ATM-mediated serine 72 phosphorylation stabilizes ribonucleotide reductase small subunit p53R2 protein against MDM2 to DNA damage

Lufen Chang; Bingsen Zhou; Shuya Hu; Robin Guo; Xiyong Liu; Stephen N. Jones; Yun Yen

doi:10.1073/pnas.0803313105

Edited by Michael Karin, University of California at San Diego School of Medicine, La Jolla, CA, and approved September 29, 2008 (received for review April 4, 2008)

Abstract

Ribonucleotide reductase small subunit p53R2 was identified as a p53 target gene that provides dNTP for DNA damage repair. However, the slow transcriptional induction of p53R2 in RNA may not be rapid enough for prompt DNA damage repair, which has to occur within a few hours of damage. Here, we demonstrate that p53R2 becomes rapidly phosphorylated at Ser⁷² by ataxia telangiectasia mutated (ATM) within 30 min after genotoxic stress. p53R2, as well as its heterodimeric partner RRM1, are associated with ATM in vivo. Mutational studies further indicate that ATM-mediated Ser⁷² phosphorylation is essential for maintaining p53R2 protein stability and conferring resistance to DNA damage. The mutation of Ser⁷² on p53R2 to alanine results in the hyperubiquitination of p53R2 and reduces p53R2 stability. MDM2, a ubiquitin ligase for p53, interacts and facilitates ubiquitination of the S72A-p53R2 mutant more efficiently than WT-p53R2 after DNA damage in vivo. Our results strongly suggest a novel mechanism for the regulation of p53R2 activity via ATM-mediated phosphorylation at Ser⁷² and MDM2-dependent turnover of p53R2 dephosphorylated at the same residue.

DNA is surprisingly reactive and is under continuous assault from daily environmental agents such as UV light, reactive chemicals, and metabolic byproducts such as reactive oxygen species. Defects in DNA damage signaling and repair can lead to mutations, ultimately resulting in cancer (1). In addition to a potential role in cancer development, damage to cellular DNA has been used for cancer therapy and is responsible for most of the toxic effects of such therapy (1, 2). Therefore, the study of genes involved in DNA damage responses could lead to a deeper understanding of cancer development and more effective treatments of malignancies.

Ribonucleotide reductase (RR) is a rate-limiting enzyme responsible for providing a balanced dNTP supply for DNA synthesis and repair (3). Unbalanced dNTP supply can lead to genetic abnormalities and cell death, underscoring the importance of the mechanisms that regulate RR activity. RR is composed of two nonidentical subunits, RRM1 and RRM2. p53R2, an analogue of RRM2 in mammalian cells, can substitute for RRM2 to interact with RRM1 and plays an important role in DNA damage induced by genotoxic stress (4, 5). p53R2 has been identified as a transcriptional target of p53 (4), whereas RRM2 is transcriptionally regulated by cell cycle associated factors, such as nuclear factor Y and E2F (6). However, the mechanism by which p53R2 activity is induced by p53 may not be rapid enough to supply dNTPs for prompt DNA repair, which can be completed within a few hours after DNA damage (1, 3). More than 90% of damaged DNA can be repaired within 8 h after non-lethal UV irradiation in HeLa cells. Yet, the p53R2 mRNA is not fully induced until 12 h after γ-irradiation in many of p53 WT cells including normal dermal fibroblast, MCF-7, LoVo, and HCT116 cell lines (4). Additionally, we have previously observed that p53R2 can interact with p53 at the protein level to regulate RR activity (7). These observations strongly suggested that the transcriptional induction of p53R2 via p53 may not be directly responsible for providing dNTPs for DNA repair after genotoxic stress.

To understand whether p53R2 activity is regulated via a posttranslational modification, we identified p53R2 as a direct substrate of ataxia telangiectasia mutated (ATM). p53R2 was phosphorylated by ATM at Ser⁷² in vitro and in vivo. ATM-dependent p53R2 Ser⁷² phosphorylation regulates cell viability and p53R2 protein stability by inhibiting p53R2 hyperubiquitination and degradation by MDM2 in response to DNA damage. The mechanism of UV-induced S72A-p53R2 degradation will be discussed.

Results

ATM Phosphorylates p53R2 Predominantly at Ser⁷².

