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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Jun 22;104(27):11388–11393. doi: 10.1073/pnas.0609467104

Induction of Cullin 7 by DNA damage attenuates p53 function

Peter Jung *, Berlinda Verdoodt *, Aaron Bailey †, John R Yates III †, Antje Menssen *, Heiko Hermeking *,‡
PMCID: PMC2040908  PMID: 17586686

Abstract

The p53 tumor suppressor gene encodes a transcription factor, which is translationally and posttranslationally activated after DNA damage. In a proteomic screen for p53 interactors, we found that the cullin protein Cul7 efficiently associates with p53. After DNA damage, the level of Cul7 protein increased in a caffeine-sensitive, but p53-independent, manner. Down-regulation of Cul7 by conditional microRNA expression augmented p53-mediated inhibition of cell cycle progression. Ectopic expression of Cul7 inhibited activation of p53 by DNA damaging agents and sensitized cells to adriamycin. Although Cul7 recruited the F-box protein FBX29 to p53, the combined expression of Cul7/FBX29 did not promote ubiquitination and degradation of p53 in vivo. Therefore, the inhibition of p53 activity by Cul7 is presumably mediated by alternative mechanisms. The interplay between p53 and Cul7 resembles the negative feedback loop described for p53 and Mdm2. Pharmacological modulation of Cul7 function may allow the sensitization of cancer cells expressing wild-type p53 to genotoxic agents used in cancer therapy.

Keywords: cul7, Fbx29, cell cycle, p21, tumor suppression

In response to genotoxic stress, numerous cell cycle checkpoints are triggered that prevent the propagation of cells with damaged genomes (1). These checkpoints mediate cell cycle arrest to provide time for DNA repair, or, in the case of too severe damage, facilitate apoptosis. A failure to correctly respond to DNA damage may lead to genomic instability, which may give rise to cancerous cells. In line with this scenario, the mutational inactivation of critical components of these checkpoints frequently occurs in cancer cells. The p53 transcription factor is a central component of the DNA damage checkpoint and mediates cell cycle arrest or apoptosis by activation of specific target genes, such as p21, 14-3-3σ, and Puma (2–4). p53 is encoded by a tumor suppressor gene that is inactivated in ≈50% of all human tumors (5). Genotoxic stress triggers rapid phosphorylation of p53 by ATM (ataxia telangiectasia mutated) and other kinases such as CHK2 (6, 7), resulting in the accumulation and activation of the p53 protein. The activity of p53 is also regulated by localization and acetylation (reviewed in ref. 7). In nonstressed cells, p53 is kept inactive by MDM2, which shields the N-terminal transactivation domain of p53, but also acts as an E3 ligase that targets p53 for proteasomal degradation (8). Because MDM2 is a direct transcriptional target of p53, both genes constitute a negative feedback loop (9). Furthermore, the degradation of the p53 protein is also tightly regulated by other E3 ligases, such as COP1, Pirh2, and p300 (10–12). ATM was recently shown to phosphorylate COP1, which promotes self-degradation of COP1 and p53 stabilization (13).

Cullin 7 (Cul7) was originally discovered as a 185-kDa protein (p185) associated with the large T antigen of simian virus 40 (SV40) (14). The C terminus of Cul7 harbors a BH3 domain, which presumably promotes apoptosis (15). Together with Skp1, Fbx29, and ROC1, Cul7 forms the SCF-ROC1 E3 ligase complex (SCF7) (16). Furthermore, Cul7 was shown to form an E3 ligase with Cul1 and the F-box protein FBX29, which confers substrate specificity (17). Association with the SCF7 complex is required for cellular transformation by SV40 large T antigen (18). Cul7 is highly homologous to PARC (PARkin-like, cytoplasmic, p53-binding protein), which negatively regulates p53 by cytoplasmic sequestration (19). PARC has been shown to heterodimerize with Cul7 (20). The two proteins have nonoverlapping functions because deletion of PARC in mice has no effect on viability (20), whereas Cul7-deficient mice exhibit neonatal lethality with reduced size and vascular defects (21).

