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J. Biol. Chem., Vol. 278, Issue 32, 29940-29947, August 8, 2003
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From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524
Received for publication, February 19, 2003 , and in revised form, April 21, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The tumor suppressors p53 and Atm and the product of the essential gene ATR are checkpoint genes of primary importance in mammals. The Atm protein is required for several aspects of the response to DNA damage, including cell cycle arrest and the kinetics of p53 activation (4, 5). Atm, Atr, and DNA-dependent protein kinase (DNA-PK)1 are members of a family of proteins that shows homology to phosphatidylinositol-3-kinases. This family includes the checkpoint proteins Mec1 and Tel1 from Saccharomyces cerevisiae, and rad3 from Schizosaccharomyces pombe. Protein kinase activity has been attributed to the members of the Atm family despite their homology to phosphatidylinositol 3-kinase family members (6–9). The mammalian Atm-like kinases have overlapping substrate specificities (8–14), yet they appear to control different aspects of the response to DNA damage. The mammalian Atm family member that has been best characterized by biochemical methods is DNA-PKcs (for review, see Ref. 15). DNA-PKcs is the catalytic subunit of a protein complex that plays a role in double strand break repair. DNA-PK is activated by single-stranded DNA (16) and double strand breaks (17). Although the catalytic subunit has been shown to bind DNA on its own, the heterodimeric protein complex Ku70·Ku80 acts as the DNA binding partner of DNA-PKcs. Thus, the DNA-PK complex is composed of DNA-PKcs and Ku70·Ku80 (15). DNA-PKcs-deficient mice display abnormalities in V(D)J recombination, a process that is essential for the rearrangement of genes to generate functional immunoglobulin and T-cell receptor genes (15). Thus, DNA-PKcs-deficient animals display severe immune deficiencies, and DNA-PK(–/–) cells display increased sensitivity to ionizing radiation and double strand break repair defects. This is despite the fact that these cells are proficient in cell cycle arrest following DNA damage and in the up-regulation of DNA damage-inducible genes (reviewed in Ref. 15). These findings suggested that either DNA-PKcs has a redundant role with Atm and Atr in the checkpoint response or the role of DNA-PKcs is specific for repair of the DNA lesions.
Chk1 (checkpoint kinase 1) was first identified in S. pombe because of its essential role for checkpoint arrest at G2/M (18, 19). In S. pombe and S. cerevisiae, the Chk1 proteins function downstream of the Atm-like proteins to regulate the checkpoint response that prevents chromosome segregation after DNA damage. In mammalian cells and S. pombe, Chk1 blocks mitotic progression via inactivation of the mitotic inducer Cdc25 (20–22).
A primary integrator of checkpoint and stress signals is the product of the tumor suppressor gene p53. p53 regulates many aspects of the DNA damage response, including cell cycle arrest, the transcriptional response, and apoptosis (reviewed in Ref. 23). Therefore, p53 function is highly regulated by different forms of post-translational modifications that include phosphorylation on multiple regulatory residues (23). Atm, Atr, and DNA-PK phosphorylate p53 on serine residues (Ser-15 by Atm, Atr, and DNA-PK and Ser-37 by DNA-PK and Atr) that have been shown to be phosphorylated in vivo following DNA damage (8–10, 12, 24). Phosphorylation of Ser-15 and -37 may be important for modulating the stability and activity of p53 (10). It has also been shown that Ser-20 plays an important role in the stabilization of p53 following DNA damage (25–27). Recently it was shown that the checkpoint kinases Chk1 and Chk2 (hCds1) phosphorylate p53 on Ser-20 (28–30).
