DNA-dependent protein kin-ase DNA-PK is another large phosphoinositide 3-kinkin-ase- 3-kinase-like kinase with the potential to phosphorylate p53 at Ser15, and has been proposed to enhan
Trang 1Structured DNA promotes phosphorylation of p53 by DNA-dependent protein kinase at serine 9 and threonine 18
Se´bastien Soubeyrand1, Caroline Schild-Poulter1and Robert J G Hache´1,2
Departments of1Medicine and2Biochemistry, Microbiology and Immunology, University of Ottawa, the Ottawa Health Research Institute, Ottawa, Ontario, Canada
Phosphorylation at multiple sites within the N-terminus of
p53 promotes its dissociation from hdm2/mdm2 and
sti-mulates its transcriptional regulatory potential The large
phosphoinositide 3-kinase-like kinases ataxia telangiectasia
mutated gene product and the ataxia telangectasia and
RAD-3-related kinase promote phosphorylation of human
p53 at Ser15 and Ser20, and are required for the activation of
p53 following DNA damage DNA-dependent protein
kin-ase (DNA-PK) is another large phosphoinositide 3-kinkin-ase-
3-kinase-like kinase with the potential to phosphorylate p53 at Ser15,
and has been proposed to enhance phosphorylation of these
sites in vivo Moreover, recent studies support a role for
DNA-PK in the regulation of p53-mediated apoptosis
We have shown previously that colocalization of p53 and
DNA-PK to structured single-stranded DNA dramatically
enhances the potential for p53 phosphorylation by
DNA-PK We report here the identification of p53 phosphoryla-tion at two novel sites for DNA-PK, Thr18 and Ser9 Colocalization of p53 and DNA-PK on structured DNA was required for efficient phosphorylation of p53 at multiple sites, while specific recognition of Ser9 and Thr18 appeared
to be dependent upon additional determinants of p53 beyond the N-terminal 65 amino acids Our results suggest a role for DNA-PK in the modulation of p53 activity resultant from the convergence of p53 and DNA-PK on structured DNA
Keywords: DNA-dependent protein kinase; p53; structured single-stranded DNA; phosphorylation
The large phosphatidylinositide 3-kinase (PI3K)-like
kinases are broad specificity serine/threonine kinases with
essential roles in regulating DNA metabolism and responses
to DNA damage Three of these kinases, DNA-dependent
protein kinase (DNA-PK), the ataxia telangiectasia mutated
gene product (ATM) and the ataxia telangectasia and
RAD-3-related kinase (ATR) [1,2] show a redundant
specificity for accessible SQ and TQ motifs in vitro that
has hindered definition of their individual roles in DNA
repair and metabolism In particular, while DNA-PK and
its associated kinase activity have been shown to be required
for double-stranded DNA (dsDNA) break repair through
the nonhomologous end-joining pathway, for V(D)J
recombination, and to play at least some role in the
regulation of other processes including transcription, DNA
replication and viral integration, demonstration of a role for
DNA-PK in specific protein phosphorylation in vivo has
remained elusive [1] We and others have hypothesized that
substrate phosphorylation by DNA-PK in vivo depends to a
large extent on mechanisms that promote the recruitment of
substrates to DNA-bound, active, DNA-PK [3–6] p53 is
a key regulatory protein that has the potential to be phosphorylated by DNA-PK, ATM and ATR as Ser15 of human p53 is efficiently phosphorylated by all three kinases
in vitro[7] Phosphorylation as well as ubiquitylation and acetylation control the activation status of p53 [8] A majority of the phosphorylation sites on p53 are clustered within the N-terminal 40 amino acids (see Fig 1) and modification at some of these sites, particularly Ser20 and Thr18, promotes the accumulation of active p53 by destabilizing the interaction of p53 with hdm2/mdm2 [9,10] Phosphorylation of other sites, such as Ser15, appear
to stimulate the transcriptional activation potential of p53, while the exact influence of phosphorylation at other sites remains to be determined [11] While p53 phosphorylation
in response to DNA damage has long made it an attractive
in vivocandidate target of DNA-PK, ATM and ATR are now believed to constitute the main effectors leading, directly as well as indirectly, to p53 phosphorylation in response to DNA damage [12,13]
Nonetheless, it has been reported that in cells lacking ATM, accumulation of p53 and phosphorylation within the N-terminus of p53 in response to treatment with agents that induce dsDNA breaks still occurs, albeit at a lower levels or with delayed kinetics [14,15] Further, in certain situations DNA-PK is essential for p53-dependent DNA damage-mediated apoptosis [16,17] In addition, DNA-PK and p53 have both been implicated in controlling the integrity of DNA replication and repair [18–24] DNA-PK reaches a maximum level during G1/early S phase, suggesting that DNA replicative structures can activate DNA-PK [25]
Correspondence to S Soubeyrand, Departments of Medicine,
Uni-versity of Ottawa, the Ottawa Health Research Institute, 725 Parkdale
Avenue, Ottawa, Ontario, Canada K1Y 4E9 Fax: +613 7615036;
Tel.: +613 7985555 ext 13705; E-mail: ssoubeyrand@ohri.ca
Abbreviations: ATM, ataxia telangiectasia mutated gene product;
ATR, ataxia telangectasia and RAD-3-related kinase; dsDNA,
double-stranded DNA; DNA-PK, DNA-dependent protein kinase;
PI3K, phosphoinositide 3-kinase; ssDNA, single-stranded DNA.
