), or in the absence of peptide (-; tracks 5 and 9). Tracks
1 and 2 represent the input extracts from either induced (+) or un-induced
(-) Flag-Strap U2OS cells. b) U2OS cells were treated with etoposide (10µM
for 12h), harvested as described and total cell extracts immunoblotted with
anti-Strap 510 antibody (track 1) or anti-phospho S203 peptide antibody
(tracks 2, 3 and 4) in the presence of non-phosphorylated peptide (+; track
3) or phospho S203 peptide (P; track 4). c) U2OS cells were treated with
ionising radiation (10Gy) and harvested together with control treated cells
after 1h, and immunoblotted with anti-phospho S203 peptide antibody (top)
or anti-α-tubulin (bottom) as described. d) U2OS cells were treated with
etoposide (10µM) or bleomycin (5µg/ml) for the indicated times, harvested
and immunoblotted with anti-phospho-ATM (S1981), to detect active ATM
(37), or anti-phospho S203.

WWW.NATURE.COM/NATURECELLBIOLOGY 3
Figure S3 Effect of siRNA knock-down of Strap on p53 and p21 levels.
U2OS cells were transfected with pHA-Strap (10µg) and pCMV-βgal (2µg)
together with either pcDNA3 (10µg) or the siRNA pSUPER vector 1, 4 or 1
and 4 together (10µg) and immunoblotted with either anti-HA monoclonal
antibody (upper) or anti-PCNA monoclonal antibody (lower). The amount
loaded was adjusted through measuring βgal activity. b, c) SAOS2 cells were
co-transfected with expression vectors for wild-type p53 (2µg), pCMV-βgal
(2µg) together with either pcDNA3 (16µg) or pSUPER-Strap (16µg) and
immunoblotted for p53 (DO-1) and PCNA respectively. The same cell extract
(as for b) was immunoblotted with anti-p21 (F-5) monoclonal antibody (c). d
and e) Flag-Strap U2OS cells (track 4) or the parental U2OS cells (tracks 1,
2 and 3) were treated with doxycycline as described, together with etoposide
(10µM) or bleomycin (5µg/ml) for 6h, harvested and immunoblotted with
anti-p21 (d), anti-PUMA (d) or anti-Bax (e). The levels of Flag-Strap is
indicated, and the PCNA level served as an internal control.

4 WWW.NATURE.COM/NATURECELLBIOLOGY
Figure S4 i) Intracellular location of Strap and S203A: Enlarged images
taken from Fig. 2 e, g, i and k, illustrating the nuclear location of wild-type
Strap (e and g), and predominantly cytoplasmic location of S203A (i and
k). ii) Strap phosphorylation at S203 is reduced by an ATM kinase-dead
mutant derivative: Either U2OS (a) or HeLa (b) cells were transfected with
expression vectors encoding wild-type Strap (10µg) together with either ATM
(a) or ATR (b) kinase dead mutants (kd; 5µg) and treated with etoposide
(Et; 10µM) or bleomycin (BLM; 5µg/ml) for either 6h (a) or as indicated (b),
and immunoprecipitated and immunoblotted with either anti-Strap phospho
S203 or anti-p53 S15 (b; lower). The bottom panel in (b) shows an anti-ATM
immunoprecipitation kinase assay (using 0.5µg p53 as the substrate).

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