Using a proteomic approach, we identified Cul7 as a p53-interacting protein. Down-regulation of Cul7 increased p53 activity. Furthermore, Cul7 expression was induced after DNA damage in a p53-independent manner and resulted in suppression of p53 activity. Therefore, Cul7 presumably plays a role in the DNA damage response by limiting p53 activity.

Results

Cul7 Interacts with p53 in Vivo.

In a proteomic screen for p53-associated proteins, we identified Cul7 as one of the most efficiently copurified proteins according to the mass-spectral sequence coverage obtained in multiple tandem affinity purifications of p53 (unpublished data). Cul7 is highly homologous to PARC, a previously identified p53 interacting protein [supporting information (SI) Fig. 6A]. To confirm the association between p53 and Cul7, we ectopically expressed both proteins in p53-deficient H1299 non-small cell lung cancer cells. By coimmunoprecipitation, we could show that Flag-tagged Cul7 specifically associates with HA-tagged p53 protein (Fig. 1A). The same result was obtained with HEK 293 cells (SI Fig. 6B). Furthermore, immunoprecipitation with a p53-specific antibody identified an association with endogenous Cul7 in the breast carcinoma cell line MCF-7 but, as expected, not in p53-deficient H1299 cells (Fig. 1B). The association between endogenous p53 and Cul7 was enhanced after induction of DNA damage by addition of the topoisomerase II inhibitor etoposide (Fig. 1B). The association of endogenous p53 with Cul7 was also observed in the osteosarcoma cell line U-2OS (SI Fig. 6C).

Fig. 1.

Fig. 1.

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Interaction between ectopic and endogenous Cul7 and p53 proteins. (A) H1299 cells were transfected with constructs encoding HA-tagged p53 and Flag-tagged Cul7. Western blot analysis of whole cell extracts (WCL) and immunoprecipitates [IP; obtained by using HA-specific, Flag-specific, or mouse preimmune serum (IgG)]. (B) Coimmunoprecipitation of endogenous p53 and Cul7 in MCF-7 and H1299 cells. Whole cell extracts (WCL) and immunoprecipitates [IP; obtained with p53-specific antibodies (DO-1 and 1801) or mouse preimmune serum (IgG)] were subjected to Western blot analysis with Cul7-specific monoclonal and p53-specific polyclonal antibodies. ∗, specific signal for Cul7.

Cul7 Protein and mRNA Accumulate After DNA Damage.

Because we found that the association between Cul7 and p53 was enhanced after treatment of cells with etoposide (Fig. 1B), we analyzed whether Cul7 mRNA and/or protein levels increase in response to DNA damage. Indeed, endogenous Cul7 protein significantly increased in MCF-7 cells as early as 10 h after treatment with etoposide and reached maximum levels after 24 h (Fig. 2A). A similar increase in Cul7 protein was observed in the osteosarcoma cell line U-2OS and also in the p53-negative cell line H1299 (Fig. 2A). The increase in Cul7 protein level was accompanied by an induction of Cul7 mRNA in MCF-7 (SI Fig. 7A) and U-2OS cells (SI Fig. 7A). In contrast, the p53-negative cell line H1299 showed no increase in Cul7 mRNA 14 h after DNA damage (SI Fig. 7A). This p53 dependence suggested that CUL7 may be a p53 target gene. However, activation of a tet-regulated p53 allele in DLD-1 and H1299 cells did not affect Cul7 mRNA expression, whereas p21 mRNA was induced as expected (SI Fig. 7B and data not shown). Furthermore, Cul7 protein increased after DNA damage in HCT116 colon cancer cells deficient for p53 with similar kinetics as in cells expressing wt p53 (Fig. 2B), whereas Cul7 mRNA was not significantly affected by DNA damage in these cell lines (SI Fig. 7C). Furthermore, the Cul7 promoter region was not responsive to p53 in a transient reporter assay (data not shown), indicating that the increase of Cul7 mRNA observed in a subset of cell lines after DNA damage is mediated by an unknown factor.

Fig. 2.

Fig. 2.