In this study, we investigated 1) the regulatory interaction between Chk1 and the members of the Atm family of kinases and 2) the functional role of the C-terminal non-catalytic domains of Chk1. We show that Chk1 was found in complexes that bind single-stranded DNA and double strand breaks and stimulated the kinase activity of DNA-PK complexes in vitro. Stimulation of DNA-PK resulted in enhanced phosphorylation of p53 on Ser-15 and -37 and required the C-terminal domains of Chk1. Chk1 stimulated DNA end joining in vitro, and Chk1 mutants lacking the C-terminal 115 amino acids acted in a dominant fashion to block DNA-PK kinase activity and DNA repair.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning, Mutagenesis, DNA, and Protein Purification—The GFP·Chk1 and GFP·Chk1KD plasmids were generated by excising Chk1 and Chk1KD cDNAs from PYS43 and PYS64, respectively, with KpnI and XmaI and ligating them to KpnI- and BspEI-cut pEYFB vector (Clontech). The GST·Chk1 C-terminal deletion mutants were generated by excising a BamHI fragment from pYS43 and pYS65 (20) and from the EYFP·Chk1 constructs. Site-directed mutagenesis was performed using the QuikChange kit (Stratagene) following the manufacturer's instructions. GST fusion proteins were expressed in Escherichia coli (BL21). Soluble proteins were concentrated using glutathione-agarose beads, or the fusion proteins were expressed in Hi5 cells and collected as previously described (20).
Immunoprecipitation, Kinase Assays, and
Pull-downs—Immunoprecipitation/kinase assays were performed as
described (8). Cells were lysed
in TGN buffer (50 mM Tris, pH 7.5, 50 mM
-glycerophosphate, 150 mM NaCl, 1% Tween, 1 mM
NaF, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 1 mM DTT, 1x protein inhibitor
mixture (20), and 10%
glycerol). Immunoprecipitation was performed in TGN buffer without glycerol.
Kinase assays were performed in kinase buffer (10 mM Hepes, pH 7.5,
50 mM -glycerophosphate, 50 mM NaCl, 10
mM MgCl2, 10 mM MnCl2, 5
µM ATP, and 1 mM DTT. GST pull-downs were conducted
as described previously
(20).
In Vitro Kinase Assays—Twenty units of DNA-PK (Promega) were
preincubated with either Chk1 or Chk1KD (40–80 pmol) in
kinase buffer (see above) containing 200 ng of sonicated calf thymus DNA, 15
µCi of [
-32P]ATP, 200 µM ATP, 1.3
mM spermidine, and 1 mM DTT. The preincubation step was
carried out at 30 °C for 15 min. The reactions were placed on ice, and
GST·p53 substrate was added. The reactions were then incubated at 30
°C for 30 min. Kinase reactions were terminated by the addition of
SDS-sample buffer with 50 mM DTT as a final concentration and
boiled. Proteins were separated by SDS-PAGE on 4–12% acrylamide gels.
Where indicated, the GST·Chk1 proteins were pretreated with 10 units of
DNase I prior to being used in the kinase reaction.
Western Blotting, Data Quantification, and Analysis—Western blot analysis was performed as previously described (20). Chk1 was detected with antibodies generated against a C-terminal peptide of Chk1 (anti-PEP antibodies; Ref. 20). p53 was detected with DO-1 (Santa Cruz Biotechnology). Phosphorylated Ser-15 and -20 on p53 were detected with phospho-specific antibodies (New England Biolabs). 32P-labeledproteins were visualized by autoradiography, and their relative densities were quantified using phosphorimaging (Molecular Dynamics) and ImageQuant. The enhanced chemiluminescence signals were quantified from exposures in the linear range using NIH image 1.6.