(Received 10 March 2004, revised 6 July 2004, accepted 2 August 2004)
Trang 2In vitro, p53 and DNA-PK both interact with
single-stranded, structured and damaged DNA [26–30] The
sequence-independent DNA binding ability of p53, which
depends on its C-terminus as well as the core domain, has
been proposed to play an important part in the initiation of
cellular responses to DNA damage [31–34]
Recently, we have shown that DNA-PK is activated from
structured single-stranded DNA (ssDNA) and hairpin
DNA ends resembling replication and recombination
intermediates [30,35] Preliminary studies indicated the
phosphorylation of p53 by DNA-PK was dramatically
enhanced by the colocalization of p53 to the ssDNA [30] In
the present study we have performed a detailed analysis of
the phosphorylation of p53 in the presence of ssDNA We
report the identification of two sites of DNA-PK
phos-phorylation in the N-terminus of p53, Thr18 and Ser9,
which are preferentially phosphorylated by DNA-PK when
p53 and DNA-PK are colocalized to ssDNA These results
reinforce the importance of colocalization for substrate
phosphorylation by PK and emphasize that
DNA-PK is a kinase with a broad specificity They also suggest a
specific role for DNA-PK in the phosphorylation of p53
from structured DNA in vivo
Materials and methods
Chemicals and recombinant proteins
Purified DNA-PK and the p53-derived peptide were
obtained from Promega (Madison, WI, USA) Wortmannin
was from Sigma (St Louis, MO, USA) and LY294002 from Calbiochem (San Diego, CA, USA) The p53wtas well as the Ser15 and Ser37 variants have been described elsewhere [11], while the other point mutants were generated by QuikChange mutagenesis (Stratagene, La Jolla, CA, USA) Mutations were confirmed by dideoxynucleotide sequen-cing The truncated p53 forms, p531)65and p53D30C, were generated by introducing nonsense mutations at positions
66 (Met) and 363 (Arg), respectively The recombinant p53s were expressed as fusion proteins from pGEX-6P1 and purified on Glutathione Sepharose 4B (Amersham Phar-macia Biotech; Piscataway, NJ, USA) and then cleaved from the GST with PreScission protease The purity of all p53 preparations was monitored by SDS/PAGE analysis Single-stranded M13 DNA (ssM13) was obtained from New England Biolabs (Beverly, MA, USA) while linearized pBluescript DNA was prepared by extraction of HindIII digested plasmid (Qiagen) from agarose gels The p53 (FL-393) and p53 pSer9 polyclonal antibodies were obtained from Santa Cruz (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA), respectively
DNA-PK kinase assays Assays were performed with 0.2 lM of p53 peptides or
100 ng of recombinant p53 at 30C for 15 min in the presence of 4.2 nM of [32P]ATP[cP] (3000 CiÆmmol)1),
10 ng of DNA and 10 units of DNA-PK in 20 lL of reaction buffer (50 mM HEPES, 100 mM KCl, 10 mM MgCl2, 0.2 mMEGTA, pH 7.5) The final ATP concentra-tion was adjusted to 50 lM where indicated Following completion of the reaction, the substrates were resolved by SDS/PAGE (8–12%) and visualized by autoradiography Quantification was performed by Phosphorimager analysis (Typhoon 8600, Molecular Dynamics) in the presence of a series of [32P]ATP[cP] standards Inhibition experiments with LY294002 (0.3–300 lM) and wortmannin (3–3000 nM) dissolved in dimethyl sulfoxide were performed as above except that the inhibitor was incubated for 5 min at 30C
in the presence of either ssM13 DNA (for p53wt and p53S15A/S37Aphosphorylation) or dsDNA (for p53 peptide phosphorylation) prior to the addition of substrate Phos-phorylation was quantified by Phosphorimager analysis of the polyacrylamide gel Phosphorylation was expressed relative to the mock-treated kinase and the resulting ratios
fit to a sigmoidal curve used to derive the IC50 The inhibition of p53 phosphorylation subsequent to DNA-PK autoinactivation was assessed as previously described [30] DNA-PK was preincubated in the presence of ssM13 with
or without 50 lMATP for 10 min at 30C Subsequently [32P]ATP[cP] in a final ATP concentration of 50 lM was added and incubation continued for 15 min
Dissection of p53 phosphorylation by DNA-PKin vitro Radiolabeled p53 was resolved by SDS/PAGE, excised and digested at 37C in situ in 400 lL of digestion buffer (50 mM NH4HCO3, pH 8.0) containing 0.050 lgÆlL)1 TPCK-treated trypsin (Worthington Biochemical Corpora-tion; Freehold, NJ, USA) for 16 h This was followed by the addition of fresh trypsin (0.025 lgÆlL)1) and redigestion for
3 h The supernatant was evaporated under vacuum and the
Fig 1 Phosphorylation of p53 by DNA-PK from ssDNA at sites
beyond Ser15 and Ser37 (A) Schematic presentation of p53
high-lighting Ser (S) and Thr (T) residues in the N-terminal 60 amino acids.
