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EMBO reports 4, 8, 787–792 (2003)
doi:10.1038/sj.embor.embor901
Published online: 1 August 2003
Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain
Ashley Craig1, Mary Scott2, Lindsay Burch1, Graeme Smith3, Kathryn Ball2 & Ted Hupp1
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1 Department of Molecular and Cellular Pathology,
Cancer Research UK Laboratories, University of Dundee, Dundee
DD1 9SY, UK
2 Department of Surgery and Molecular Oncology,
Cancer Research UK Laboratories, University of Dundee, Dundee
DD1 9SY, UK
3 KuDOS Pharmaceuticals Limited,
Milton Road, Cambridge CB4 0WG,
UK
To whom correspondence should be addressed
Ted Hupp Tel: +44 1382 496430; Fax: +44 1382 633952;
t.r.hupp@dundee.ac.uk
Received 26 February 2003; Accepted 11 June 2003; Published online 1 August 2003.
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Abstract
The tumour suppressor p53 is a tetrameric protein that is
phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2
(CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide
fragments of p53 containing the BOX-I motif, indicating that undefined
determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived
from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation
of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites.
CHK2 can be fully activated in trans by the two p53 DNA-binding-domain
peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53
at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is
predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase
activity that is predominantly activated in trans towards Ser 20. Cell
division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53
DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the
smallest of the two CHK2 binding sites, and mutating certain amino acids in the
BOX-V peptide prevents CHK2 activation. A database search identified a p53
BOX-I-homology motif in p21WAF1 and although CHK2 is inactive
towards this protein, the p53 DNA-binding-domain peptides induce
phosphorylation of p21WAF1 at Ser 146. This provides evidence
that CHK2 can be activated allosterically towards some substrates by a novel
docking interaction, and identify a potential regulatory switch that may
channel CHK2 into distinct signalling pathways in vivo.
EMBO reports 4, 8, 787–792 (2003)
doi:10.1038/sj.embor.embor901
Published online: 1 August 2003
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Introduction
The tumour-suppressor protein p53 is activated as a transcription
factor in response to a variety of genotoxic and metabolic stresses, resulting
in either cell-cycle arrest or apoptosis (Vogelstein et
al., 2000). Well-characterized gene products that mediate the
tumour-suppressor function of p53 include the cyclin kinase inhibitor
p21WAF1 and the pro-apoptotic BCL2 antagonist BAX. Several
functional domains of p53 are involved in promoting transactivation, including
an amino-terminal activation domain that binds p300, a core sequence-specific
DNA-binding domain, a tetramerization domain and a carboxy-terminal regulatory
domain, the phosphorylation and acetylation of which regulates p53-dependent
transcription. DNA-damage-activated protein kinases phosphorylate up to three
clustered sites in the BOX-I transactivation domain of p53. These protein
kinases form part of the evolutionarily conserved ataxia-telangiectasia-mutated
(ATM)–checkpoint kinase 2 (CHK2) DNA-damage signalling cascades that
activate p53 (Wahl & Carr, 2001). CHK2 can
phosphorylate p53 at Thr 18 or Ser 20 (Shieh et al.,
2000). Phosphorylation at the Thr 18 site attenuates MDM2 (mouse
double minute 2) binding to p53 (Craig et al.,
1999; Schon et al., 2002) and
relieves p53 from negative control by MDM2. In addition, phosphorylation at Thr
18 or Ser 20 by CHK2 stabilizes p300 binding to the LxxLL activation domain of
p53, and promotes DNA-dependent acetylation of p53 by p300 (Dornan et al., 2003). Therefore, phosphorylation of
the p53 activation domain seems to act as a switch to convert p53 from an
MDM2-binding protein to a p300-binding protein, leading to enhanced
DNA-dependent acetylation of p53 at promoters (Lane &
Hupp, 2003).
The role of CHK2 in modifying the p53 pathway is controversial.