DNA Binding and Immunoprecipitation Assays—Nuclear extracts and DNA cellulose binding assays were carried out as previously described (31) except that the nuclear extracts were dialyzed against buffer Z (31) containing 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, and 1x protein inhibitor mixture (20). 12 µg of nuclear extract were incubated with DNA cellulose for 15 min at 30 °C in complete kinase buffer in a 50-µl volume. After preincubation, p53 was added and incubated an additional 30 min. For the DNA end binding assays, 50 pmol of each 78-mer oligonucleotide (one oligonucleotide was biotinylated) were annealed and incubated with 25 µl of streptavidin magnetic beads according to the manufacturer's instructions (Dynal). The DNA end binding assay was carried out by incubating 30–80 µg of nuclear extract with either annealed oligos bound to magnetic beads or magnetic beads alone as control and incubated at room temperature for 45 min-1 h in kinase buffer. ATP was added to the reaction buffer (20 µM) where indicated. The beads were washed eight times with kinase buffer and the proteins separated on 4–12% gradient gels (Invitrogen) and processed for Western analyses as above. The immunoprecipitation assays were carried out with 1 mg/ml nuclear extract in buffer Z containing 20 micromolar ATP and protease inhibitors as above. The extracts were precleared with protein A-Sepharose beads and incubated with 2 µg of antibody at 4 °C overnight. The proteins were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences) using the following antibodies: Chk1 anti-PEP antibody (20) or Santa Cruz Biotechnology (C19 or G4); DNA-PKcs (Ab1) and (Ab2), Oncogene Research; Ku70 (M19), Santa Cruz Biotechnology.
Immunoprecipitation of FLAG·Chk1 from 293T cells was carried out by first enriching for chromatin-associated proteins. 293T cells transfected with either CMV-FLAG·Chk1 or pCDNA3.1 were treated or not with 5 mM HU for 18 h, collected and washed with phosphate-buffered saline, and resuspended in solution A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, protease inhibitors). Cytoplasmic proteins were separated from nuclei by low-speed centrifugation (4,800 rpm for 4 min at 4 °C using the St-Micro rotor, Sorvall Super T21). Isolated nuclei were washed with solution A, resuspended in solution A containing 1 mM CaCl2 and 150 units of micrococcal nuclease (Sigma), and incubated at 4° for 1 h before stopping by adding EGTA 1 mM as final concentration. Sodium chloride was added to 300 mM final concentration and incubated for 10 min on ice. The nuclear preparation was then sonicated twice for 15 s and cleared by centrifugation at 13,200 rpm (same rotor as above) for 15 min at 4 °C. The extracts were diluted in Buffer Z and precleared with Protein A-Sepharose followed by incubation with FLAG-affinity resin at 4 °C overnight. The proteins were precipitated by gentle centrifugation, washed extensively, and separated on a 4–12% gradient SDS-PAGE gels.
DNA Repair Assays—Extracts were prepared and used for
end-joining assays as previously described
(32) with the following
modifications. 10 ng of pDEA-7Z digested with BsaI were incubated
with 15 µg of total cell extract in a 10-µl volume for 90 min at 37
°C. GST and GST·Chk1
proteins were added at approximately
equal concentrations at the beginning of the reaction. The reactions were
treated with 10 µg of Proteinase K for 30 min at 37 °C followed by
phenol chloroform extraction. The deproteinated DNA products were separated on
0.7% agarose gels containing 1 µg/ml ethidium bromide, transferred to
nitrocellulose, hybridized with a 32P-labeled RsaI
fragment from pDEA-7Z, and visualized by autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used phosphorylation-defective p53 mutated proteins and anti-phospho-specific antibodies to determine the residues phosphorylated by DNA-PK and Chk1. Mutation at Ser-37, a site known to be phosphorylated by both DNA-PK and Chk1, resulted in considerable loss of the enhanced phosphorylation of p53 mediated by DNA-PK and Chk1 (see Fig. 1, c–f). However, phosphorylation of this mutated p53 was still enhanced when both DNA-PK and Chk1 were present (Fig. 1f). These data suggest that DNA-PK and Chk1 show synergistic activity toward more than one site on p53. Phosphorylation of Ser-15 was slightly reduced in the mutated Ser-37 (Fig. 1c, lane 4), and the mutation of Ser-15 caused a mild reduction of 32P incorporation into GST·p53 (1–80). Mutations at both Ser-15 and -37 in GST·p53 (1–80) resulted in undetectable phosphorylation by the kinases whether they were present alone or together (Fig. 1e, last lane). These results indicate that DNA-PK and Chk1 act synergistically to phosphorylate p53 on serines 15 and 37, with Ser-37 being the major phospho-acceptor of the enhanced phosphorylation observed in these assays. Recently it has been shown that phosphorylation of Ser-20 and not Ser-15 regulates stability of p53 following DNA damage (27). In our assays, mutation of Ser-20 also decreased the enhanced levels of 32P incorporation and phospho-Ser-15 as measured by antibody staining. This reduction may indicate that Ser-20 is required for Ser-15 or -37 to be phosphorylated, or it may be that a change in the protein at residue 20 affects the adjacent residues and indirectly affects Ser-15 and -37 phosphorylation.