The filled arrowheads indicate the position of trypsin cleavage, whereas
the open arrowhead indicates the location of a CNBr cleavage site.
(B,C) Phosphorylation of recombinant p53 (B) Purified recombinant
p53 wt and the indicated variants were phosphorylated by DNA-PK in
the presence of 40 n M [ 32 P]ATP[cP] (3 CiÆlmol)1) in the absence of
DNA (–), or in the presence of 10 ng of ssM13 DNA (ss) or
SmaI-linearized pBluescript (ds) DNA The phosphorylated p53s were
resolved by SDS/PAGE and quantified by Phosphorimager analysis.
Phosphate incorporation is indicated (values below the lanes; fmol P).
(C) Phosphorylation of p53 wt and p53 S15A/S37A as in (B) but in the
presence of 50 l M [ 32 P]ATP[cP] Phosphate incorporation is indicated
(values below the lanes; pmol P).
Trang 3pellet resuspended in a denaturation buffer (6M urea,
25 mM Tris/HCl, pH 8.0) The sample was then loaded
onto a 40% alkaline acrylamide gel as described previously
[35] and resolved for 6000 VÆh)1at 3 W For CNBr digests,
the protein was trypsinized as above, evaporated to dryness
and incubated in 100 lL of CNBr in formic acid
(100 mgÆmL)1) for 90 min at 20C The samples were then
vacuum-dried and the pellets resuspended in denaturation
buffer and submitted to electrophoresis as above Each
analysis was confirmed by obtaining two to five repetitions
with reproducible results
For phosphoamino acid mapping, the trypsinized p53
fragments were resolved by 40% PAGE and eluted in H2O
An aliquot was then evaporated to dryness and 5.5MHCl
was added for 1 h at 110C The hydrolysate was
evaporated, mixed with unlabeled pSer, pThr and pTyr
standards and then applied onto 10· 10 cm plastic backed
cellulose thin layer chromatography plates (Merck,
Darms-tadt, Germany) Phosphomanino acids were resolved by
two consecutive ascending chromatographies in ethanol/
acetic acid/H2O (1 : 1 : 1, v/v/v; 80 min) and 2-propanol/
HCl/H2O (7 : 1.5 : 1.5, v/v/v; 180 min) The
phospho-amino acids were then visualized by spraying the plates
with 0.25% (v/v) ninhydrin/acetone
Results
Phosphorylation of p53 by DNA-PK from ssDNA
at novel sites for DNA-PK
While p53 has been reported to be phosphorylated
exclu-sively on Ser15 and Ser37 by DNA-PK in the presence of
double-stranded linear DNA, to date no study has
evalu-ated the impact of structured DNA or DNA colocalization
on the kinase specificity [11,36] To begin detailed analysis of
the phosphorylation of p53 colocalized to ssDNA with
DNA-PK, we compared the phosphorylation of
recombin-ant p53 with the phosphorylation of S15A and S37A
substituted p53 (p53S15A, p53S37A) in the presence of ssM13
and linearized double-stranded plasmid DNA (Fig 1)
In the presence of ssDNA, substrate phosphorylation
occurs in competition with DNA-PK autophosphorylation
and autoinactivation [30] Previously we demonstrated that
this potent autoinactivation of DNA-PK linked in cis can be
minimized when assessing phosphorylation of heterologous
DNA-PK substrates by performing the kinase reactions at
the limiting ATP concentration of 40 nM[30]
At 40 nMATP, p53 was phosphorylated 11 ± 2.5 (n¼
5) times more efficiently by DNA-PK in the presence of the
optimal amount of ssM13 than in the presence of an
equimolar amount of linearized double-stranded plasmid
DNA (Fig 1B) Unexpectedly, p53 substituted at Ser15 and
Ser37 remained a strong substrate for DNA-PK in the
presence of ssM13, with 20 ± 5% (n¼ 4) of the phosphate
incorporation of p53wt Indeed p53S15A/S37Awas
phosphor-ylated three times more efficiently in the presence of ssM13
than was p53wt in the presence of linear plasmid DNA
(Fig 1B, lanes 3, 11)
To determine whether this additional phosphorylation
of p53 arose due to the limiting concentration of ATP in
the assay, we repeated the experiment at the usual ATP
concentration employed for DNA-PK, 50 l (Fig 1C)
Phosphorylation of p53 in the presence of ssM13 was reduced to 3.0 ± 0.4 (n¼ 4) times the efficiency of p53 phosphorylation in the presence of linear double-stranded DNA This high remaining level of p53 phosphorylation by DNA-PK in the presence of ssM13 DNA has previously been shown to be directly attributable to colocalization of p53 to the ssM13 with DNA-PK, which allows for rapid p53 phosphorylation prior to a DNA-PK autoinactivation [30]
Before pursuing the sites of this new phosphorylation it was important to ensure that the phosphorylation observed was mediated directly by the DNA-PK rather than a minor contaminant of the DNA-PK preparation Although our SDS/PAGE analysis indicated that the DNA-PK was about 90% pure, the potential contribution of contaminating kinases had to be excluded Consequently, we titrated the sensitivity of phosphorylation of p53S15A/S37A and the classical p53-derived DNA-PK peptide substrate containing only Ser15 to the DNA-PK inhibitors wortmannin and LY294002 The IC50 values for p53S15A/S37A closely matched the values obtained for the p53 peptide, confirming that both activities were due to a single enzyme species, namely DNA-PK (Fig 2A) Notably, these values exclude