Original studies indicated that CHK2 forms stable complexes with p53 in cells
(Falck et al., 2001), deletion of
CHK2 reduces the specific activity of p53 (Wu et
al., 2002), and CHK2 and p53 cooperate in the DNA-damage response
(Hirao et al., 2000; Takai et al., 2002). Sequestration of CHK2 to
spatially restricted nuclear regions impairs its ability to stimulate p53
activity (Lukas et al., 2003). However,
neither mutation of the murine p53 Chk2 site nor deletion of Chk2 alter
p53 stabilization after damage (Takai et al.,
2002; Wu et al., 2002). The CHK2
phosphorylation site at Ser 20 is one of the least conserved sites on p53, the
transactivation domain of p53 does not have a consensus CHK2 phosphorylation
site, and the p53 activation-domain fragment is not a CHK2 substrate (O'Neill et al., 2002), questioning whether CHK2 is a
p53 kinase. In this report, we have reconstituted the minimal components
required for CHK2 to function as a p53 kinase, and we resolve the issue of how
CHK2 can target a protein that does not have a CHK2 consensus phosphorylation
site. The phosphorylation of p53 by CHK2 requires kinase docking to a
protein–protein interaction module in the DNA-binding domain of p53.
These data have led to the identification of an 'activated' CHK2 consensus-site
that expands the types of potential CHK2 substrates and highlights the growing
realization that protein-kinase docking sites can be an important component of
kinase specificity (Biondi & Nebreda,
2003).
Results
Peptide consensus sites for CHK2 that have homology to CDC25, but not
to the BOX-I activation domain of p53, have been developed (Fig.
1A). Small peptides from the p53 activation domain were not
phosphorylated by CHK2, but CHK2 was an effective kinase for tetrameric p53,
and phosphorylated Thr 18 and Ser 20 (Fig. 1B). These
data suggest that CHK2 is activated towards p53 by other determinants in the
tetramer. Overlapping peptides from p53 (Fig. 1C) were
assayed for CHK2 protein binding to identify such CHK2-docking sites. Two
high-affinity CHK2 binding regions were identified in the BOX-II (peptide 5)
and BOX-V (peptide 18) domains of p53, with a more localized binding site
present in the BOX-V domain (Fig. 1C). If CHK2 requires
docking to these two sites in the DNA-binding domain of p53 to promote
BOX-I-domain phosphorylation, then the peptides might competitively inhibit
CHK2 phosphorylation. Surprisingly, however, both of the BOX-II-domain and
BOX-V-domain peptides stimulated, rather than inhibited, phosphorylation of p53
at Ser 20 (Fig. 1D, compare lane 2 with lanes 5 and 8).
The BOX-I peptide that contained the phosphorylation site for CHK2 did not
inhibit p53 phosphorylation (Fig. 1D, compare lanes 2 and
3), consistent with the fact that this small peptide is not phosphorylated by
CHK2 (O'Neill et al., 2002; see below). The
Thr 18 kinase activity of CHK2 was also stimulated by the BOX-II-domain and
BOX-V-domain peptides (Fig. 1E, compare lane 1 with lanes
2 and 3). Using [32P ]ATP as a substrate, the
BOX-II-domain and BOX-V-domain peptides stimulated phosphorylation of p53
(Fig. 1F, compare lane 1 with lanes 2 and 3, lower band)
under conditions in which CHK2 autophosphorylation was unaffected (Fig. 1F, lanes 1–3, upper band). These data indicate that
the kinase activity of CHK2 is stimulated by the BOX-II-domain and BOX-V-domain
peptides. The human CHK2 used in these assays was purified from insect cells
and the enzyme was highly phosphorylated at the ATM-activating site of Thr 68,
indicating that CHK2 was in an activated conformation (see
supplementary information
online).
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Figure 1
Checkpoint kinase 2 docking to the DNA-binding domain of p53.
(A) Low homology between p53 and checkpoint kinase 2 (CHK2) substrates.
Consensus phosphorylation sites have been defined for both CHK1 and CHK2
(CHK1-tide and CHK2-tide; O'Neill et al.,
2002). The homology of these consensus-site peptides to cell division
cycle 25 (CDC25) and p53 are as indicated. The key residues required for p300
and MDM2 (mouse double minute 2) binding to the p53 BOX-I domain (Dornan et al., 2003) are shown in blue. Green shading
indicates homolgous sites, red shading indicates phosphoacceptor sites.
(B) CHK2 modifies two sites on p53. An immunochemical assay using
phospho-specific monoclonal antibodies to the BOX-I phosphorylation sites
(Craig et al., 1999) was used to assay CHK2.