To rule out that the DNA-PK protein fraction (Promega) contained other p53 kinases that could be enhanced by the recombinant Chk1, such as Atr, the proteins in the DNA-PK fraction (20-fold of the amount used in each reaction or 400 units) were analyzed by immunoblotting for the presence of DNA-PK, Atr, Atm, replication protein A (Rpa) 2, and Chk1. Although we observed a strong signal for DNA-PK and Ku80, we did not detect Atm, Atr, or Chk1 signals in the fraction used as a source of DNA-PK/Ku in our assays (Supplementary Fig. 2).3 However, the possibility exists that other p53 kinases are present at such low levels that they are below the level of detection by the antibodies used. Atm does not phosphorylate p53 on Ser-37, which is the major phospho-acceptor residue in our enhancement assay, indicating that it is not Atm that is being activated in our assay.
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The C-terminal Domain of Chk1 but Not Its Catalytic Activity Is
Required for the Stimulation of DNA-PK—To address the direction of
signaling between Chk1 and DNA-PK, we found that addition of wortmannin (a
phosphatidylinositol kinase inhibitor) during or after the preincubation step
resulted in a dramatic reduction in p53 phosphorylation (data not shown),
whereas wortmannin had no effect on phosphorylation of p53 by Chk1. This
suggested that Chk1 was enhancing the phosphorylation of p53 by DNA-PK. To
further investigate the role of Chk1 in this synergistic interaction, we
tested a catalytically defective Chk1 protein (Chk1KDD130A)
(20) for its ability to act
synergistically with DNA-PK. We found that Chk1KD had a comparable
ability to that of wild-type Chk1 in the stimulation of p53 phosphorylation by
DNA-PK (Fig. 2). This effect
was observed whether we used the first 80 amino acids of p53
(Fig. 2b) or the
full-length p53 protein (Fig.
2a) fused to GST as the substrate. These results
indicated that the role of Chk1 is not dependent on catalytic activity and
suggested that Chk1 could be exerting a conformational change on the DNA-PK
complex. The Chk1 kinases from different species share conserved motifs in
their C-terminal domain (Ref.
20). The high conservation
between the Chk1 kinases suggests that they may be regulated by common
mechanisms and that the C-terminal tail is likely to contain regulatory
domain(s). To address whether regions outside the kinase domain were required
for the effect on DNA-PK activity, we generated mutants of Chk1 that lacked
the C-terminal 115 amino acids in both the catalytically proficient and
catalytically defective mutant of Chk1
(Fig. 2, b and
c). Deletion of the C-terminal region of Chk1 abrogated
the enhancement of p53 phosphorylation by DNA-PK
(Fig. 2c, compare
lanes 2 and 4, 3 and 5). In fact, the
GST·Chk1 mutant, which retained some catalytic activity as
measured by phosphorylation of
Cdc25C,4 inhibited the
DNA-dependent phosphorylation of p53 by DNA-PK at the concentrations tested
(Fig. 2c, compare
lanes 1 and 4).
Thus Chk1 stimulates the kinase activity of DNA-PK via its C-terminal domains. Although the role of DNA-PK in p53 phosphorylation in vivo is not clear, our data suggest that high levels of Chk1 or the catalytically inactive Chk1D130A (Chk1KD) mutant could stimulate the kinase activity of DNA-PK.
