Stability of Checkpoint Kinase 2 Is Regulated via Phosphorylation at Serine 456*

Checkpoint kinase 2 (Chk2), a DNA damage-activated protein kinase, is phosphorylated at Thr-68 by ataxia telangiectasia mutated leading to its activation by phosphorylation at several additional sites. Using mass spectrometry we identified a new Chk2 phosphorylation site at Ser-456. We show that phosphorylation of Ser-456 plays a role in the regulation of Chk2 stability particularly after DNA damage. Mutation of Ser-456 to alanine results in hyperubiquitination of Chk2 and dramatically reduced Chk2 stability. Furthermore, cells expressing S456A Chk2 show a reduction in the apoptotic response to DNA damage. These findings suggest a mechanism for stabilization of Chk2 in response to DNA damage via phosphorylation at Ser-456 and proteasome-dependent turnover of Chk2 protein via dephosphorylation of the same residue.

The human Chk2 protein is 543 amino acids in length and includes three important domains (Fig. 1A). An SQ/TQ cluster domain containing five SQ and two TQ motifs encompasses residues 19 -69 at the N terminus and includes Thr-68, the most well characterized site of phosphorylation by ATM (2,(23)(24)(25). The forkhead homology-associated (FHA) domain (residues 112-175) likely modulates protein-protein interactions in trans as well as potential interactions with other domains of the protein in cis (6,26). Two clinically significant mutations occur in this region, R145W and I157T (27,28). The Chk2 kinase domain is within the C terminus of the protein extending from residue 220 to 486 (25).
In cells that have not been exposed to DNA damage, Chk2 exists as a monomer (4,26). The Chk2 FHA domain, which consists of an 11-strand ␤-sandwich, forms a phospho-threonine binding module (29). In response to DNA damage, the region surrounding phosphorylated Thr-68 in one Chk2 molecule binds in trans to the FHA domain of another Chk2 molecule resulting in dimerization (5,6,26,30).
Many questions remain regarding how Chk2 is activated in response to different types of DNA damage. Genetic studies indicate that IR-induced Chk2 kinase activity is reduced in ATM-deficient cells (2,14,31). In vivo studies have shown that mutation of Thr-68 to alanine significantly reduces IR-stimulated Chk2 kinase activity (24). However, the T68A mutation does not completely abolish IR-induced Chk2 activity suggesting that optimal activation of Chk2 is a more complex process requiring multiple phosphorylation events, including autophosphorylation at Thr-383 and Thr-387 and Ser-516 (5). The latter site, Ser-516, was identified as an autophosphorylation site, which, when mutated, results in reduced Chk2 kinase activity and a dramatic decrease in ionizing radiation-induced apoptosis in cells expressing an S516A mutant (5,7).
In mammalian cells Chk2 is a stable protein that is expressed throughout the cell cycle (32). Aberrantly low levels of Chk2 protein have been found in subsets of human breast carcinomas (33,34), testicular tumors (35), and lymphomas (36). It is thought that the underlying cause for these low levels of Chk2 protein are somatic mutations or polymorphic variants of Chk2, which make the altered Chk2 proteins less stable and more likely to be degraded.
Heterozygous germ line mutations have been observed in a subset of patients with Li Fraumeni syndrome, a familial cancer predisposition syndrome typically associated with a germ line mutation in TP53 (27). The CHK2 mutations identified in Li Fraumeni syndrome kindred include a truncation mutation, 1100delC, in which the kinase domain is disrupted and therefore has no kinase activity and a missense mutation within the FHA domain, R145W, that leads to rapid degradation of the Chk2 protein and thereby reduced expression and reduced kinase activity (6,27,28). CHK2 germ line mutations have also been linked to familial breast, colon, and prostate cancer syndromes, and CHK2 somatic mutations have been identified in breast, colon, bladder, lung, and prostate cancers, further suggesting that CHK2 may act as a tumor suppressor gene (37).
In an effort to identify new biologically relevant phosphorylation sites in Chk2, we used MALDI-TOF mass spectrometry to map several candidate phosphorylation sites on Chk2. Here we show that a novel phosphorylation site in the Chk2 kinase domain, Ser-456, is important for stabilization of Chk2 protein in response to DNA damage and dephosphorylation of this site may facilitate Chk2 turnover in a proteasome-dependent manner.