The addition of CHK2 to reactions with p53 tetramers (compare lane 1 with lane
2) results in the specific phosphorylation of p53 at Ser 20 and Thr 18, but not
at Ser 15. (C) Two peptides derived from the core domain of p53 bind to
CHK2. (Ca) Overlapping peptides derived from p53 were tested for binding
to CHK2. The five conserved BOX domains of p53 are shown in black. The CHK2
phosphorylation sites are in the BOX-I domain, whereas the CHK2-docking
peptides are in the BOX-II and BOX-V domains. (Cb) Direct binding of
CHK2 was tested for all p53 peptides, and relative binding of CHK2 to the
BOX-II and BOX-V peptides in an ELISA (enzyme-linked immunosorbent assay)
format (ELISA method as in Dornan et al.,
2003) is shown as relative light units (RLUs). (D,E)
The BOX-II-domain and BOX-V-domain peptides stimulate CHK2 activity. Kinase
reactions containing p53 were assembled without CHK2 (lane 1) or with CHK2
(lanes 2–9). The indicated overlapping peptides from the BOX-I, BOX-II
and BOX-V domains were added, and reaction products were analysed for p53
phosphorylation at Ser 20 (D) and Thr 18 (E) using an
immunochemical blotting assay. (F) The BOX-II-domain and BOX-V-domain
peptides stimulate CHK2 activity. Kinase reactions containing
[32P]ATP, full-length p53 and CHK2 were assembled without
peptide (lane 1) or with the indicated peptides from BOX-II and BOX-V (lanes 2
and 3). Reaction products were analysed for p53 phosphorylation after
electrophoresis by autoradiography. The upper band shows CHK2
autophosphorylation and the lower band shows p53 phosphorylation. (G)
The minimal adjacent locations of the BOX-II-domain and BOX-V-domain peptides
(in the S2' and H2 motifs) within the core domain of p53 are shown in
green. (H) The entire BOX-II and BOX-V peptide sequences that bind CHK2
(C) extend further into the core domain and reside within the same plane
(shown in red, including part of the S10  -sheet, the S2 and S2'
-sheets and the helix H2).
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The BOX-II-domain and BOX-V-domain peptides are not contiguous in the
primary sequence of p53 (Fig. 1C), but reside on the same
face in the crystal structure of p53, which may form the basis for a
three-dimensional docking site for CHK2 (Fig. 1G,H).
Protein kinases, including phosphoinositide-dependent protein kinase 1 (PDK1),
mitogen-activated protein kinase (MAPK), and glycogen synthase kinase 3 (GSK3)
also have high-affinity docking sites that are distinct from the substrate
phosphorylation site (Biondi & Nebreda, 2003).
Two mechanisms may account for CHK2 stimulation after binding the BOX-II-domain
and BOX-V-domain peptides (Fig. 2A): first, CHK2 docking
to a high-affinity site might tether the kinase to p53, allowing it to
phosphorylate a low-affinity, non-consensus phosphorylation site. Second, CHK2
might be activated as a p53 kinase after docking by an allosteric or
induced-fit mechanism. The effect of these CHK2-docking peptides in
trans on the phosphorylation of a small glutathione-S-transferase
(GST)-tagged N-terminal fragment of p53 (amino acids 1–66;
p53N1–66) that lacks the CHK2-docking sites was examined
(Fig. 2B). A [32P]ATP kinase assay
showed some basal CHK2-dependent 32P incorporation into
p53N1–66 (Fig. 2B, lane 1). However,
the inclusion of BOX-II-domain or BOX-V-domain peptides stimulated the
phosphorylation of p53N1–66 (Fig.
2B, compare lane 1 with lanes 2 and 3). Under these conditions, the
basal autophosphorylation of CHK2 was unaffected (Fig.
2B, compare lane 1 with lanes 2 and 3; CHK2 band). The inclusion of both
CHK2-docking peptides together induced stoichiometric phosphorylation of
p53N1–66 by CHK2 (Fig. 2C, compare
lanes 3 and 4 with lane 5), without altering the basal autophosphorylation of
CHK2 (Fig. 2C, compare lane 1 with lane 5, CHK2 band).
This multi-peptide addition effectively reconstituted CHK2 activity towards a
p53 fragment that lacks the CHK2-docking sites, and indicates that allosteric
effects mediate CHK2 phosphorylation of p53 (Fig. 2A,
model 2). The specific activity of CHK2 and 'peptide-activated' CHK2 was tested
towards its classic substrate, CDC25C. A [32P]ATP kinase
assay showed equivalent phosphorylation of CDC25C by either CHK2 (Fig. 2D, lane 2) or 'peptide-activated' CHK2 (Fig. 2D, compare lane 2 with lanes 3–5).
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Figure 2
Checkpoint kinase 2 is activated in trans by p53
DNA-binding-domain peptides. (A) Two models account for the stimulation
of checkpoint kinase 2 (CHK2) by the BOX-II-domain and BOX-V-domain peptides.