Fig 2 DNA-PK directly mediates phosphorylation of p53 S15A/S37A (A)
IC 50 values for the inhibition of p53 phosphorylation Kinase reactions with p53 S15A/S37A (100 ng) or the synthetic p53-derived peptide (0.5 lg) were performed with DNA-PK in the presence of increasing amounts of Wortmannin (3–3000 n M ), LY294002 (0.3–300 l M ) or an equivalent amount of dimethyl sulfoxide The reaction products were quantified by Phosphorimage analysis of polyacrylamide gels The
IC 50 values are the mean of two interpolations from two independent inhibition profiles (B) Autoinactivation of DNA-PK prevents r p53 S15A/S37A phosphorylation DNA-PK was preincubated for 10 min either with (lanes 1, 3) or without (lanes 2, 4) 50 l M ATP in the presence of ssM13 [32P]ATP[cP] was then added and the ATP con-centration raised to 50 l M in all the samples and kinasing of p53 wt
(lanes 1, 2) or p53 S15A/S37A (lanes 3, 4) was performed by standard assay On the left is a Phosphorimager analysis of a representative gel and on the right is a graphical display of the Phosphorimager quan-tification of two independent determinations (± SD) expressed as the ratio of p53 phosphorylation following a preincubation with ATP over
a control preincubation without ATP.
Trang 4phosphorylation of p53S15A/S37A by minor contaminating
amounts of ATM or ATR in the kinase preparation, as
these kinases are not inhibited by the concentrations of
wortmannin and LY294002 employed [37–40]
To further ascertain that DNA-PK is directly involved in
p53S15A/S37A phosphorylation, we took advantage of the
rapid autoinactivation of DNA-PK that occurs on ssM13 in
the presence of 50 lM ATP [30] It was reasoned that a
contaminating kinase should remain unaffected by this
rapid, DNA-dependent, inactivation and that p53
phos-phorylation should then proceed normally On the contrary,
preincubation of the DNA-PK for 10 min in the presence of
50 lM ATP and ssM13 prior to addition of p53 and
[32P]ATP[cP], led to phosphorylation of both p53wt and
p53S15A/S37Aby 80% (Fig 2B) Hence DNA-PK directly
targets p53S15A/S37A
To begin analysis of p53 phosphorylation by DNA-PK in
the presence of ssM13 in greater detail, tryptic digests of
p53wtphosphorylated in the presence of 40 nMand 50 lM
ATP were resolved on a 40% alkaline polyacrylamide gels
(Fig 3) Alkaline PAGE allows separation of peptides
according to a combination of charge and size; the presence
of additional negative charges, such as those induced by
phosphorylation or by substitution of Ser with Asp or Glu,
enhances peptide migration
Trypsin digestion of p53 is expected to lead to the
separation of Ser15 and Ser37 onto two peptides containing
amino acids 1–24 and 25–65, respectively (Fig 1A) p53
phosphorylation at 40 nM ATP in the presence of ssM13
resulted in the resolution of two major tryptic
phosphopep-tides (A and B) on alkaline gels (Fig 3A) Two pepphosphopep-tides
with the same corresponding migrations were also observed
following trypsin digestion of an N-terminal p53 peptide (aa
1–65) phosphorylated by DNA-PK (Fig 3B) A third
peptide whose appearance varied in intensity through the
course of the study, designated A¢, was observed in both
instances This peptide likely reflects an alternative cleavage
product of peptide A as both bands were abrogated by the
Ala37 substitution (Fig 4B) In summary, these data
suggested that the additional phosphorylation of p53 detected at 40 nMATP occurred in the N-terminus of p53 within the two peptides containing Ser15 and Ser37 Interestingly, at 50 lM ATP, two additional phospho-peptides, with intensity approaching that of peptide B as well as a somewhat weaker band were detected within full-length p53 (Fig 3C, peptides C, D and E, respectively) Additionally, the signal yielded by peptides A broadened and decreased in resolution These results suggested that the activity of DNA-PK at the higher ATP concentration was increased to include additional sites within p53 Import-antly, although weaker in intensity, highly similar tryptic profiles were obtained in the presence of dsDNA ends (data not shown), indicating that although colocalization stimu-lated phosphorylation of p53 it did not appear to induce the exposure of new sites on p53
DNA-PK phosphorylates p53 at Thr18 and Ser9 The relative simplicity of tryptic peptide digestion pattern
of p53 phosphorylated at 40 nMATP prompted us to first characterize the additional p53 phosphorylation under these conditions To identify peptides A and B, p53 phosphorylated by DNA-PK from ssM13 DNA at 40 nM ATP was treated with CNBr which cleaves p53 tryptic peptide 25–65, but not 1–24 (Fig 1A) CNBr treatment of the tryptic digest converted peptide A to a higher mobility peptide, without affecting the intensity or mobility of peptide B (Fig 4A) This identified peptide A as contain-ing amino acids 25–65 of p53 and peptide B as containcontain-ing amino acids 1–24