We hypothesized that p53R2 activity can be regulated at the posttranslational level in response to DNA damage. Using in vivo ³²P-orthophosphate labeling, we have observed that ³²P can be incorporated into p53R2 induced by UV irradiation (Fig. 1A), suggesting that p53R2 is a phosphorylated protein. The phosphatidylinositol-3 kinase-like (PIKK) family of kinases, including ATM and ATM- and Rad3-related (ATR), are important in sensing DNA damage and initiating the subsequent signaling cascades (1, 8). To explore if a signaling cascade exists between p53R2 and these PIKKs, we first examined whether p53R2 can be directly phosphorylated by PIKKs in vitro. Bacterially expressed full-length His-tagged p53R2 fusion proteins were used as a substrate in an in vitro kinase reaction with immunoprecipitated ATM from KB cells after UV irradiation. Recombinant p53R2 protein was strongly phosphorylated by the immunoprecipitated ATM at 30 min after UV exposure (Fig. 1B). Immunoprecipitated ATR or DNA-PKcs was not able to phosphorylate p53R2 (data not shown). Furthermore, c-Jun N-terminal kinase immunoprecipitated from the same cell lysates efficiently phosphorylated its known substrate c-Jun (9) but not p53R2 [supporting information (SI) Fig. S1]. These results strongly suggested that p53R2 is a specific substrate for ATM. Examination of p53R2 amino acid sequences revealed two ATM consensus sites at Ser⁷²K and Ser¹¹²Q. Alanine substitutions of these potential phosphorylation sites within p53R2 identified Ser⁷² as a major site for phosphorylation by immunoprecipitated ATM. Compared with WT-p53R2, S72A-p53R2 (i.e., p53R2 with a single substitution at Ser⁷²) is less phosphorylated by immunoprecipitated ATM complex. Substitution at Ser¹¹² (S112A-p53R2) on p53R2 had no significant effect on its level of phosphorylation. A combination of both mutations (S72A/S112A-p53R2) did not result in any further reduction of phosphorylation compared with the S72A-p53R2 mutant (Fig. 1C).

Fig. 1.

p53R2 is a substrate of ATM kinase. (A) UV induced an increased ³²P-labeling of p53R2. HEK293 cells were transiently transfected with Myc-tagged p53R2 or empty vector. Eighteen hours after transfection, cells were metabolically labeled with ³²P-orthophosphate and later stimulated with (+) or without (−) UV irradiation (20 J/m²). After 30 min, radiolabeled p53R2 was immunoprecipitated with anti-Myc antibody and evaluated by Phosphoimager (Upper) or Western blot (Lower) by anti-p53R2 antibody. (B) Bacterially expressed p53R2 recombinant proteins were subjected to in vitro ATM immunocomplex kinase assay (KA) using the ATM immunoprecipitated from KB cells at the indicated times after UV (20 J/m²) treatment in the presence of [γ-³²P]ATP. The resulting mixture was resolved by a NuPAGE gel and visualized by autoradiography and Coomassie blue staining (CB). (C) Identification of individual ATM phosphorylation site. WT p53R2 and Ala substitution mutants at Ser⁷² (S72A), Ser¹¹² (S112A), or both residues (S72A/S112A) were subjected to in vitro ATM kinase assay, and visualized by autoradiography and Coomassie staining as described earlier.

Ser 72 Phosphorylation by ATM Stabilizes p53R2 Protein Against UV Irradiation.