Chk1 Binds DNA and Double Strand Ends and This Interaction Does Not Require the Catalytic Activity of DNA-PK— Although we did not detect binding of Chk1 purified from bacterial cells to double-stranded oligonucleotides by gel retardation assays under phosphorylation permissive conditions (see above), the regulatory interactions between DNA-PK complexes and Chk1 suggested that Chk1 may interact with these proteins in the presence of DNA. To investigate whether Chk1 may complex with proteins bound to DNA, we examined the ability of Chk1 from nuclear extracts prepared from the glioblastoma cell lines MO59J and MO69K to be precipitated using DNA cellulose beads under phosphorylation-permissive conditions. Chk1 from nuclear extracts bound to the DNA cellulose beads in a DNA-PKcs- and ATM-independent manner (Fig. 3a). Because DNA cellulose beads contain both double- and single-stranded DNA, we next determined the form of DNA bound by Chk1-containing complexes by performing the binding reactions with biotinylated double- or single-stranded 78-mers bound to magnetic streptavidin beads (Dynal). Chk1 from MO59J (lacking functional DNA-PKcs) and MO59K (functional DNA-PKcs) glioblastoma cells was precipitated with both DNA cellulose (data not shown) and DNA ends (Fig. 3b), indicating that the catalytic activity of the DNA-PK was not required for Chk1 to interact with both forms of DNA. We also observed that the interaction of Chk1 with dsDNA cellulose did not require Atm, a checkpoint kinase that has been shown to bind DNA (35).5 Ku70 also bound the double-stranded oligonucleotides in the absence of functional DNA-PKcs. Chk1 from the same extracts was also precipitated by single-stranded oligonucleotides bound to streptavidin beads (Fig. 3b). Furthermore, the interaction of Chk1 from nuclear extracts derived from MO59J cells with DNA was increased after treatment with the replication inhibitor HU (Fig. 3b, lanes 11 and 12, 13 and 14). These results indicate that Chk1 complexes with proteins that bind strand breaks and single-stranded DNA and suggest a potential mechanism of Chk1 activation and/or possible role in the processing of DNA damage lesions. Although binding of Chk1 to DNA did not require the catalytic activity of the DNAPK, we cannot rule out the possibility that the DNA binding activity of Chk1 could be mediated by the catalytically inactive DNA-PKcs in MO59J cells and/or enhanced by other proteins involved in DNA strand break or ssDNA recognition or processing, such as the Ku70, Ku86, or Rpa. As previously documented, although DNA-PK and Ku bound ssDNA they preferentially interacted with double-stranded DNA (Fig. 3b) (16, 36).
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Chk1 and Ku70 Are in the Same Protein Complexes in Vivo— The interaction between Chk1 and DNA-PKcs could also be detected by pull-down analyses with GST·Chk1. GST·Chk1 can complex with DNA-PKcs from HeLa cell nuclear extracts prepared from cells treated with HU (Fig. 4a). To determine whether the in vitro interactions we observed between Chk1 and proteins from the DNA-PK complex were occurring in vivo, we analyzed the proteins present in Chk1 and Ku70 complexes by co-immunoprecipitation assays. Nuclear extracts prepared from HeLa cells were incubated under phosphorylation-permissive conditions with antibodies against Chk1 or Ku70 (Fig. 4b). The complexes were precipitated with protein A-Sepharose beads and subjected to Western analyses using antibodies against Chk1 and Ku70. Ku70 co-precipitated with Chk1 (Fig. 4b, lane 2), and Chk1 was present in the Ku70 immunocomplexes (Fig. 4b, lane 5). Ku80 was also observed in the Chk1 and Ku70 complexes (Fig. 4C and data not shown). To determine whether the interaction between Chk1 and Ku could be enhanced in S phase and/or by a checkpoint signal, we also performed the immunoprecipitation reactions using extracts from HeLa cells that had been treated with 5 mM HU for 24 h. Although we detected phosphorylated Chk1 in the complexes precipitated by the Chk1 antibody, we did not observe an increase in the amount of Ku70 present in the Chk1 complexes (Fig. 4b, compare lanes 2 and 3). Chk1 and Ku have many roles in the cell, which predicts, in turn, that they would have many different partners. Thus, it is not surprising that only a small amount of Chk1 associates with Ku. This is not different from the observation that only a small fraction of those proteins are found in a complex at any given time, despite the abundance of Ku70/80 and DNA-PKcs in the cell. One possible explanation is that the DNA lesion itself stimulates assembly of proteins on DNA breaks, and as such we would not expect to identify a large number of these complexes in the cell. We have recently observed that Chk1 proteins associate with chromatin (37). We carried out immunoprecipitation experiments from chromatin-enriched nuclear extracts prepared from 293T cells expressing FLAG·Chk1. Using this approach, we observed co-immunoprecipitation of Ku80 with FLAG·Chk1, and this association was not changed following treatment with HU (Fig. 4c). Thus, we conclude that Chk1 and Ku70/Ku86 are present in the same protein complex, supporting the regulatory interactions we had observed between these proteins in vitro and suggest a role for this complex in signaling and/or repair.