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Increase of Cul7 protein in response to DNA damage. (A) Detection of Cul7 protein levels in MCF-7, U-2OS, and H1299 cancer cell lines by Western blot analysis. The respective cell lines were treated with etoposide (20 μM) for the indicated periods. (B) Detection of Cul7 protein levels in p53-deficient and wild-type HCT116 colon cancer cell lines (28) by Western blot analysis. Cells were treated with 20 μM etoposide for the indicated periods. (C) Detection of Cul7 protein in MCF-7 cells expressing wild-type p53 by Western blot analysis. Cells were treated for the indicated periods with 20 μM etoposide alone or in combination with 5 mM caffeine. Caffeine was added 45 min before etoposide.

Interestingly, treatment with 5 mM caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases (PIKK) such as ATM and ATR (ATM- and Rad3-related) (22), effectively blocked the accumulation of Cul7 protein in MCF-7 cells and p53-negative H1299 cells after DNA damage (Fig. 2C and SI Fig. 8A). In the absence of DNA damage, caffeine treatment had no significant effect on Cul7 expression level (SI Fig. 8A Lower). Furthermore, caffeine also prevented the increase in Cul7 mRNA after DNA damage (SI Fig. 8B). However, down-regulation of ATM and/or ATR by RNA interference had no effect on the induction of Cul7 by DNA damage (data not shown). Therefore, other PIKK family members may be involved in the induction of Cul7 protein and mRNA during the DNA damage response.

Increased p53 Activity and G1 Arrest After Knockdown of CUL7.

To study the effect of Cul7 down-regulation on p53 activity we used the pEMI (plasmid for episomal microRNA expression) vector system (23). MCF-7 cell lines stably harboring pEMI constructs, which allow conditional expression of Cul7-specific microRNAs and mRFP from a bidirectional promoter, were generated. A cell line displaying homogenous induction of mRFP 48 h after addition of doxycycline (DOX) to medium is shown in Fig. 3A. The maximum knockdown of Cul7 was achieved 72 h after induction of microRNA expression whereas a nonsilencing microRNA had no effect on Cul7 protein levels (Fig. 3B). Interestingly, p21 protein levels increased after pEMI-mediated Cul7 knockdown in untreated and in DNA damaged cells. The amount of p53 protein was not significantly affected by the down-regulation of Cul7. Expression of a nonsilencing microRNA in a control cell line did not affect p53 and p21 protein expression. The effect on p21 expression after Cul7 ablation was also observed with pools of pEMI-vector containing U-2OS cells (SI Fig. 9A), ruling out clonal or cell type-specific effects. We noticed that MCF-7 cells with down-regulation of Cul7 displayed an increase in size and showed a change in morphology indicative of a cell cycle arrest, which is consistent with the induction of the CDK-inhibitor p21 (Fig. 3 A and B). Indeed, when we analyzed the DNA content of these cell populations by flow cytometry, MCF-7 cells with a knockdown of Cul7 showed an increased fraction of cells in G1-phase and a decrease of cells in S-phase (Fig. 4A). When cells exhibiting a knockdown of Cul7 were treated with etoposide an increase of cells arrested in G1 resulted when compared with cells expressing a nonsilencing microRNA. Cells without microRNA induction or cells expressing a nonsilencing microRNA showed a significant increase in the 4N DNA content after DNA damage indicating a predominant G2/M-arrest (Fig. 4A and SI Fig. 9B). To determine whether the effects of Cul7 down-regulation were p53-dependent, we transfected MCF-7 pEMI-Cul7microRNA cells with p53-specific or nonsilencing short interfering RNAs (siRNAs) before induction of the Cul7-specific microRNA (Fig. 4B). Indeed, down-regulation of p53 partially rescued the effect of Cul7 knockdown resulting in an increased proportion of cells that were arrested in G2/M and a decrease of cells in G1 in response to DNA damage compared with controls (Table 1). Taken together, these results show that Cul7 limits the activity of p53.

Fig. 3.

Fig. 3.