To confirm whether p53R2 is phosphorylated at Ser⁷² in cells following genotoxic stress, a polyclonal antibody against phosphorylated Ser⁷² of p53R2 (anti-pS72-p53R2) was generated. The anti-pS72-p53R2 antibody recognized WT-p53R2, but not the S72A-p53R2 mutant, after co-transfection with ATM plasmids. WT-p53R2 alone, without co-transfection with ATM plasmids, was not detected by this antibody (Fig. 2A). Moreover, this antibody also detected an endogenous phosphorylated form of p53R2 induced by cisplatin, UV radiation, and ionizing radiation (IR; Fig. 2B). We next examined the phosphorylation state of p53R2 in ATM-deficient fibroblasts derived from patients with ataxia-telangiectasia (A-T) after reconstitution without (pEBS7) or with (pEBS7-YZ5) ATM expression plasmids (10). Re-introduction of the ATM gene into A-T cells restored Ser⁷² phosphorylation of p53R2, as well as Ser¹⁹⁸¹ phosphorylation of ATM induced by UV irradiation (Fig. 2C). Surprisingly, p53R2 expressed in A-T cells (pEBS7) was rapidly degraded following UV exposure, whereas RRM1 and actin proteins remained unchanged (Fig. 2C). p53R2 proteins in ATM reconstituted cells were resistant to UV-induced degradation. The S72A-p53R2 mutant transiently transfected in 293 cells also underwent UV-induced protein degradation, whereas WT-p53R2 was stable (Fig. 2D). Similar results were obtained in pulse-chase experiments (Fig. 2E). UV induced rapid decay of S72A-p53R2, but failed to alter the amount of radiolabeled WT-p53R2 (Fig. 2E). p53R2 degradation induced by UV in A-T cells can be blocked in cells pretreated with the proteasome inhibitor MG-132 (Fig. S2), suggesting that UV-induced S72A-p53R2 decay is through the proteasomal pathway. Interestingly, polyubiquitinated forms of the S72A-p53R2 mutant were detected, but little WT-p53R2 ubiquitination was seen after co-expression with a ubiquitin expression plasmid in 293 cells following UV exposure (Fig. 2F).

Fig. 2.

In vivo Ser⁷² phosphorylation stabilizes p53R2 against UV. (A) 293 cells were transfected with Myc-tagged WT-p53R2, S72A-p53R2, or ATM expressing plasmids 30 min after UV exposure (20 J/m²) and subjected to immunoblotting analysis using anti-pS72-p53R2 or anti-Myc antibodies. (B) KB cells treated without or with UV (20 or 80 J/m²), cisplatin (20 μM), or IR were immunoprecipitated with anti-p53R2 antibodies and immunoblotted with anti-pS72-p53R2 antibodies. (C) UV induced destabilization of p53R2 proteins in ATM-deficient cells. A-T cells complemented without (pEBS7 vector) or with ATM expressing plasmids (pEBS7-YZ5) were stimulated by UV (20 J/m²) and subjected to immunoblotting analysis. (D) Ser⁷² phosphorylation is required for p53R2 protein stabilization to UV. 293 cells were transfected with Myc-tagged WT-p53R2 or S72-p53R2 together with or without ATM expression plasmids. After 24 h, cells were stimulated by UV (20 J/m²) and subjected to immunoblotting analysis using anti-p53R2. (E) S72A-p53R2 has a shorter half-life after exposure to UV. 293 cells were transfected with Myc-tagged WT- or S72A-p53R2, and protein half-life was determined by pulse-chase analysis. (F) S72A-p53R2 mutant is polyubiquitinated. 293 cells were transfected with HA-ubiquitin plus Myc-tagged WT-, S72A-p53R2, or empty vector. After 24 h, cells were exposed to UV and ubiquitination was examined by immunoprecipitation with anti-Myc antibodies and immunoblotting with HA antibodies.

MDM2 Interacts with and Facilitates Ubiquitination of S72A Mutant p53R2 In Vivo.

MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation, interacts with p53 dynamically in response to UV irradiation (11). Given that p53R2 is a newly identified ATM substrate, we next tested whether MDM2 is involved in UV-induced ubiquitination and degradation of unphosphorylated p53R2. In transient transfection experiments, we found that co-expressing MDM2 promoted UV-induced polyubiquitination of S72A-p53R2 that was seen only in cells treated with the proteasome inhibitor MG132 (Fig. 3A). To further investigate if MDM2 contributes to the degradation of p53R2, we examined endogenous p53R2 levels in MDM2-knockout mouse embryonic fibroblasts (MEFs) (12) in which ATM proteins were knocked down by siRNA. As shown in Fig. S3, p53R2 accumulated more in p53^−/−MDM2^−/− MEFs than in p53^−/− MEFs after UV irradiation. To further characterize the functional interaction between MDM2 and p53R2, we examined whether these two proteins interact. We first examined the interaction between endogenous p53R2 and MDM2 (Fig. 3B). Using co-immunoprecipitation (co-IP) assays, we observed that MDM2 was present in the immunocomplexes of p53R2 before UV treatment (Fig. 3B). The interaction between MDM2 and p53R2 was disrupted in ATM proficient cells (YZ5) at later time points after UV exposure (Fig. 3B, Left). In contrast, endogenous p53R2 in ATM-deficient cells (pEBS7) remained associated with MDM2 after UV exposure (Fig. 3B, Right). This interaction was also detected between exogenously expressed MDM2 with WT-p53R2 or S72A-p53R2 in 293 cells before exposure to UV (Fig. 3C). However, lower levels of MDM2 proteins dissociated from S72A mutant p53R2 compared with WT-p53R2 proteins following UV exposure. These results suggest that hyperubiquitination and degradation of S72A mutant p53R2 might result from a higher binding affinity with MDM2.