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Chk1 Stimulates DNA-PK-dependent End Joining by Whole Cell
Extracts—The regulation of the kinase and DNA binding activities of
the DNA-PK complex, which is involved in DNA repair, by Chk1 suggested that
Chk1 would also enhance the DNA-PK-mediated end joining of double strand
breaks. We next examined whether Chk1 could have an effect on DNA-PK-dependent
DNA end processing and whether this effect required the C-terminal domains
(contained in the last 115 amino acids) of Chk1. For this, we examined the
ability of extracts to process 5'-overhangs generated by restriction
digest of plasmid DNA (pDEA-7Z) with the enzyme BsaI
(32). To visualize the DNA
fragments, they were transferred to nitrocellulose and hybridized with
-32P-labeled 1.1-kb RsaI probe derived from
pDEA-7Z. We detected the appearance of a labeled product of the correct
molecular weight for a dimer
(32) and higher molecular
weight products when the DNA was incubated with cell extracts. The processing
of the DNA ends was dependent on the DNA-PKcs because we observed
little to no processing when the DNA was incubated with extracts from MO59J
cells (Fig. 5a,
DNA-PKcs–). To show that the factor missing in
the MO59J required for this reaction was the DNAPKcs, we
reconstituted the processing reactions in the extracts from MO59J cells by
addition of purified DNA-PK fraction (Promega)
(Fig. 5a). We then
chose a concentration of DNA-PK that would restore low levels of end joining
to extracts from MO59J cells (50 units) and tested whether Chk1 could
stimulate the end-joining activity under those conditions. Addition of Chk1
had a synergistic effect with DNA-PK on end joining of both cohesive and blunt
ends (Fig. 5b) when
DNA-PK was added to the MO59J extracts, further supporting the interaction
between the checkpoint kinase and the proteins regulating DNA end joining. To
examine the role of C-terminal domains of Chk1 in stimulating DNA-PK-dependent
end joining, we examined whether addition of Chk1 C-terminal to the
reaction had any effect on end processing in extracts from MO59K (DNAPK+)
cells. Chk1 protein blocked processing of the DNA ends in a
dose-dependent manner, whereas addition of molar excess amounts of GST had
little effect on the reaction (Fig.
5c). These results support our model derived from
previous experiments indicating a functional interaction between Chk1 and the
proteins in the DNA-PK complex and the importance of the C terminus of Chk1
for this interaction.
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Cells with defects in the DNA-PKcs display a sensitive phenotype
to ionizing radiation (38,
39). We examined the effect of
expression of Chk1 or Chk1 proteins on the appearance of cells with
abnormal, pignotic, or fragmented nuclei following treatment with ionizing
radiation (Fig. 6). We observed
an 
2-fold increase in the number of cells with pignotic or fragmented DNA
6 h following treatment with 10 Gy of ionizing radiation in HeLa cells
expressing Chk1 compared with cells expressing Chk1 or an empty vector.