According to the anchoring model (model 1), the BOX-II-domain and BOX-V-domain
peptides only anchor CHK2 and should have no effect in trans on CHK2
activity towards the p53 fragments. Alternatively, according to the allosteric
model (model 2), the BOX-II-domain and BOX-V-domain peptides can activate CHK2
towards the p53 activation-domain fragment that lacks the CHK2-core domain
sites. (B) The BOX-II-domain and BOX-V-domain peptides activate CHK2.
Kinase reactions containing [32P]ATP,
p53N1–66 and CHK2 were assembled without peptide (lane 1)
or with the indicated peptides from BOX-II and BOX-V (lanes 2 and 3). Reaction
products were analysed after electrophoresis by autoradiography for
p53N1–66 phosphorylation and CHK2 autophosphorylation. The
upper band shows CHK2 autophosphorylation and the lower band shows
p53N1–66 phosphorylation. (C) The BOX-II-domain and
BOX-V-domain peptides synergistically activate CHK2. Kinase reactions contained
[32P]ATP and CHK2 only (lane 1), or contained
[32P]ATP, CHK2 and p53N1–66 either
without peptide (lane 2), with the indicated peptides from BOX-II and BOX-V
(lanes 3 and 4), or with both peptides from BOX-II and BOX-V (lane 5). Reaction
products were analysed after electrophoresis by autoradiography for
p53N1–66 phosphorylation (lower band) and for CHK2
autophosphorylation (upper band). (D) The BOX-II-doman and BOX-V-domain
peptides do not affect CHK2 phosphorylation of CDC25. Kinase reactions
contained [32P]ATP and CHK2 only (lane 1), or contained
[32P]ATP, CHK2 and CDC25C either without peptide (lane
2), with the indicated peptides from BOX-II and BOX-V (lanes 3 and 4), or with
both peptides from BOX-II and BOX-V (lane 5). Reaction products were analysed
for CDC25 phosphorylation (lower band) and CHK2 autophosphorylation (upper
band) after electrophoresis by autoradiography. (E) The BOX-II-domain
and BOX-V-domain peptides activate CHK2 phosphorylation of p53 at Thr 18.
Kinase reactions containing p53N1–66 and CHK2 were
assembled without peptide (lanes 1 and 4) or with peptides (lanes 2, 3, 5 and
6). Reaction products were analysed for p53N1–66
phosphorylation at Ser 20 (lanes 1–3) or Thr 18 (lanes 4–6) using
an immunochemical blotting assay. Protein band 'a' is unphosphorylated
p53N1–66 or p53N1–66 phosphorylated
at Ser 20, whereas protein band 'b' is a kinase supershift that indicates Thr
18 phosphorylation. (F) The BOX-II-domain and BOX-V-domain peptides
activate CHK2 phosphorylation of p53(S15A)N1–66 at Ser 20
and Thr 18. Kinase reactions containing p53(S15A)N1–66 and
CHK2 were assembled without peptide (lanes 1 and 4) or with peptides (lanes 2,
3, 5 and 6). Reaction products were analysed for p53 phosphorylation at Ser 20
(lanes 1–3) or Thr 18 (lanes 4–6) phosphorylation using an
immunochemical blotting assay. (G) Mutation of the BOX-V peptide
attenuates its activity as a CHK2 activator. Reactions were assembled with
p53N1–66, [32P]ATP, CHK2 and the
BOX-V or BOX-II peptides, as indicated in the figure. BOX-V peptides containing
alanine mutations at the indicated codons (270–277) were added, and the
relative phosphorylation of p53 and CHK2 is indicated.
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The activation of CHK2 by BOX-II-domain and BOX-V-domain peptides
towards p53N1–66 was examined using the immunochemical Ser
20 and Thr 18 phosphorylation assay. CHK2 showed basal Ser 20 kinase activity
(Fig. 2E, lane 1), whereas no residual activity was
detected towards Thr 18 (Fig. 2E, lane 4). The BOX-II and
BOX-V peptides stimulated Ser 20 phosphorylation (Fig.
2E, lanes 2 and 3) and activated Thr 18 kinase activity (Fig. 2E, lanes 5 and 6). A conformational change induced by
activated CHK2 on p53N1–66 phosphorylation at Thr 18 is
inferred by the mobility shift from band 'a' to band 'b' in Fig.