Fig. 3.

MDM2 ubiquitinates and interacts with p53R2. (A) MDM2 induced S72A mutant p53R2 ubiquitination in response to UV. 293 cells were transiently transfected with the plasmids encoding Myc-tagged WT- or S72A- p53R2 along with ATM and MDM2. The cells were preincubated with or without MG-132 for 30 min, followed by stimulation with or without UV (20 J/m²) for 6 h. After treatment with UV, the cell lysates were immunoprecipitated with anti-Myc. Ubiquitination of WT- or S72A-p53R2 was examined by immunoblotting with a specific ubiquitin antibody. p53R2 protein levels were examined by anti-p53R2 antibody. (B) Enhanced interaction between endogenous MDM2 and unphosphorylated p53R2. Cell lysates isolated either from pEBS7-YZ5 (ATM proficient) or MG132-pretreated pEBS7 (ATM deficient) cells at the indicated times after UV stress (20 J/m²) were immunoprecipitated with anti-p53R2 antibody, followed by immunoblotting with anti-MDM2 antibodies. (C) Enhanced interaction between exogenous MDM2 and S72A-p53R2 mutant. 293 cells, after transient transfection with plasmids encoding Myc-tagged WT- or S72A-p53R2 together with ATM and MDM2, were preincubated with MG-132 for 30 min before exposure to UV (20J/m²). At the indicated times, cells were lysed and immunoprecipitated with Myc-tagged antibody, followed by immunoblotting with MDM2 or Myc-tagged antibodies.

p53R2 and RRM1 Associate with ATM.

To demonstrate a physical interaction between ATM and p53R2, we performed co-IP experiments. In support of the aforementioned observation that ATM is a direct p53R2 kinase, endogenous ATM and p53R2 were co-immunoprecipitated in UV-irradiated cells (Fig. 4A). ATM was brought down by p53R2 specific antibodies as early as 15 min after UV irradiation (Fig. 4A). This interaction was further verified by a reciprocal co-IP assay using ATM-specific antibody. However, when ATM antibody was used, p53R2 was already associated with ATM before UV irradiation (Fig. 4B). Their interaction was clearly enhanced around 30 min after exposure to UV. In addition to p53R2 polypeptide, RRM1 (which has a molecular mass of 80 kDa) was also detected in the ATM immunoprecipitated complex. These results suggested that RRM1 and p53R2 can form a complex with ATM in vivo. However, p53R2 was not detected in ATR (Fig. 4C) or DNA-PKcs immunocomplexes (Fig. 4D).

Fig. 4.

Interaction between ATM and p53R2. (A) Co-IP of endogenous ATM by anti-p53R2 antibody. Cell lysates were isolated from KB cells at the indicated times after UV stress (20 J/m²) and immunoprecipitated by anti-p53R2 antibody, followed by immunoblotting with anti-ATM or anti-p53R2 antibodies. (B) Same as in A except that ATM antibody was used for co-IP, followed by immunoblotting with anti-ATM, anti-p53R2, and anti-RRM1 antibodies. (C) Same as in A except that ATR antibody was used for co-IP, followed by immunoblotting with anti-ATR or anti-p53R2 antibodies. (D) Same as in A except that DNA-PKcs antibody was used for co-IP, followed by immunoblotting with anti- DNA-PKcs or anti-p53R2 antibodies.