This phenotype could be explained if the cells failed in the checkpoint
response, as has been documented for conditional embryonic stem cells after
removal of the remaining wild-type Chk1 allele
(40). The phenotype could also
be because of a defect in DNA repair. When the cells expressing Chk1
were treated with nocodazole after ionizing radiation, we did not observe an
increase in the mitotic index of these cells 8 h after
radiation.6 These
results suggest that the effect of Chk1 on the radiosensitivity of HeLa
cells is not because of a defect in the G2/M checkpoint-induced
cell cycle arrest.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are two major pathways that regulate the repair of double strand breaks in mammalian cells. The pathway that mediates recombination between homologous sequences is regulated by proteins, including Rad51, -52, -54, -55, and -57 (42). The pathway that mediates the joining of non-homologous sequences (NHEJ) is regulated by the DNA-PK/Ku complex, Xrcc4, DNA ligase IV, Rad50, and nucleases such as Mre11 and Fen1 (reviewed in Refs. 43 and 44). Rad50 and Mre11, which form a complex involved in processing of double strand breaks, have also been implicated in checkpoint signaling in yeast and mammals (42, 45, 46). Extensive biochemical and genetic studies have implicated DNA-PK in recombination and double strand break repair, yet little is known about the regulation of this complex or about the signaling pathways that dictate the mechanism that the cell uses to repair DNA lesions. There is evidence for a requirement of DNA-PK in lesion repair during G1/S and S phases of the cell cycle. Additionally, DNA-PK function is required to stop DNA replication in vitro following UV radiation (47).
We reconstituted DNA damage signaling in vitro with DNA-PK complexes in order to address the regulatory and substrate relationship between Chk1 and this member of the Atm family of kinases. During these studies, we determined that Chk1 activates the DNA binding and kinase activities of the DNA-PK/Ku complex. We show that Chk1 stimulates DNA-PK to phosphorylate p53 on Ser-15 and Ser-37, with Ser-37 being the major phospho-acceptor. Although the role of DNA-PKcs in the regulation of p53 remains controversial (10, 24, 48–50), the data that are used to argue that DNA-PK does not phosphorylate p53 in vivo are derived from studies carried out with cells lacking DNA-PKcs. The only conclusion that could be reached from those studies was that DNA-PKcs is not required for such signaling to take place. However, those studies do not rule out the possibility that DNA-PKcs participates in signaling to p53. There are three kinases of the ATM family that phosphorylate p53 in vitro: Atm, Atr, and DNA-PK, which could argue for overlapping roles or redundancy. Chk1 also phosphorylates p53 on Ser-15 and -37 (30) and Chk1 and Chk2 phosphorylate Ser-20 (27, 29, 30). The studies described here used p53 as readout to determine that Chk1 stimulated the kinase activity of DNA-PK in vitro. In addition, we describe an interaction between Chk1 and DNA-PK that results in the phosphorylation of p53 on residues that are not phosphorylated by Atm or Chk2 (Ser-37) and that are postulated to regulate the transcriptional role of p53 or be involved in the protein stability following ultraviolet radiation (51–53).
Evidence to support the regulation of DNA-PK complexes came from studies that showed that interaction of DNA-PK with Rpa or Ku is regulated by DNA damage during S phase and involves the DNA-PK-dependent phosphorylation of the p34 subunit of Rpa (54). The damage-induced activation of DNA-PK in cells, which leads to the phosphorylation of p34, is abrogated by 7-hydroxystaurosporine (UCN-01), a drug that inactivates the G2/M checkpoint and has been shown to inactivate Chk1 but not DNA-PK in vitro (54–56). Furthermore, the activation of DNA-PK by Chk1 also leads to enhanced phosphorylation of p34.7
Unlike the catalytic activity, the C-terminal domains of Chk1 are required to enhance kinase activity of DNA-PK. A dominant deletion mutant of Chk1 that fails to activate DNA-PK also blocks DNA-PK-dependent processing of double strand breaks in vitro, corroborating an interaction between the proteins that process the lesions and Chk1. It is of interest to note that deletion of the C-terminal 115 amino acids of Chk1 does not result in increased kinase activity.8 In this regard our mutant behaves differently from other published C-terminal deletion mutants that display high kinase activity. The reason for the different results could be that, unlike other deletion mutants (30, 57), we only removed 115 amino acids from the C terminus. It is possible that our mutated protein contains regulatory domains that block promiscuous interaction of the catalytic domain with all substrates.