EXPERIMENTAL PROCEDURES
Mass Spectrometry-C-terminally FLAG-tagged Chk2 wildtype (WT) protein was immunopurified from recombinant baculovirus-infected sf9 insect cells as described previously (4,11). Immunopurified Chk2-FLAG was concentrated to 0.2 mg/ml with Centricon YM-30 (Millipore) and loaded onto 24 ml of Superdex 200 (GE Healthcare) equilibrated with buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 20% glycerol, and 1 mM dithiothreitol to fractionate dimeric and monomeric forms of Chk2 (4). After the peak (ϳ5 g) of each dimer and monomer fraction was concentrated to 10 l with Centricon YM-30, proteins were denatured with 6 M guanidine hydrochloride at 37°C for 30 min. The SH group of proteins was reduced with 100 mM dithiothreitol, and the buffer was exchanged with 50 mM NH 4 HCO 3 . The sample was treated with 300 mM iodoacetamide at room temperature for 1 h, and the buffer was exchanged with 50 mM NH 4 HCO 3 again. After concentrating the sample to ϳ20 l, 300 ng of sequencinggrade trypsin (Promega) was added, and the mixture was incubated at 37°C for overnight. To enrich the phosphopeptides in the tryptic digest, the sample was bound to and eluted from Zip-Tip MC (Millipore) immobilized with Fe 3ϩ ions according to the manufacturer's protocol.
Reflective MALDI mass spectra of the tryptic digest of proteins were recorded on an Applied Biosystems (Forster City, CA) Voyager delayed extraction time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3-ns pulse). The accelerating voltage was 16 kV, the delay time was 200 ns, the grid voltage was set to 80% of the accelerating voltage, and the guide wire voltage was set to 0.03% of the accelerating voltage. The spectrum was an average of 256 laser shots. Typically, the peptide mixture (1 l) was mixed with 1 l of matrix (10 mg/ml of ␣-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid) and spotted onto a 100-well plate. Phosphopeptides were identified by comparing expected peptide masses of tryptic digestion of Chk2 (MS-Digest program) with observed masses. After phosphopeptides were identified, the sample spot on a 100-well plate was phosphatase-treated by applying 1 l of 0.1 unit of calf intestine phosphatase (New England Biolabs) in 100 mM NH 4 HCO 3 and incubated at 37°C for 30 min. The spot was allowed to dry out, and 1 l of matrix was applied before the mass spectrum was retaken.
Expression Plasmids, Mammalian Cell Lines, and Culture Conditions-A mammalian expression plasmid encoding HA-Chk2 was kindly provided by Dr. Junjie Chen (Mayo Clinic, Rochester, MN). FLAG-tagged WT Chk2 in a pcDNA3 vector has previously been described (38). N-terminally FLAG 3tagged Chk2 was cloned into NotI/XbaI restriction sites in a p3ϫFLAG-CMV7.1 expression vector. The QuikChange II site-directed mutagenesis kit (Stratagene) was used to introduce point mutations encoding S456A, S456D, S456F, T68A, S120A, S260A, S379A, and D347A into the various expression constructs described. A mammalian expression plasmid encoding HA-USP28 was kindly provided by Dr. Stephen Elledge (Harvard Medical School, Boston, MA).
HCT15 colorectal cancer cells were obtained from ATCC and maintained in RPMI 1640 medium with 10% fetal bovine serum. HCT15 cell derivatives expressing WT or D347A kinase-dead (KD) HA-Chk2 were kindly provided by Dr. J. Chen (Mayo Clinic, Rochester, MN). To establish additional stable lines expressing HA-tagged WT and mutant Chk2 proteins, HCT15 cells were transfected with plasmids encoding WT or mutant Chk2 and pcDNA3 as a neo marker. Clones were selected in 800 g/ml G418, and stable cell lines were maintained in RPMI 1640 medium with 10% fetal bovine serum supplemented with 400 g/ml G418. H1299 lung carcinoma and HEK 293 (transformed human embryonic kidney) cell lines were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. To establish 293 stable lines expressing FLAG 3 -tagged WT and mutant Chk2, cells were transfected with plasmids encoding WT or mutant Chk2 and pcDNA3 as a neo marker. Clones were selected in 600 g/ml G418, and 293 FLAG 3 -Chk2 stable cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum supplemented with 400 g/ml G418. For plasmid transfection, cells were plated in 6-well dishes at Ͼ90% confluence and transfected with 4 g of DNA and 5 l of Lipofectamine 2000 (Invitrogen) per well. When indicated, cells were treated with the radiomimetic compound neocarzinostatin (NCS, 500 ng/ml, Kayaku Co., Tokyo, Japan) for 2 h and/or 50 M MG132 (Calbiochem) for 2-5 h.