Substitution of Ser37 with Ala in full-length p53 elimin-ated the signal from peptides A/A¢ while conversion of Ser15 to Ala strongly interfered with, but did not abrogate, fragment B phosphorylation (Fig 4B) Together these results identify the presence of a new DNA-PK phosphory-lation site in amino acids 1–24 of p53 The presence of additional phosphorylation site(s) within tryptic peptide B was also observed in the context of a polypeptide spanning
aa 1–65 following Ser15 and Ser37 substitutions, despite
a > 95% reduction in total phosphorylation (Fig 4C)
In addition to Ser15, peptide B contains Ser6, Ser9, Thr18 and Ser20 as well as an additional Ser (at position 1) that comigrates upon cleavage of the GST tag (Fig 1A) Phosphoamino acid analysis of peptide B from Ala15/37-substituted p53 revealed the predominant presence of phosphothreonine (Fig 4D, top), thereby establishing Thr18 (the only threonine residue in amino acids 1–24) as
a third major DNA-PK phosphorylation site within the N-terminus of p53 Interestingly a similar analysis of the wild-type protein showed proportionally less but significant Thr18 phosphorylation demonstrating that phosphoryla-tion does indeed occur at this site in the Wt context (Fig 4D, bottom)
While at limiting ATP concentrations p53 was almost exclusively phosphorylated on Ser15, Thr18 or Ser37, at the saturating and physiologically relevant ATP concentration
of 50 lM, additional radiolabeled tryptic p53 peptides were observed (Fig 3C, bands C and D) The introduction of T18E or S15D mutations shifted the migration of these phosphopeptides indicating that they were phosphopeptide B-derived (data not shown)
A
B
A B
A
B C D E
40 nM ATP
50 µM ATP
40 nM
ATP
Fig 3 Tryptic analysis of p53 phosphorylation Alkaline PAGE
ana-lysis of the phosphorylation of tryptic peptides of p53 wt (A,C) and
p531)65(B) phosphorylated by DNA-PK in the presence of ssM13
(A,C) or linearized pBluescript DNA (B) and 40 n M (A,B) or 50 l M
[ 32 P]ATP[cP] (C) Aliquots of 2000 cpm from the tryptic digests of
phosphorylation reaction were resolved through 40% alkaline PAGE.
Tryptic phosphopeptides were labeled A–E on the basis of increasing
mobility Peptide A¢ is a subordinate cleavage product of peptide A as
discussed in the text.
Trang 5To identify the remaining three bands originating from
p53 tryptic peptide 1–24, we assessed the effect of additional
substitutions on the phosphorylation of p53 at 50 lMATP
(Fig 4E) As mentioned above, the recombinant p53 used
in the mapping contained a serine residue at position 1 as
which was replaced with Ala Substitution of this Ala
reduced the peptides migrating in the range B-E from 3 to 2
indicating that it was indeed phosphorylated (Fig 4E, top,
lanes 1 and 2) Within that context, substitution of Ser9, but
not Ser6 nor Ser20, with Ala resulted in the loss of the
remaining higher mobility peptide, leaving a single peptide,
presumably phosphorylated at Thr18 (Fig 4E, lane 4)
Finally, mutation of both Thr18 and Ser9 to Ala in the
context of the Ser1/15 mutation abrogated fragment B (data
not shown), consistent with phosphorylation of both Thr18
and Ser9 Introduction of the single Thr18 and Ser9
mutations in the wild-type protein background resulted in
the abrogation of one band, further indicating that these
sites are genuine targets in the wild-type protein (Fig 4E,
lanes 6–8) and not artifacts due to Ser15 mutation
Finally, to confirm the presence of the nonconsensus p53 phosphorylation in the context of a wild-type protein, western blot analysis of p53wtphosphorylated by DNA-PK was performed Because of a lack of a suitable pThr18 antibody, we focused on Ser9 phosphorylation Ser9 phosphorylation was observed only in the presence of both DNA-PK and p53wt (and not in the alanine-substituted control p53), indicating that Ser9 was targeted by DNA-PK
in the context of the wild-type protein (Fig 4F)
Perhaps not surprisingly, in view of the lack of effect on total phosphorylation by the S15A and S37A single mutations (Fig 1B), initial attempts at comparing total phosphorylation of p53wt and p53S9A/T18V revealed no significant difference (data not shown) Consequently, the proportional significance of these sites on total phosphory-lation was rather estimated in the context of the wild-type protein by quantifiying the tryptic profiles of phosphoryl-ated p53wt; this approach had the additional advantage of circumventing potential artifacts arising from the introduc-tion of mutaintroduc-tions Taking into consideraintroduc-tion that the fastest
Fig 4 Phosphorylation of p53 on Thr18 and Ser9 (A) Alkaline PAGE analysis of CNBr cleavage of tryptic phosphopeptides derived from p53 wt