S72A-p53R2 Exerts a Dominant-Negative Effect in Cell Cycle Progression and Increases Cellular Sensitivity to DNA Damage Stress.

We next evaluated the effect of p53R2 phosphorylation on cell cycle progression following DNA damage. Stable cell lines overexpressing Myc-tagged WT-, S72A-p53R2, or vector control were generated. Expression of the transgenes was assessed by immunoblotting analysis using a Myc-tagged specific antibody (Fig. S4A). All cell lines had a normal cell cycle distribution before UV exposure (Fig. 5A). Following UV exposure, WT-p53R2 or vector control expressing cells exhibited a time-dependent decrease in G1 phase accompanied by an increase in S phase cells (0, 3, and 12 h) and a later increase in cells in G2/M phase (24 and 36 h). UV-induced G2/M arrested cells were slightly increased in WT-p53R2 transfected cells compared with vector transfected cells. However, such G2/M phase accumulation was not detected in S72A-p53R2 expressing cells. Instead, these cells accumulated predominantly in the S phase and decreased in the G1 phase. Most of the S72A-p53R2 expressing cells failed to enter the G2/M phase (Fig. 5A). We also monitored the dynamic response of these cells to UV under an RT-CES system. There was no difference in cell growth among these cells without UV treatment (Fig. S4B). However, S72A-p53R2 expressing cells resulted in a delayed growth curve compared with WT-p53R2 expressing cells, whereas vector control expressing cells had a growth curve between WT- and S72A-p53R2 expressing cells (Fig. S4B).

Fig. 5.

Cells overexpressing S72A-p53R2 are more sensitive to genotoxic stress. Overexpressing S72A-p53R2 increased cellular sensitivity to genotoxic stress. (A) KB cells stably expressing WT-p53R2, S72A, or control vector were exposed to UV (20 J/m²) and their cell cycle profiles were determined by propidium iodide staining and FACS analysis at the indicated time points. Only surviving cells that remained attached to the plates were analyzed. (B) KB cells stably expressing WT-p53R2, S72A, or control vector were analyzed for cell viability by MTT assay 72 h after exposure to UV, IR, or cisplatin at the indicated doses.

Aberrant cell cycle progression may trigger a cell death response. We next used MTT assay to analyze the viability of those cells after exposure to genotoxic stress, including UV, IR, and cisplatin. As expected, S72A-p53R2 expressing cells are the most susceptible to all three genotoxic treatments (Fig. 5B). Cells overexpressing p53R2 with a mutation at Ser¹¹² had a genotoxic sensitivity similar to that in cells expressing WT-p53R2 (Fig. 5B). These results strongly indicate an important role of ATM-dependent Ser⁷² phosphorylation of p53R2 in protecting cells from genotoxic-induced cell death.

Discussion

Our results demonstrate that ATM phosphorylates p53R2 at Ser⁷² in response to genotoxic stress and that this modification is essential for maintaining p53R2 protein stability. Mutation of Ser⁷² to alanine rendered p53R2 both hyperubiquitinated and less stable than WT-p53R2 after UV irradiation. The role of ATM signaling has been implicated in the regulation of p53R2 protein expression (13), but the underlying mechanism has not been clear. Here we present direct evidence that ATM not only phosphorylates but also interacts with p53R2. The phosphorylation of p53R2 at Ser⁷² occurs at a time point that corresponds to the enhanced binding between p53R2 and ATM. The timing of phosphorylation at this site is also closely linked to the change in stability of p53R2 in A-T cells following UV irradiation. Therefore, we hypothesized that ATM-dependent phosphorylation at Ser⁷² in p53R2 potentially contributes to its stability against UV induced degradation.