2E. Together with the results from the 32P kinase assay
above, these data indicate that the predominant site of phosphorylation by
activated CHK2 is at Thr 18. Although there is no contaminating Ser 15 kinase
in CHK2 (Fig. 1B), p53(S15A)N1–66
was also used as a substrate, as this eliminates any contribution of residual
Ser 15 phosphorylation in driving Thr 18 phosphorylation. The BOX-II and BOX-V
peptides activated CHK2 towards both the Ser 20 and Thr 18 sites of p53
(protein band 'b' in Fig. 2F; compare lanes 2 and 3, and
5 and 6, respectively, with lanes 1 and 4). However, the Ser 15 residue seems
to be important for CHK2 phosphorylation at Ser 20, as CHK2 has no basal Ser 20
kinase activity towards p53(S15A)N1–66. CHK2
immunoprecipitated from irradiated human cells is modified on Thr 68 (see
supplementary information online)
and can also be activated towards the Thr 18 site of
p53N1–66 by the BOX-V peptide (see
supplementary information online),
indicating that wild-type CHK2 has biochemical characteristics similar to
recombinant CHK2.
The related enzyme CHK1 was also tested for the presence of a
p53-docking activity to determine whether the allosteric effector site could be
expanded to a related CHK2 family member (see
supplementary information online).
CHK1 shows constitutive Thr 18 kinase activity towards
p53N1–66 or p53(S15A)N1–66, whereas
CHK1 is not active as a Ser 20 kinase on p53 fragments unless bound by the
BOX-II or BOX-V peptides. These data indicate that the allosteric effect of the
p53 DNA-binding domain peptides can be extended to two
calcium–calmodulin-kinase superfamily members: CHK2 has basal Ser 20
kinase activity and is predominantly activated towards Thr 18, whereas CHK1 has
constitutive Thr 18 kinase activity and is predominantly activated towards Ser
20.
As the BOX-V-domain peptide is the most potent CHK2 and CHK1 activator
under limiting conditions (Fig. 2F; and see
supplementary information online)
and represents a relatively small CHK2-docking interface (Fig.
1C), the effects of individual amino-acid mutations on the BOX-V-peptide
activation of CHK2 were examined. The mutation of amino acids to alanine at
positions 270, 272, 273, 274 and 277 in the S10 -sheet (Fig. 2G, compare lane 4 and lanes 6–8 with lane 1)
attenuated CHK2 activation by the BOX-V peptide. This region is adjacent to the
binding site of the RNA-MDM2 isoform (Shimizu et al.,
2002) and suggests that this domain may be a multiprotein-binding
site. Although mutation of the BOX-V docking sites of CHK2 prevents p53
phosphorylation (Fig. 2G), mutating single amino acids on
full-length p53 did not block phosphorylation by CHK2 (data not shown),
presumably because of the relatively large docking interface of CHK2 on p53
(Fig. 1H).
The ability of CHK2 to alter its phosphorylation activity depending on
enzyme docking opens the door to identifying novel BOX-I-domain-like CHK2
substrates. CHK2 cannot phosphorylate the minimal BOX-I-domain peptide (amino
acids 12–27; Fig. 3B, left bar), but the specific
activity of CHK2 was increased by more than 16-fold by the inclusion of
BOX-II-domain and/or BOX-V-domain peptides (Fig. 3B,
right bars). These data indicate that CHK2 can be stimulated towards a minimal
BOX-I-peptide motif. A database search for proteins with homology to the p53
BOX-I domain has identified the cyclin-dependent kinase (CDK) inhibitor
p21WAF1 (Fig. 3A). This region of
p21WAF1 is a regulatory domain, the phosphorylation of which at
Ser 146 by atypical protein kinase Cs (PKCs) can regulate the half-life of the
protein in insulin-stimulated cells (Scott et al.,
2002). The Ser 146 residue of p21WAF1 and the Thr 18
position of p53 are equivalent in the homology line-up. We examined whether
CHK2 was inactive towards p21WAF1 and whether BOX-II-domain and
BOX-V-domain peptides activated CHK2 towards the Ser 146 site of
p21WAF1. Although CHK2 activity was latent towards
p21WAF1 protein (Fig. 3C, lane 2), the
BOX-II and BOX-V peptides activated CHK2 towards p21WAF1 (Fig. 3C, compare lane 2 with lanes 3 and 4). The combined
addition of both BOX-II-domain and BOX-V-domain peptides synergistically
activated CHK2 towards p21WAF1 (Fig. 3C,
compare lanes 3 and 4 with lane 5). Thus, proteins with BOX-I-homology-domain
motifs can be CHK2 substrates after docking-mediated kinase activation.