Antibodies and Immunoblotting-Cell lysates were prepared as described previously (38). For immunoprecipitations, equal amounts of protein were incubated for 12-16 h at 4°C with the indicated antibodies in the presence of phosphatase inhibitors (Phosphatase Inhibitor Cocktails 1 and 2, Sigma). 10 l of a 50/50 protein A-Sepharose (GE Healthcare) slurry and 20 l of a 50/50 protein G-Sepharose (GE Healthcare) slurry were added for an additional hour before washing extensively in TEGN buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P40, 400 mM NaCl, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture containing 1 M benzamidine, 3 mg/ml leupeptin, 100 mg/ml bacitracin, and 1 mg/ml ␣ 2 -macroglobulin). Bound protein was eluted by boiling in SDS sample buffer. Immunoprecipitates and lysates were separated on 7.5 or 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Pro-tran, Schleicher and Schuell). Membranes were blocked for 1 h in Tris-buffered saline containing 5% nonfat milk powder and 0.1% Tween 20 and then probed with primary antibody overnight in 2.5% milk (or bovine serum albumin for phospho-specific antibodies) and 0.1% Tween 20 Tris-buffered saline. After washing, membranes were incubated for 1 h with horseradish peroxidase-linked secondary antibody (Sigma) in Tris-buffered saline with 0.1% Tween 20. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences). Anti-Chk2 rabbit polyclonal antibody (Protein Sciences) was used at 1:100 for immunoprecipitation and 1:1000 for Western blotting. FLAG-Chk2 and HA-Chk2 fusion proteins were immunoprecipitated with anti-FLAG M2 monoclonal antibody (Sigma) and anti-HA monoclonal antibody (Covance), respectively. Polyclonal antibody to phospho-Chk2 (Ser-456) was produced by immunizing rabbits with a synthetic phospho-peptide (keyhole limpet hemocyanin-coupled) corresponding to residues surrounding Ser-456 in human Chk2. Antiserum was purified using protein A and peptide affinity chromatography. Affinity-purified phospho-specific and nonphospho-specific fractions were collected. The phospho-specific fraction was used at 1:200 for immunoblotting. When indicated transferred nitrocellulose membranes were treated with alkaline phosphatase (calf intestine phosphatase (CIP), 3 units/ml, New England Biolabs) or buffer alone for 16 h at 37°C prior to blocking. Phosphorylated p53 (Ser-20) was detected using a phospho-specific antibody (Cell Signaling Technology), and p53 was detected using the monoclonal antibody 1801.
Half-life Experiments-To determine the half-life of WT and S456A mutant Chk2 proteins, two clones of asynchronously growing HCT15 cells stably expressing HA-tagged WT or S456A mutant Chk2 were treated with 25 g/ml cycloheximide (Sigma) and harvested as described above at 0, 2, 4, 6, and 8 h post treatment. For half-life determination following DNA damage, cells were treated with 500 ng/ml NCS for 2 h and washed with fresh medium prior to cycloheximide treatment. For half-life determination after proteasome inhibition, cells were treated with 50 M MG132 (Calbiochem) for 2 h (in the presence or absence of NCS) and washed with fresh medium prior to cycloheximide treatment. 50 g of whole cell extract for each time point was separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-HA antibody for Chk2 levels, and with anti-actin antibody (Sigma) as a loading control. Densitometric analyses of immunoblots were quantified using Kodak Image software.
Ubiquitination of Chk2-H1299 cells were co-transfected either with plasmids encoding FLAG-Chk2 (wild type or mutants) and HA-ubiquitin or HA-Chk2 (wild type or mutants) and FLAG-ubiquitin. At 24 h post-transfection cells were treated with 50 M MG132 and 500 ng/ml NCS as indicated for 4 h. FLAG-or HA-tagged Chk2 was immunoprecipitated using anti-FLAG M2 or anti-HA monoclonal antibodies, respectively. Immunoprecipitates were subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with anti-FLAG or anti-HA antibody for the FLAG-or HA-tagged ubiquitin. In reciprocal experiments FLAG-ubiquitin was immunoprecipitated, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with anti-HA antibody for HA-Chk2. For detecting ubiquitination of endogenous Chk2, H1299 and RKO cells were transfected with FLAG-ubiquitin, and endogenous Chk2 was immunoprecipitated with anti-Chk2 antibody.
FACS Analysis-Equivalent numbers of 293 or HCT15 parental and stable cell lines were plated in 60-mm dishes. At 24 h after plating, 20 M etoposide (VP16, Calbiochem) was added. After 24 or 48 h (as indicated) cells (floating and attached) were collected and prepared for FACS analysis as previously described (38). Cell cycle profiles were obtained using a BD Biosciences FACSCalibur flow cytometer and CellQuest software.