Its stability suggests that p53R2 is more functionally important than its RRM2 homologue when exposed to genotoxic stress. Unlike RRM2, p53R2 does not contain a KEN box sequence, which can be targeted by the Cdh1-APC E3 complex for proteolysis during G0/G1 arrest and late mitosis (14, 15). However, we observed that endogenous p53R2 in A-T cells or S72A-p53R2 expressed in 293 cells was sensitive to UV exposure (Fig. 2) even though their mRNA levels remained unchanged (data not shown). Here we demonstrate that MDM2 promotes ubiquitination and degradation of S72A-p53R2. p53R2 protein accumulates in MDM2-deficient and ATM knockdown cells after UV exposure (Fig. S3). Our data suggest that hyperubiquitination and degradation of S72A mutant p53R2 after UV stress might be a result of its strong binding with MDM2 (Fig. 3 B and C). MDM2 is well known for its role as a p53 ubiquitin ligase (11). We found that p53R2, p53, and ATM can form a trimeric protein complex (Fig. S5), but whether p53R2 and p53 compete against each other as targets for MDM2 ubiquitination remains to be investigated.

p53R2 has been implicated in the regulation of cell cycle progression induced by stress from DNA damage. p53R2-null cells or cells treated with the RR inhibitor hydroxyurea prevented G2/M arrest and resulted in cell death (4). Similarly, cells overexpressing S72A-p53R2 exhibited abnormal cell cycle progression and enhanced sensitivity to genotoxic stress (Fig. 5). As demonstrated in Fig. 4, p53R2 constitutively interacts with RRM1. Therefore, overexpressed S72A-p53R2 might out-compete WT-p53R2 in binding RRM1 and disturb cell cycle progression. Measuring RR activity by the quantification of total dNTP revealed that overexpressed S72A-p53R2 reduced basal and UV-induced dNTP levels compared with WT-p53R2 and mock vector (Fig. S6).

Recent studies demonstrate that ATM regulates the function of p53R2 by providing dNTP for mitochondrial DNA synthesis (13, 16). However, there seems to be no significant difference in the content of mtDNA in cells overexpressing WT- or S72A-p53R2 before or after UV exposure (Fig. S4C). We previously identified a potential role of p53R2 in scavenging reactive oxygen species (ROS) and protecting mitochondrial membrane potential against oxidative stress-induced damage (17). It will be of interest to determine whether the enhanced sensitivity of S72A-p53R2 expressing cells to genotoxic stress results from abnormal accumulation of reactive oxygen species.

In this study we demonstrate a direct link between p53R2 and ATM. Cells deficient either in ATM or p53R2 exhibit defects in DNA damage repair and cell cycle progression. p53R2 and RRM1 are normally expressed at low basal levels in resting cells, and both proteins can be translocated into the nucleus immediately upon genotoxic stress (4, 5). Interestingly, p53R2 is detected in the MRE 11 complex (NBS1/Rad50/MRE11; Fig. S7), a DNA damage complex involved in double-strand break repair and DNA damage checkpoint pathways (18), further suggesting that p53R2 is part of the DNA damage complex. Similar to NBS1, p53R2 can pre-associate with ATM before UV stress (19). Their interaction is enhanced after UV exposure. The increase in complexed ATM and p53R2 might be the result of conformational changes induced in the protein complex upon DNA damage. Recent evidence demonstrated that ATM at site-specific DSBs requires functional NBS1 protein, ATM kinase activity, and auto-phosphorylation of ATM at Ser 1981 (20). Therefore, ATM may immediately recruit the pre-associated RRM1 and p53R2 proteins along with the MRE11 complex to the DNA damage sites to supply dNTP during the acute phase of DNA damage repair. Weak or indirect interactions might explain why binding between p53R2 and ATR or DNA-PKcs was not observed (Fig. 4 C and D). Interestingly, ATR's binding partner ATR-interacting protein (21), but not DNA-PKcs regulatory subunits Ku 70/80, can specifically interact with p53R2 (Fig. S8). This result suggested that p53R2 may also be involved in ATR-mediated DNA damage repair.

Defects in DNA damage repair may cause genomic instability and predispose cells to cancer. Epidemiological studies have linked ATM mutations to a high risk of breast cancer (22). Moreover, a number of polymorphisms in the gene encoding p53R2 have been identified in esophageal squamous cell carcinoma and in colon carcinoma cells (23–25), but none of these mutations are associated with altered p53R2 activity. A possible identification of Ser⁷² mutation in breast cancer will further support the role of p53R2 in maintaining genomic integrity against cancer development. ATM and other proteins in its pathways have been studied as potential therapeutic targets to increase the sensitivity of cancer cells to DNA-damage agents (26). The development of p53R2 small molecular inhibitors to block Ser⁷² phosphorylation could be a potential therapy against malignancy.