Chk1 Complexes at DNA—We speculate that the proteins that mediate Chk1 binding to dsDNA and ssDNA are different. However, the possibility exists that the double-stranded DNA is processed by helicases and nucleases in the extract to generate ssDNA regions that are then bound by proteins that target Chk1 to ssDNA. We detected small amounts of Rpa2, a component of the Rpa single-stranded DNA binding complex in the dsDNA pull-downs, suggesting that this could be the case.9 There are several mechanisms that could lead to a double strand break during DNA replication that would require immediate checkpoint signaling and repair. Examples include a single strand nick in the leading or lagging strand template or at a stalled replication fork. We propose that proteins such as the Ku and Rpa complexes are involved in the decision of the cell to repair a lesion that is encountered during DNA replication and that Chk1 could play a regulatory role in this process as previously suggested (16, 54). Whether the activation of DNA-PK acts as an amplification signal to increase the signaling to p53 to signal repair or apoptosis (50) and whether this synergism results in increased repair of double strand breaks in vivo remains to be determined.
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FOOTNOTES |
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The on-line version of this article (available at
http://www.jbc.org)
contains two supplementary figures.
Both authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of Molecular Genetics,
Biochemistry, and Microbiology, University of Cincinnati College of Medicine,
231 Albert Sabin Way, Cincinnati, OH 45267-0524. Tel.: 513-558-3275; Fax:
513-558-8474; E-mail:
Yolanda.Sanchez{at}uc.edu.
1 The abbreviations used are: DNA-PK, DNA-dependent protein kinase;
PKcs, PK catalytic subunit; Chk1, checkpoint kinase 1;
Chk1KD, CHK1 kinase dead; HU, hydroxyurea; DTT, dithiothreitol;
GST, glutathione S-transferase; ssDNA, single-stranded DNA; dsDNA,
double-stranded DNA; Rpa, replication protein A; EYFP, enhanced yellow
fluorescent protein; GFP, green fluorescent protein.
2 D. M. Goudelock and Y. Sanchez, unpublished data.
3 D. M. Goudelock and Y. Sanchez, unpublished results.
4 D. M. Goudelock and Y. Sanchez, unpublished data.
5 D. M. Goudelock and Y. Sanchez, unpublished results.
6 K. Jiang and Y. Sanchez, data not shown.
7 D. M. Goudelock and Y. Sanchez, unpublished results.
8 D. M. Goudelock and Y. Sanchez, unpublished results.
9 D. M. Goudelock and Y. Sanchez, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CiteULike 
Complore 
Connotea 
DNA as a molecular weight marker. The
products were transferred to nitrocellulose and probed with a
32P-labeled fragment of pDEA-7Z. a, reconstitution of end
joining in MO59J extracts. DNA-PK complexes (10 and 150 units) were added to
extracts from MO59J, and the joining of BsaI-cut plasmid was
monitored by Southern analyses. b, Chk1 stimulates end-joining
reactions. Bacterially produced GST·Chk1 was added with or without 50
units of DNA-PK complexes to the reaction to determine the effect on formation
of dimers in extracts from M059J cells (5 and 15 µg of extract for
reactions using cohesive or blunt ends, respectively). The labeled DNA added
to the reaction was digested with either BsaI or SmaI to
determine the effect of Chk1 on ligation of cohesive or blunt ends,
respectively. c, Chk1