Identification of Ser-456 as a Chk2 Phosphorylation Site-
Even though phosphorylation at Thr-68 is a prerequisite for the initial dimerization and activation of Chk2, our results suggest that it is not needed for maintenance of Chk2 dimerization or kinase activity (4). Thus, phosphorylation at other regions might be involved in those functions. To identify additional phosphorylation sites on Chk2, tryptic digests of recombinant baculovirus-derived FLAG-tagged human Chk2 were analyzed by reflective MALDI-TOF mass spectrometry, and molecular masses of ions observed in the 700-to 3000-Da range were compared with that of expected tryptic peptides, including phosphorylation at serine and threonine residues. The sequence coverage was ϳ80% of expected masses in the specified molecular weight range. After analyses of all mass peaks, we identified a previously characterized site (Ser-516) as well as four additional prospective phospho-peptides at Ser-120, Ser-260, Ser-379, and Ser-456. Note that Ser-379 and Ser-456 are conserved in both mice and S. cerevisiae (Fig.  1B). The phospho-peptide peaks disappeared (loss of 80 Da) after treatment with CIP showing that Chk2 was phosphorylated at these sites (Fig. 1C, compare the upper panels for Ser-456 and Ser-516 with the lower panels, and data not shown for Ser-120, Ser-260, and Ser-379). Treatment with buffer control did not abolish the phospho-peptide mass peaks (data not shown).
Recently King et al. reported that Ser-120, Ser-260, and Ser-379 are phosphorylated in bacterially expressed Chk2 and are likely novel Chk2 autophosphorylation sites (39). Because Ser-456 had not been previously identified, we chose to further characterize this site.
Chk2 Ser-456 Is Phosphorylated in Human Cells-To confirm and analyze Ser-456 phosphorylation we generated a Chk2 Ser-456 phospho-specific antibody. The phospho-specific antibody (pS456) recognized wild-type ectopic Chk2 that was stably expressed in an HCT15 cell line (WT1, see section below for further description) in the presence and absence of DNA damage ( Fig. 2A). Nevertheless, there was significantly increased recognition by the pS456 antibody 4 -8 h after treatment with NCS versus untreated when compared with total levels of Chk2 protein. Detection by the pS456 antibody was subsequently diminished relative to total Chk2 protein by 16 h post NCS treatment suggesting DNA-damage inducible phosphorylation followed by dephosphorylation at this site ( Fig. 2A). To further our analysis we looked at a 293 cell line (WT-6) stably expressing FLAG 3 -tagged WT Chk2. The pS456 antibody recognized FLAG 3 -WT Chk2 both in the absence and presence of DNA damage with enhanced recognition 4 -6 h after treatment with NCS ( Fig. 2B and data not shown). Furthermore, treatment with alkaline phosphatase (CIP) greatly reduced recognition by the pS456 antibody ( Fig. 2B; compare lanes 7 and 8 with lanes 10 and 11) but did not reduce detection by a preparation of the antibody that had been bound to and eluted from a column consisting of non-phosphorylated Chk2 peptide spanning Ser-456 (Fig. 2B, compare lanes 1 and 2 with lanes  4 and 5). Interestingly, the pS456 antibody recognized kinase-dead D347A Chk2 to an approximately similar extent as WT Chk2 indicating that Ser-456 is not an autophospho-rylation site in vivo (Fig. 2C). As further evidence of its phospho-specificity, the pS456 antibody did not detect S456A or S456F mutant forms of Chk2, but displayed relatively increased recognition of the phospho-mimicking Chk2 mutant S456D (Fig. 2C).
Despite numerous attempts, all preparations of our pS456 antibody had to be used at very high concentrations and could not recognize levels of endogenous Chk2 that are present at even lower levels than ectopically expressed Chk2 in our various stable cell lines. Therefore, it was not possible to determine under what specific conditions endogenous Chk2 is phosphorylated at Ser-456. Nonetheless, both the mass spectrometry data and the facts that the anti-pS456 antibody (a) failed to recognize Chk2 mutated at Ser-456 to A or F; (b) lost reactivity with Chk2 after CIP treatment; and (c) displayed recognition of Chk2 that changed as a function of time after DNA damage have led us to conclude that Ser-456 is a bona fide phosphorylation site on Chk2. We surmise that some aspect of the region surrounding Ser-456 contributes to the difficulty of detecting phosphorylation with various antibody preparations as has been shown previously for p53 (40). Indeed, further analysis by mass spectrometry has revealed that Lys-458, which is within the epitope region recognized by our pS456 phosphospecific antibody, is modified by acetylation (data not shown).
We used limited molecular dynamics simulations to model phosphorylation at Ser-456 based on the recently reported crystal structure of the Chk2 kinase domain (41). The model predicts that phosphoserine 456 is near the surface of the Chk2 protein, where it is readily accessible for modification or interaction with other residues (Fig. 2D). Taken together we conclude that Chk2 can be phosphorylated on Ser-456 in insect and human cells.