phosphorylated by DNA-PK in the presence of ssM13 and 40 n M [32P]ATP[cP] The migration of tryptic phosphopeptides A and B are indicated by arrows (B) Tryptic phosphopeptide profiles of p53 wt , p53 S15A , p53 S37A and p53 S15A/S37A phosphorylated by DNA-PK in the presence of 40 n M
[ 32 P]ATP[cP] and ssM13 DNA (C) Tryptic phosphopeptide profiles of p531)65(Wt 1–65, 2 l M ) and S15A/S37A substituted p531)65(S15A/S37A 1–65, 2 l M ) phosphorylated by DNA-PK as in (B) Phosphate incorporation (pmol) is indicated at the bottom below the exposure (D) Tryptic phosphopeptide B of DNA-PK phosphorylated p53 Wt or p53 S15A/S37A was eluted from a 40% alkaline PAGE gel and hydrolyzed in HCl Phosphoamino acids were resolved by TLC in the presence of phosphoserine and phosphothreonine markers Assignment of phosphorylation was made by superimposition of autoradiographs and ninhydrin staining, with the position of phosphoserine (pSer) and phosphothreonine (pThr) migration indicated to the left of the phosphorimage (E) Alkaline PAGE analysis of tryptic phosphopeptides derived from recombinant p53s following incubation with DNA-PK in the presence of ssM13 and 50 l M [ 32 P]ATP[cP] (F) Western blot analysis of p53 Wt or p53 S9A/T18V
phosphorylation by DNA-PK DNA-PK was incubated with the indicated p53 species in the presence of 50 l M ATP and assessed for total phosphorylation (top) or pSer9 phosphorylation (bottom) by Western blotting The amino acid substitutions within full-length recombinant p53 are listed at the top of each lane in the panels In panel (E) and (F), p53 substituted at Ser1 with Ala is highlighted by asterisks.
Trang 6band (Fig 3C or Fig 5B, band E) has four phosphate
groups, with a progressive reduction of one band per
phosphate removed, one can estimate the contribution of
Ser9 and Thr18 on total phosphorylation Conservatively
assuming that all of the B-E bands are phosphorylated at
Ser15 and that C-E are also phosphorylated on pSer1,
leaving D and E as containing phosphorylation on Ser9 and
Thr18, phosphorylation at the latter sites would account for
10% ± 2% of total phosphorylation Taking the least
conservative approach, i.e inferring that pSer15/pSer1
phosphorylation correspond to the two lowest intensity
fragments, would increase this value to 18% ± 2% Thus
phosphorylation at these two sites probably accounts for
8–20% of total p53 phosphorylation
Phosphorylation of p53 at Ser9 andThr18 is preferentially
enhanced within full-length p53
Initial experiments comparing p53 phosphorylation by
DNA-PK with the phosphorylation of a p53 peptide
containing only the N-terminal 65 amino acids (p531)65)
indicated that p531)65was a noticeably poor substrate for
DNA-PK in the presence of ssDNA at 40 nM ATP
(Fig 3B) Similarly, at 50 lM ATP, p531)65
phosphoryla-tion occurred with an efficiency less than 1% that of p53wt
(Fig 5A) By contrast, phosphorylation by DNA-PK in the
presence of dsDNA increased the absolute phosphorylation
of the p53 peptide 30-fold, while decreasing phosphate incorporated into p53wtby 2.5-fold Together, these data suggested that p53 phosphorylation was affected by the nature of the DNA cofactor and by the remainder of p53 beyond amino acid 65
Previously, we have demonstrated that the efficiency of phosphorylation of recombinant p53 by DNA-PK in the presence of ssDNA correlated directly with the ssDNA binding ability of p53 [30] In the present experiments, however, the reduction in the efficiency of phosphorylation
of the p53 peptide could not entirely be accounted for by the loss of ssDNA binding (Fig 5A) Phosphorylation of a mutated version of p53 lacking the C-terminal 30 amino acids (p53D30C) that is unable to interact with ssDNA [27], occurred with an efficiency that was only eightfold lower than p53wtin the presence of ssM13, leaving the level of phosphorylation of p53D30C 30-fold higher than that of p531)65(0.37/0.011 pmol)
To investigate whether DNA binding and the presenta-tion of full-length p53 also influenced the recognipresenta-tion of individual phosphorylation sites by DNA-PK, we com-pared the pattern of tryptic phosphopeptides obtained from p53wt, p53D30C, and the amino acid 1–65 peptide phosphor-ylated by DNA-PK in the presence of ssM13 DNA (Fig 5B) Interestingly, while the ratio between peptides A/A¢ and B showed little variation between substrates, the level of phosphorylation of peptides C-D was markedly decreased for p53D30C and was undetectable for the p53 peptide (Fig 5B), even upon prolonged exposure of the gels
In order to better discriminate the contribution of structural elements within p53 that may promote its phosphorylation at Ser9 and Thr18 from the direct contri-bution of p53 DNA binding to structured DNA, we quantified the absolute levels of phosphorylation of p53wt with p53S15A/S37Ain the presence of dsDNA Utilization of dsDNA minimizes DNA binding by p53 and resulted in more similar total phosphorylation levels (Fig 5A) Substi-tution of Ser15 and Ser37 with Ala in full-length recombin-ant p53-reduced phosphate incorporation to 35% of the level of both p53wtand p53D30Cat 50 lMATP, confirming that colocalization to DNA was not required for the phosphorylation of Ser9 and Thr18 by DNA-PK In contrast, DNA-PK was essentially unable to effect phos-phorylation of p531)65, S15A/S37A Thus, these data indicate that phosphorylation of p53 at Ser9 and Thr18 by