Materials and Methods

Cells and UV Radiation.

Cells were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin. UV irradiation was performed with an UVC lamp (G8T5; General Electric). Dose rates were measured with a UVX Radiometer (UVP). For UV irradiation as well as for mock treatment, the growth medium was aspirated and the cell layer covered with a small amount of PBS solution. After treatment was completed, the growth medium was replenished.

[³⁵S]Methionine/Cysteine Pulse Chase Labeling.

Pulse-chase experiments were performed as described previously (27).

Kinase Assays.

ATM was immunoprecipitated from KB cells treated with or without UV irradiation and were used in kinase assays as previously described (28). His-tagged recombinant p53R2 (5) or GST-cJun (1–79 aa) (28) proteins were added to the ATM immunocomplex G protein beads for the kinase reaction.

Immunoblotting Assays.

Immunoblotting analysis was performed as described (27). IRDye 680-conjugated rabbit or IRDye 800-conjugated mouse Ig-specific secondary antibodies (LI-COR) were used. Blocking buffer (LI-COR) was used to pretreat blots and dilute antibodies. The blots were imaged on an Odyssey Infrared Imaging System (LI-COR). Ubiquitination assays were performed as described previously (27).

In Vivo³²P Metabolic Labeling.

In vivo labeling of ³²P-labeled polypeptides was performed as described previously (29).

Cell Cycle Assays.

KB stable cells expressing WT, S72A mutant p53R2, or empty vector were collected for fixation in methanol at the indicated time after UV irradiation. Following fixation, cells were washed and resuspended in PBS solution with 25 μg/ml and 50 μg/ml RNase 30 min before FACScan analysis. Analysis of the cell cycle results were performed using the ModFit LT program.

SI.

For real-time cell proliferation monitoring system, real-time quantitative PCR, RNA interference, and dNTPs pool assays, see SI Materials and Methods.

Acknowledgments

We thank E.Y. Lee for the ATM plasmids and D.K. Ann for ATM deficient and proficient cells. This work was supported by National Cancer Institute grant R01CA72767.

Footnotes

¹To whom correspondence may be addressed. E-mail: lchang{at}coh.org or yyen{at}coh.org
Author contributions: L.C. and Y.Y. designed research; L.C., B.Z., S.H., R.G., and X.L. performed research; S.N.J. contributed new reagents/analytic tools; L.C. analyzed data; and L.C. and R.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0803313105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA

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1. Berkovich E,
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3. Kastan MB
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OpenUrl Abstract/FREE Full Text
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1. Renwick A,
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OpenUrl Abstract/FREE Full Text
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1. Yamaguchi T,
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OpenUrl Abstract/FREE Full Text
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1. Deng ZL,
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1. Chang L,
2. et al.
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1. DiDonato J,
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OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 105 (47)

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[81] et al.

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Smeds J,
Kumar R,
Hemminki K
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OpenUrl Abstract/FREE Full Text

[83] Smeds J,

[84] Kumar R,

[85] Hemminki K

[86] ↵
Yamaguchi T,
et al.
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OpenUrl Abstract/FREE Full Text

[87] Yamaguchi T,

[88] et al.

[89] ↵
Deng ZL,
et al.
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OpenUrl PubMed

[90] Deng ZL,

[91] et al.

[92] ↵
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OpenUrl CrossRef PubMed

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[94] Lim DS

[95] ↵
Chang L,
et al.
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OpenUrl CrossRef PubMed

[96] Chang L,

[97] et al.

[98] ↵
Kamata H,
et al.
(2005) Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 120:649–661.
OpenUrl CrossRef PubMed

[99] Kamata H,

[100] et al.

[101] ↵
DiDonato J,
et al.
(1996) Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation. Mol Cell Biol 16:1295–1304.
OpenUrl Abstract/FREE Full Text

[102] DiDonato J,

[103] et al.

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ATM-mediated serine 72 phosphorylation stabilizes ribonucleotide reductase small subunit p53R2 protein against MDM2 to DNA damage

Abstract

Results

ATM Phosphorylates p53R2 Predominantly at Ser⁷².

Ser 72 Phosphorylation by ATM Stabilizes p53R2 Protein Against UV Irradiation.