WT and S456A Mutant Chk2 Proteins Have Similar Protein
Kinase Activity-The sporadic colorectal cancer cell line HCT15 carries two mutations (R145W and A247D) on two different Chk2 alleles (28). Endogenous Chk2 is virtually undetectable in HCT15 cells (28). Additionally, HCT15 cells show no measurable Chk2 kinase activity before or after DNA damage, thereby making them suitable for examining the properties of ectopically expressed mutant forms of Chk2 (42). To examine Chk2 variants we constructed stable cell lines expressing HA-tagged WT and S456A mutant Chk2 in HCT15 cells. Interestingly, all seven isolated S456A-expressing clones had significantly less total Chk2 protein levels than four clones expressing WT Chk2 (data not shown).
We examined the kinase activity of the HA-Chk2 variants from untreated or NCS-treated HCT15 stable cell extracts using autoradiography to detect both Chk2 autophosphorylation and phosphorylation of GST-Cdc25c-(200 -256) (Fig. 3A, upper panel). Both WT and S456A Chk2 proteins had markedly more kinase activity than D347A kinase-defective Chk2. The kinase activity of HA-S456A mutant Chk2 in two stable HCT15 clones (456-7 and 456-8) after NCS treatment was slightly less than WT HA-Chk2 purified from two HCT15 clones (WT-0 and WT-5). However, because there was comparatively less S456A mutant Chk2 protein this indicated that, on a per mole basis, S456A mutant Chk2 has similar kinase activity to WT Chk2 (Fig. 3A, lower panel) and that phosphorylation at Ser-456 is not required for protein kinase activity. Additionally, immunofluorescence staining revealed that S456A Chk2 localizes to the nucleus in a manner indistinguishable from WT Chk2 before and after DNA damage (Fig.  3B). Relevant to experiments in the rest of this report, these data also indicate that S456A Chk2 is not likely to be globally unfolded, because this mutant is active as a protein kinase.
WT Chk2 Is Stabilized Following DNA Damage in HCT15 Cells; S456A Is Less Affected-That S456A mutant Chk2 was consistently present at lower levels than WT Chk2 led us to investigate the stability of mutant Chk2 protein by performing cycloheximide chase experiments. Cultures of HCT15 cells (two clones each) expressing WT or mutant forms of Chk2 were treated with cycloheximide and harvested at different times after treatment (Fig. 4A). The half-life of WT Chk2 was ϳ3 h in these cells. Mutant Chk2 (S456A) had a significantly shorter half-life, i.e. ϳ2 h, whereas that of the phospho-mimicking S456D mutant, at just under 4 h, was greater than that of wild-type Chk2. To examine whether DNA damage affects the turnover of Chk2 Densitometric analysis was carried out using Kodak Image software. The graph represents the ratio of phosphorylated Chk2 to total Chk2 at each time point. B, 293 cells stably expressing FLAG 3 -tagged WT Chk2 (WT-6) were treated with or without NCS (500 ng/ml) for 4 h then harvested. Whole cell extract (80 g) from each sample was resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were cut into segments (through lanes 3, 6, and 9), which were treated or untreated with CIP for 16 h at 37°C as indicated. Western blots were then probed with anti-pS456 antibody or as a control the non-phospho-specific fraction of anti-pS456 antibody that bound to and was eluted from a non-phosphopeptide column and then re-aligned for ECL detection (top panel). The blot segments were then stripped and re-probed with an anti-FLAG M2 antibody (bottom panel). C, H1299 cells transfected with constructs expressing WT, S456A, S456F, S456D, or D347A versions of Chk2 were treated with NCS (500 ng/ml) for 2 h then harvested. Lysates (600 g) were immunoprecipitated with anti-FLAG M2 antibody, and the immunoprecipitates were resolved by SDS-PAGE and blotted with anti-pS456 antibody (top panel). Note that the blot was cut between S456D (lane 4) and D347A (lane 5) to remove irrelevant lanes. The blot was then stripped and re-probed with anti-FLAG M2 antibody (bottom panel). D, simulation of Chk2 phospho-serine 456 based on the ADP-bound Chk2 kinase domain structure, 2CN5. Minimized average structure (600 -700 ps) from a 1-ns trajectory (n ϭ 3), performed at 37°C (310.15K). The GROMACS 3.3.1 molecular dynamics simulation package was used with the Gromos 43a1p forcefield (user contributed) and spc216 water model. OCTOBER 12, 2007 • VOLUME 282 • NUMBER 41 protein, cultures were pre-treated with NCS for 2 h and then washed before treatment with cycloheximide. Here the half-life of WT Chk2 was increased to Ͼ8 h (similar to the Asp mutant after such treatment), whereas the half-life of S456A Chk2 increased to ϳ4 h (Fig. 4B). Thus, although DNA damage extended the half-life of both wild-type and mutant Chk2 proteins in HCT15 cells, WT Chk2 (or S456D Chk2) was significantly more stabilized than was S456A Chk2 under either condition.