DNA-PK is dependent upon specific determinants within the remainder of the p53 protein that are not directly related to its ability to bind DNA structures
Discussion
Our results demonstrate the phosphorylation of p53 at two sites, Ser9 and Thr18, which have not previously been appreciated as potential targets for DNA-PK in vitro Importantly, phosphorylation at Ser9 and Thr18 showed a strong preference for the colocalization of p53 and
DNA-PK on ssDNA This may explain why these sites have not been previously recognized as bona fide DNA-PK targets Indeed, typical DNA-PK activity assays involve dsDNA ends in combination with peptides or polypeptides span-ning the N-terminal portion of p53 Another ancillary
Fig 5 Phosphorylation of p53 within the novel N-terminal sites is
dependent on binding to ssDNA and full-length p53 The
phosphoryla-tion of p53 wt , p53 D30C and p531)65by DNA-PK in the presence of
50 l M [32P]ATP[cP] is compared (A) Comparison of total phosphate
incorporation (pmol) in the presence of ssM13 and linear pbluescript
dsDNA Data shown is representative of the results of three
inde-pendent experiments (B) Alkaline PAGE analysis of tryptic
phos-phopeptide labeling of the three forms of p53 phosphorylated by
DNA-PK in the presence of ssM13 The position of migration of
phosphopeptides A–E is indicated to the left of the panel (C) The
contribution of phosphorylation of p53 at Ser15 and Ser37 to the total
phosphorylation of p53 by DNA-PK in the presence of dsDNA was
determined by comparing32P incorporation into wild-type and
Ala-substituted recombinant p53s Following incubation with DNA-PK,
the p53 polypeptides were resolved by SDS/PAGE and phosphate
incorporation was quantified by Phosphorimager The results are
expressed as a ratio of the phosphorylation of the alanine-substituted
p53 variant (hatched bars) over its serine equivalent (100%, solid bars).
Data represent the mean ± SD of three determinations performed in
duplicates.
Trang 7explanation resides in the relatively low phosphorylation
level of these sites We have estimated that phosphorylation
at these sites may account for 10-20% of total
phosphory-lation of the wild-type protein at 50 lMATP Clearly this
does not account for the 35% phosphorylation remaining
observed in the absence of both Ser15 and Ser37 This
discrepancy suggests that Ala mutations may either
intro-duce potential novel sites elsewhere in p53 or somehow
facilitate phosphorylation of Ser9 and Thr18
The absence of Ser9/Thr18 phosphorylation in p531)65
suggests that the overall conformation of p53 or
determi-nants beyond the N-terminal 65 amino acids are important
for phosphorylation at Ser9 and Thr18 These results also
suggest that the conformation change induced by the
binding of p53 to ssDNA and DNA ends facilitates the
presentation of Ser9 and Thr18 in a manner that makes
them attractive substrates for DNA-PK This may be
mediated in part by the core domain of p53 which although
insufficient, has been shown to be required for
sequence-independent binding [33] Alternatively, a second possibility
is that full-length p53 becomes involved in a protein–protein
interaction with DNA-PK that promotes p53 [41]
While p53 has been known to interact with linear and
ssDNA for several years, the functional implications of this
activity have been uncertain Binding of p53 to ssDNA is
known to stimulate sequence-specific DNA binding and may
play a role in promoting tetramerization of the protein [27]
Our present and previous results [30] show that
colocaliza-tion of p53 and DNA-PK to such DNAs promote a close to
10-fold enhancement of p53 phosphorylation Thus
colocal-ization of DNA-PK and p53 to DNA would likely be
important for regulation of p53 by DNA-PK in vivo
Phosphorylation was highly specific as several other sites
in the N-terminus of p53, including Ser6 and Ser20 were not
recognized by DNA-PK Phosphorylation of Thr18
appeared to be preferred to phosphorylation at Ser9 in vitro,
as it was the only additional site detected at limiting ATP
concentrations It is interesting that in human p53wt, Ser9
follows a Pro residue, as it has been suggested previously
that such Pro-Ser/Thr might in fact form a variant
consensus site for DNA-PK [42] By contrast the two
additional Ser-Gln dipeptides in p53, at amino acids 99–100
and 166–167 were not recognized in full-length p53, as
assessed by a lack of a shift in fragment A migration (data
not shown), even though a peptide containing Ser99 is