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Figure 3
p53-core-domain peptides induce checkpoint-kinase-2-dependent
phosphorylation of p21WAF1. (A) Homology of the p53
BOX-I-homology domain to p21WAF1. A database search of proteins
with homology to the BOX-I domain of p53 identified p21WAF1. The
Thr 18 and Ser 20 phosphoacceptor sites of p53 (bold) correspond to the Ser 146
and Thr 148 phosphoacceptor sites of p21WAF1. (B) The
BOX-II-domain and BOX-V-domain peptides activate checkpoint kinase 2 (CHK2)
phosphorylation of a BOX-I domain peptide. Kinase reactions contained the p53
BOX-I peptide, [32P]ATP and CHK2 (all bars), either
without CHK2-docking peptides (left bar) or with the BOX-II-domain and
BOX-V-domain peptides, individually or together (II + V; right bars). Reaction
products were spotted onto filter paper; kinase activity is represented as
picomoles of 32P incorporation (Scott et
al., 2002). (C) CHK2 activation towards
p21WAF1. The BOX-II-domain and BOX-V-domain peptides
synergistically activate CHK2 phosphorylation of p21WAF1. Kinase
reactions contained [32P]ATP and CHK2 only (lane 1), or
[32P]ATP, CHK2 and p21WAF1 with or without
the indicated peptides from BOX-II and BOX-V (lanes 2–5). Reaction
products were analysed after electrophoresis by autoradiography for
p21WAF1 phosphorylation (Scott et al.,
2002).
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Conclusion
The substrates of protein kinases have classically been defined by the
ability of a small fragment derived from the full-length protein to serve as an
effective substrate in a phosphotransferase reaction. However, growing evidence
indicates that protein kinases have high-affinity docking sites that catalyse
substrate phosphorylation. Such docking sites have been observed previously for
CDK2 (Luciani et al., 2000), PDK1, MAPK and
GSK3 (Biondi & Nebreda, 2003). The docking site
for a protein kinase consists of a small, linear sequence of amino acids that
forms the basis for an anchoring motif that can control substrate
phosphorylation. A docking site usually has no sequence homology to the
substrate of the kinase, can be attached to the substrate itself or on an
adaptor protein, and can itself be a site of phosphorylation. CHK2 action after
DNA damage is related, at least in part, to the direct phosphorylation of p53.
However, p53 does not have a canonical CHK2 phosphorylation site (O'Neill et al., 2002). The identification of a
CHK2-docking site in p53 identifies an allosteric mechanism for CHK2 that may
have widespread significance for understanding CHK2 kinase function, substrate
specificity determination and cell-cycle checkpoint regulation.
Methods
Reagents, proteins, antibodies and enzyme assays.
All chemicals were from Sigma unless otherwise indicated. Tetrameric
forms of p53 were expressed as previously reported (Luciani
et al., 2000). The CHK2 and CHK1 complementary
DNAs were a gift from S. Elledge. The p53N1–66 fusion
protein (GST-tagged) was generated by amplifying the cDNA encoding the
N-terminal 66 amino acids of p53 and subcloning this into the
isopropyl--D-thiogalactoside (IPTG)-inducible expression vector pGEX-2TK
(Pharmacia). CHK2 and CHK1 were cloned into pFASTBAC-HTb (which
contains a His6 tag) and His-tagged CHK2 and CHK1 were expressed in
and purified from Sf9 cells using Ni2+ agarose and ion-exchange
chromatography. Peptides derived from the p53 coding region were obtained from
Chiron Mimitopes. p53 tetramer or peptide kinase assays were carried out as
described previously (Luciani et al., 2000),
adding empirically determined amounts of CHK2 and p53 (400 ng),
p53N1–66 and p53(S15A)N1–66 (1  g)
or CDC25 (1 g). Immunoblotting for Thr 18 and Ser 20 phosphorylation was
performed using the monoclonal antibodies FPS18 and FPS20 (Craig et al., 1999). Antibodies to CHK2 and the
Thr-68-modified CHK2 were obtained from Santa Cruz Biotechnology. p21 protein
was purified as indicated and the Ser-146-phospho-specific antibody was used as
a probe for p21 phosphorylation (Scott et al.,
2002).
Supplementary information is available at EMBO reports
online (http://www.emboreports.org).
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Acknowledgements
K.L.B. is supported by a Cancer Research UK Senior Fellowship and by
the Association for International Cancer Research. T.R.H. is supported by a
Programme Grant from Cancer Research UK, a UK Medical Research Council Career
Establishment Grant, and the Association for International Cancer Research.
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