Chk2 Phosphorylation and Protein Stability
In a separate series of experiments we found that levels of HA-tagged WT Chk2 from HCT15 stable cells were increased after treatment with NCS or etoposide compared with protein from untreated cells (Fig. 4C). By contrast, HAtagged S456A mutant Chk2 could not be stabilized by this treatment, and in fact was less stable than S456A Chk2 from untreated cells (Fig. 4C).
To extend these experiments showing differential turnover rates of Chk2 variants, HCT15-stable clones expressing either WT or S456A Chk2 were treated with the proteasome inhibitor MG132 in the presence or absence of NCS for 2 h then cultures were washed before adding cycloheximide. In the presence of MG132 the half-lives of both versions of Chk2 increased: S456A from 2 to 6 h and WT Chk2 from 3 to 6 h (Fig. 5A). In cells treated with the DNA-damaging agents NCS and MG132, the half-life of S456A Chk2 increased from ϳ3 h to Ͼ6 h while that of WT Chk2 was also Ͼ6 h as it is in the absence of MG132 (Fig. 5B). These data indicating that WT and S456A Chk2 proteins are regulated by the proteasome were extended by experiments described below.
WT Chk2 Is Ubiquitinated in Various Cell Lines; S456A Mutant Chk2 Is Hyperubiquitinated-Because S456A Chk2 is both unable to be phosphorylated at Ser-456 and is rapidly turned over, we hypothesized that it might be ubiquitinated to a greater extent than WT Chk2. We transfected FLAG-tagged Chk2 constructs encoding serine to alanine mutants at all four of the phosphorylation sites identified in our study (S120A, S260A, S379A, and S456A; see Fig. 1) as well as WT Chk2 and T68A into H1299 cells along with HA-tagged ubiquitin. Twenty-four hours post transfection the cells were treated concurrently with NCS and MG132 for 4 h. The S456A mutant was strikingly hyperubiquitinated compared with either WT Chk2 or the three other point mutants (Fig. 6A). The hyperubiquitination of the S456A mutant was even greater after MG132 treatment suggesting that this mutant is preferentially targeted for proteasome-mediated degradation compared with WT Chk2 (Fig. 6B). Supporting the likelihood that the observed hyperubiquitination was not simply a result of the alanine mutation, a serine to phenylalanine (S456F) mutation at the site was as hyperubiquitinated as the S456A mutant (Fig. 6C). It is also noteworthy that, when a phospho-mimicking Chk2 mutant (S456D) was used, in the same experiments its ubiquitination pattern was similar to WT Chk2 (Fig. 6, B and C). To rule out possible artifactual results caused by the FLAG epitope tag on the Chk2 protein, the experiment was repeated using HAtagged versions of S456A and wild-type Chk2 co-transfected along with FLAG-tagged ubiquitin. The same hyperubiquitination phenotype was observed under these conditions as well (Fig. 6B). Also the fact that there was less ubiquitination of WT and S456D versions of Chk2 after NCS treatment is consistent with those forms of Chk2 being more stable after DNA damage (Fig. 6B).
In a reciprocal experiment H1299 cells were co-transfected with HA-Chk2 (wild type and mutant) and FLAGubiquitin and immunoprecipitated with anti-FLAG antibody (Fig. 6C). In this case, the FLAG-tagged ubiquitin was immunoprecipitated, and the Western blot was probed with anti-HA antibody to detect HA-Chk2. Multiple ubiquitinated Chk2 polypeptides were observed for the S456A

Chk2 Phosphorylation and Protein Stability
mutant as well as a single species at approximately the size of Chk2 that was also seen with WT Chk2, indicating that the S456A mutant is more capable of binding to ubiquitin and forming polyubiquitin chains than WT Chk2 (Fig. 6C). The hyperubiquitination phenotype of the S456A mutant was observed in several different cell lines, including U2OS and 293 ( Fig. 6D and data not shown). Furthermore, we observed ubiquitination of endogenous Chk2 in H1299 (Fig. 6E) and RKO cell lines (data not shown). Because the deubiquitinating enzyme USP28 has been shown to stabilize Chk2 protein after DNA damage (43), based on our findings we hypothesized that USP28 may play a role in the deubiquitination of Chk2. In fact, ectopically expressed USP28 caused a dramatic loss of ubiquitination of endogenous Chk2 in H1299 cells (Fig. 6F), and USP28 can deubiquitinate overexpressed WT, S456D, and S456A mutant forms of Chk2 (data not shown).
Taken together, our results from the cycloheximide chase and ubiquitination experiments indicate that, in the absence of a DNA damage response, WT Chk2 is turned over more rapidly by the proteasome. Further, we postulate that, in the hours immediately after a DNA damage event has occurred (when induction of phosphorylation at Ser-456 is observed), WT Chk2 is stabilized and is not turned over at its resting frequency.