recognized by DNA-PK [43] This apparent discrepancy
reiterates how important the molecular environment of the
target site is in determining the specificity of the kinase
DNA-PK is not the only candidate kinase for
phos-phorylation of p53 at Ser9 and Thr18 Previous work has
shown that casein kinase 1 also has the potential to
phosphorylate p53 at Ser9 and Thr18 [44,45] For casein
kinase 1, phosphorylation of p53 at Ser9 and Thr18 was
dependent on prior phosphorylation of Ser6 and Ser15
Phosphorylation was also readily observed with N-terminal
peptides of p53 By contrast, phosphorylation of Ser9 and
Thr18 by DNA-PK was dependent on full-length p53 but
was independent of phosphorylation at other sites in p53 It
was also independent of the addition of IC 261, a specific
casein kinase 1 inhibitor [46] The checkpoint kinases Chk1
and Chk2 have also been associated with phosphorylation
at several N- and C-terminal sites of p53 in vitro including
Ser20 [47] Here again DNA-PK differs as no phosphory-lation of Ser20 was detected in our assay Of significant interest, Chk1 directly stimulates the ability of DNA-PK to phosphorylate p53 [48] While the authors focused most of their study on a truncated version of p53 and did not evaluate the stimulation in the presence of ssDNA, it will be interesting to evaluate the impact of Chk1 on the specificity
of DNA-PK toward full-length p53
The gatekeeper function of p53 depends principally on its ability to monitor progression of cells through the cell cycle, and to induce cell cycle arrest or direct a cell towards apoptosis in response to a variety of stresses [12] Numer-ous reports have demonstrated that phosphorylation of N-terminal domain of p53 is essential to the accumulation
of p53 and potentiates p53 acetylation and its transactiva-tion functransactiva-tion [49] Identificatransactiva-tion of the kinases involved
in vivohas been challenging and it has become obvious that there is currently no simple one site-one kinase model to fit the experimental evidence Rather, p53 phosphorylation probably involves a complex network of kinases whose interactions between themselves and p53 depend upon the exact nature of the stress and the cell type involved For instance while Chk1 and Chk2 were long held as kinases acting immediately upstream of p53, two recent reports have questioned their implications in p53 stabilization, at least in certain cancer cells, and it has been suggested that a yet-to-be identified kinase(s) is(are) involved instead [50,51] Currently, several lines of evidence point to a role of DNA-PK in the apoptotic branch of the p53 pathway Indeed, activation of DNA-PK in response to ionizing radiation is directly linked to the activation of the latent cellular population of p53 that directs cells towards DNA damage-induced apoptosis [16] Further, the presence of shortened telomeres that result from telomerase deficiency fail to induce apoptosis in the absence of DNA-PKcs [52] Thus despite the overlap between the large PI3K-like kinases in their ability to phosphorylate p53 in vitro, p53 phosphorylation by DNA-PK might occur under appro-priate circumstances Achanta et al has provided evidence that DNA-PK may also play an important role in the p53-dependent induction of apoptosis that follows nucleoside-induced arrest of DNA synthesis [41] They also showed that p53 and DNA-PK colocalize in the nuclei of nucleo-side-treated cells and could be coimmunoprecipitated Our results offer the intriguing possibility that the accumulation
of stalled replication intermediates, which contain ssDNA regions, may directly facilitate the phosphorylation of p53
by DNA-PK [53]
In conclusion, our results broaden the previously recog-nized specificity of DNA-PK towards p53 to include two new sites, Ser9 and Thr18 It will be important to next determine whether DNA-PK plays a role in mediating the phosphorylation of these sites in response to dsDNA breaks and to explore whether the action of DNA-PK on p53 occurs in response to other forms of cellular stress, such as replication blocks induced by nucleoside analogues or topoisomerase poisons Given the similarities in substrate selection by DNA-PK, ATM and ATR, it will also be interesting to assess whether Ser9 and Thr18 can also be targeted by these kinases, particularly because Ser15 phos-phorylation in vivo is not required to mediate cell cycle regulation following ionizing radiation [54]
Trang 8We are grateful to Dr Lambert (National Institutes of Health,
Bethesda, Maryland) for providing plasmids encoding p53 wt , p53 S15A ,
p53 S37A and p53 S15A/S37A mutants as GST fusion proteins This work
was supported by a grant from the Canadian Institutes for Health
Research to RJGH SS was supported by a fellowship from Canadian
Institutes for Health Research while RJGH is an Investigator of the
Canadian Institutes for Health Research.
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