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Effects of acute inactivation of Cul7. (A) MCF-7 cell lines with inducible expression of mRFP and Cul7-microRNA (MCF-7-EMI-Cul7-miRNA) were compared with cells expressing a nonsilencing microRNA (MCF-7-EMI-nonsilencing). Seventy-two hours after addition of DOX (500 ng/ml), mRFP expression and cell morphology were analyzed by life cell microscopy. (Magnification: ×200.) (B) Down-regulation of Cul7 enhances transactivation of p21 by p53. Shown is Western blot analysis of Cul7, p53, and p21 protein levels in MCF-7-EMI-Cul7-microRNA cells and the control cell line MCF-7-EMI-nonsilencing. DOX (500 ng/ml) was added for 72 h, and etoposide (20 μM) was added for the indicated periods.

Fig. 4.

Fig. 4.

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Cul7 inactivation augments p53 activity. (A) DNA content analysis by flow cytometry after Cul7 knockdown in MCF-7 cells. Cells were treated with 500 ng/ml DOX for 72 h. DNA damage was induced by addition of etoposide (20 μM). MCF-7 cells with and without induction of Cul7-specific or a nonsilencing microRNA were treated with etoposide for the indicated periods. The experiment was repeated twice, and representative results are provided. The % cell cycle distributions are average results of two independent experiments. 2N, G1 phase; 4N, G2/M phase. SI Fig. 9B shows a similar analysis 24 h after etoposide treatment. (B) Concomitant knockdown of Cul7 and p53 in MCF-7 cells. MCF-7 EMI-Cul7microRNA cell line was transfected with p53-specific (p53si) or nonsilencing (NonS) siRNAs. Twenty hours later, Cul7 knockdown was induced by addition of DOX for 72 h. Cul7, p53, and β-actin protein levels were analyzed by Western blotting.

Table 1.

DNA content analysis by flow cytometry after Cul7 and p53 knockdown in MCF-7 cells

Cul7-specific miRNA induction Hours + etoposide Cell cycle phase Nonsilencing siRNA p53-specific siRNA
− 0 G1 54.48 50.20
S 18.99 20.40
G2/M 23.09 27.00
+ 24 G1 59.60 47.27
S 8.04 7.85
G2/M 27.00 38.88
+ 48 G1 53.47 41.10
S 8.85 8.85
G2/M 31.50 46.27
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The MCF-7-EMI-Cul7microRNA cell line was transfected with p53-specific or nonsilencing siRNAs. Twenty hours later, Cul7 knockdown was induced by addition of 500 ng/ml DOX 48 h before etoposide treatment for the indicated periods. The %-cell cycle distributions are average results of three independent experiments.

Ectopic Expression of Cul7 Inhibits p53 Function.

Because the Cul7 protein level increased in response to DNA damage we analyzed the effect of ectopic Cul7 expression on endogenous p53 and p21 protein levels. Induction of ectopic Cul7 expression by addition of DOX inhibited the increase in p53 and p21 levels after etoposide treatment (Fig. 5A; for vector controls see SI Fig. 10A). Furthermore, coexpression of Cul7 led to a reduced transactivation activity of p53 in a transient reporter assay (SI Fig. 10B). When cells ectopically expressing Cul7 were treated with the DNA damaging agent adriamycin, a topoisomerase II inhibitor, an increase in apoptosis was observed (SI Fig. 10 C and D). This effect presumably resulted from an inability of cells to stably arrest. Taken together, these results demonstrate that Cul7 modulates the stabilization and activity of p53 during the DNA damage response.

Fig. 5.

Fig. 5.

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p53 accumulation is inhibited by Cul7. (A) Western blot analysis of MCF-7 cells stably transfected with pRTS-1 episomal expression vector (39) encoding FlagCul7. See SI Fig. 10B for pRTS-1-Luciferase (pRTS-1-Ctrl) vector control experiment. Cul7 expression was induced by addition of 500 ng/ml DOX for 48 h. Cells were treated with etoposide (20 μM) for the indicated periods. (B) H1299 cells were transfected with plasmids encoding p53, Cul7, and FBX29. Shown is Western blot analysis of whole cell extracts (WCL) and immunoprecipitates (IP; obtained with a HA-specific antibody). (C) In vivo ubiquitination assay for p53 in H1299 cells. Cells were transfected with pMT107 [His-tagged ubiquitin (38)], pcDNA3-p53-VSV, and plasmids expressing MDM2 or FBX29 plus Cul7, as indicated. For details, see Materials and Methods. Circle with an X, background signal originating from anti-VSV antibody. Bottom lane, Western blot analysis of whole cell lysate.