Reduced Apoptosis in Cells Expressing S456A Chk2-Chk2 activity has been linked to the induction of apoptosis in response to IR or treatment with cytotoxic drugs in several previous studies, and reduced levels of Chk2 protein have been associated with decreased amounts of apoptosis (31,38,43). Because the S456A mutation results in a version of Chk2 protein that is significantly less stable than WT Chk2 after DNA damage, we sought to examine what, if any, downstream effects the S456A mutation might have specifically in regards to the role of Chk2 in apoptosis. Two clones each of FLAG 3 -tagged WTand S456A-expressing cells were treated with etoposide and harvested at subsequent time points. Both S456A clones had significantly smaller populations of cells with sub-G 1 content of DNA even at 24 h (Fig. 7A). The sub-G 1 numbers for both S456A clones were much closer to numbers observed with parental 293 cells than WT expressing clones (Fig. 7A). This same trend was observed in HCT15 cells stably expressing S456A mutant Chk2 versus WT though the difference in raw numbers was more subtle in these cells (supplemental Fig. S1).
Chk2 has been shown to phosphorylate p53 at Ser-20 (10 -12, 14). To study specific downstream effects of the S456A mutation, which may account for the observed lower levels of apoptosis, we examined Ser-20 phosphorylation on p53 in cells stably expressing WT or S456A Chk2 in response to DNA damage. We saw no significant difference in Ser-20 phosphorylation between WT or mutant Chk2 at 6 and 24 h after etoposide treatment (Fig. 7B). This finding is similar to results of kinase assays performed using cdc25c as a substrate and further suggests that S456A Chk2 retains kinase activity despite being significantly less stable than WT Chk2.

DISCUSSION
In this study we report a novel phosphorylation site in the Chk2 kinase domain, Ser-456, which plays an important role in stabilizing Chk2 protein in response to DNA damage. A serine to alanine mutation at this site, S456A, while not affecting kinase activity or subcellular localization, renders Chk2 both hyperubiquitinated and less stable than wild-type Chk2 particularly after DNA damage. Chk2 proteins can be stabilized in the presence of MG132, further suggesting that regulation of phosphorylation at Ser-456 may not only play a role in stabilizing Chk2 but dephosphorylation may represent a mechanism for regulating Chk2 turnover at times when it is no longer necessary to maintain high levels of Chk2 in the cell (for example a return to steady state levels after DNA damage response). This is not the first observation of a mutation in the kinase domain of Chk2 that does not affect kinase activity, but rather protein stability. A CHK2 somatic mutation identified in a lung cancer patient consisting of a valine for aspartic acid substitution at amino acid 311 (D311V) in the kinase domain was shown to have only modestly reduced kinase activity compared with wild type but was observed to be significantly less stable than wild-type Chk2 protein when expressed in various cell types (44,45). The authors of that study suggested that reduced protein expression caused by the D311V mutation and loss of the corresponding wild-type allele may have impeded Chk2 function at the G 1 checkpoint, perhaps contributing to cancer development (45).
It should be noted that reports vary as to whether Chk2 levels are increased or decreased after DNA damage (5,43,46). It was reported that Chk2 protein levels are decreased in HeLa cells after IR (5) and that Chk2 protein is degraded in response to cisplatin treatment in an ATM-independent manner (46). By contrast, Chk2 protein levels are increased after IR treatment of H460 cells (43). Such differences could reflect differences in cell types, DNA damage signals, kinetics, or other factors.
Kinetics data in HCT15 cells showed that induction of phosphorylation at Ser-456 occurs within a few hours following a DNA damage event and lasts until ϳ12 h later, at which point phosphorylation status at this site apparently returns to basal levels. The timing of phosphorylation at this site is closely linked to the approximate change in stability of wild-type Chk2 that was observed in the presence versus absence of DNA damage. Therefore, we hypothesize that phosphorylation of Ser-456 prevents rapid degradation of Chk2. Chk2 dephosphorylated at Ser-456 is instead targeted for degradation in a proteasome dependent manner.
Recently, Zhang et al. (43) reported that the deubiquitinating enzyme USP28 is responsible for stabilization of Chk2 and 53BP1 in response to DNA damage. Our observation, that USP28 deubiquitinates endogenous and overexpressed Chk2 proteins, suggests a mechanism for the stabilization of Chk2 after DNA damage. USP28 has been shown to mediate the stability of other proteins through deubiquitination, including the MYC transcription factor (47). Interestingly USP28 binds to MYC via an interaction with the F-box protein FBW7 (47). As with USP28 and MYC, we were not able to see a direct interaction between USP28 and Chk2. However, it will be very interesting to determine if this interaction is mediated via binding to an as yet unidentified Chk2 E3 ligase.
It is also likely that other, perhaps undiscovered, phosphorylation sites in Chk2 may contribute to its increase in stability following exposure to DNA damage. If phosphorylation at Ser-456 were solely FIGURE 6. S456A mutant Chk2 is hyperubiquitinated compared with WT Chk2. A, S456A mutant Chk2 is more ubiquitinated than WT Chk2 or other phosphorylation site point mutants. H1299 cells were co-transfected with constructs expressing FLAG-tagged WT Chk2 or individual phosphorylation site point mutants as indicated (2.6 g each) and HA-ubiquitin (1.3 g). At 24 h post transfection cells were treated with NCS (500 ng/ml) and MG132 (50 M) for 4 h. Immunoprecipitations were performed with anti-FLAG antibody followed by immunoblotting with anti-HA antibody to detect ubiquitinated proteins. Bottom panel, the blots were stripped and reprobed with anti-FLAG antibody. B, H1299 cells were transfected with constructs expressing HA-tagged versions of WT, S456A, or S456D Chk2 (2.6 g each) and FLAG-ubiquitin (1.3 g). After 24 h cells were treated as indicated with NCS and/or MG132 for 4 h then harvested and immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-FLAG antibody. C, H1299 cells were co-transfected with either FLAG-tagged WT, S456A, S456D, or S456F versions of Chk2 along with HA-ubiquitin or HA-tagged versions of Chk2 with FLAG-ubiquitin as indicated. After 24 h, cells were treated with MG132 (50 M) for 4 h then harvested and immunoprecipitated with anti-FLAG antibody. D, S456A Chk2 is more ubiquitinated than WT Chk2 in 293 stable cell lines. 293 clonally derived cell lines stably expressing FLAG 3 -tagged WT (WT-6) or S456A (456-1) Chk2 proteins were transfected with increasing quantities of HA-ubiquitin (0 -4 g). Drug treatment, immunoprecipitation and immunoblotting were performed as described in A. Bottom panel, the blots were stripped and reprobed with anti-FLAG antibody for FLAG 3 -Chk2. E, endogenous Chk2 is ubiquitinated in H1299 cells. H1299 cells were transfected with or without FLAG-ubiquitin (2 g) and 24 h later were treated with MG132 (50 M) for 4 h. Ubiquitination of Chk2 was analyzed by immunoprecipitation of H1299 lysates (1 mg) with anti-Chk2 antibody and immunoblotting with anti-FLAG antibody for FLAGtagged ubiquitin. F, endogenous Chk2 is deubiquitinated by USP28 in H1299 cells. H1299 cells were transfected for 24 h with (or without) FLAG-ubiquitin (2 g) in the presence or absence of HA-USP28 (3 g). Cells were treated with MG132 for 4 h, and ubiquitination of Chk2 was analyzed as in E. OCTOBER 12, 2007 • VOLUME 282 • NUMBER 41 responsible for the stabilization of Chk2 protein after DNA damage, it would be expected that the phospho-mimicking S456D mutant would be stabilized equally in the presence and absence of DNA damage. In fact S456D, although more stable than WT in the absence of DNA damage, was stabilized significantly more after NCS treatment suggesting that likely other factors or sites may contribute to overall Chk2 protein stability.

Chk2 Phosphorylation and Protein Stability
The idea that Chk2 may be turned over as a means of attenuating a DNA damage signal is supported by examples of other proteins that are degraded by ubiquitin-mediated proteolysis. The Chk1 kinase, which plays an important role in S phase checkpoint signaling in response to genotoxic stress, has been shown to undergo polyubiquitination and proteolytic degradation possibly to promote checkpoint termination (48). Interestingly, Chk1 degradation is triggered by phosphorylation at Ser-345, a known ATR target site.
Although there are numerous examples of substrates requiring phosphorylation as a prerequisite for ubiquitination (49), there also exist several proteins that must be dephosphorylated to undergo ubiquitination. For example, the pro-apoptotic tar-get Bcl2 is protected from ubiquitination and degradation by mitogen-activated protein (MAP) kinase-mediated phosphorylation. Inhibition of MAP kinase activity or activation of mitochondrial PP2A protein phosphatase in turn leads to dephosphorylation and proteasome-dependent degradation of Bcl2 (50).
We propose that Ser-456 is a novel site on Chk2 whose phosphorylation status must be maintained to stabilize Chk2 in response to some forms of DNA damage. Dephosphorylation of Ser-456 would represent a powerful mechanism for returning Chk2 protein levels to their steady states following resolution of DNA damage. Future studies, including the identification of an E3 ligase for Chk2, will hopefully provide further insight into the mechanism by which Chk2 levels are regulated in the cell.