Cul7/Fbx29 Does Not Exhibit E3-Ligase Activity Toward p53.

The SCF7-E3 ligase complex contains the F-box protein FBX29 (16), which recruits the substrates for ubiquitination. Next, we analyzed whether FBX29 forms a complex with p53 and Cul7. In a coprecipitation assay with ectopically expressed proteins, p53 associated with FBX29 in a Cul7-dependent manner in H1299 cells (Fig. 5B). Similar results were obtained for endogenous p53 in MCF-7 cells ectopically expressing Cul7 and FBX29 (SI Fig. 11A). Furthermore, ectopic FBX29 was coprecipitated with p53 in a Cul7-dependent manner (Fig. 5B and SI Fig. 11B). In line with the inability of p53 to directly associate with FBX29, p53 does not serve as a substrate for Cul7-mediated ubiquitination/degradation: ectopic expression of Cul7 in combination with FBX29 did not promote ubiquitination of p53 in vivo (Fig. 5C). The same result was obtained when proteasomal activity was inhibited by MG132 to increase the sensitivity of the ubiquitination assay (SI Fig. 11C). Coexpression of the E3 ligase MDM2 served as a positive control and led to an efficient mono- and polyubiquitination of p53 and decreased p53 levels (Fig. 5C and SI Fig. 11C). Therefore, other mechanisms than increased ubiquitination and subsequent proteasomal degradation are presumably responsible for the inhibition of p53 by Cul7.

Discussion

Our analysis revealed that Cul7 is induced by DNA damage in a caffeine-sensitive manner and negatively regulates p53 function presumably by direct interaction. While this manuscript was in preparation, a direct interaction between ectopic p53 and Cul7 was reported by others (24–26), which confirms our findings. Unlike the interaction between PARC and p53 (19), the Cul7/p53 interaction does not seem to influence p53 localization (24). Cul7 is a central component of an SCF-ROC1 E3 ligase complex (SCF7), consisting of Skp1, Cul7, FBX29, and ROC1 (16). We found that the substrate recognizing component of this complex, Fbx29, does not directly interact with p53, but associates indirectly with p53 in a Cul7-dependent manner. In agreement with our findings, the Cul7 domains responsible for p53 and FBX29 binding are not overlapping (25, 27).

Furthermore, we provide evidence that endogenous Cul7 acts as a negative regulator of p53 because acute down-regulation of Cul7 by a conditional RNA interference approach led to increased p21 protein levels, which augmented a DNA damage-induced G1 arrest in a p53-dependent manner. In addition, p53 accumulation and activity after DNA damage was compromised by ectopic Cul7 expression. Disruption of p53 function was previously described to sensitize human cancer cells to apoptosis induced by genotoxic drugs (28) and the p53/p21 axis was shown to be critical for sustained cell cycle arrest after DNA damage (29). In agreement with Cul7 acting as a negative regulator of p53 function, ectopic expression of Cul7 increased the apoptotic fraction of MCF-7 cells after exposure to genotoxic drugs, presumably by preventing the establishment of a stable p53-mediated cell cycle arrest.

Previously, a Cul7-mediated mono- and di-ubiquitination of p53 has been observed in vitro by using immunoprecipitated complexes of Cul7 and ectopic p53 from H1299 cells. However, the biochemical consequences of this p53 modification remained elusive (24). We could not confirm a ubiquitination of p53 by Cul7/FBX29 in vivo in H1299 cells. Because several E3 ligases have been shown to mono- and di-ubiquitinate p53 (11, 13–15), it is possible that immunoprecipitates of Cul7/p53 contain other E3-ligases responsible for the in vitro ubiquitination of p53 observed by Andrews et al. (24). The association of Cul7 with p53 was shown to occur with oligomeric forms of p53 (26) and tetrameric p53 is less efficiently imported into the nucleus than its monomeric form (31). Ectopic Cul7 interfered with p53 activation in response to DNA damage when cytoplasmic and nuclear p53 levels are increased. Therefore, Cul7 might associate with p53 in the cytoplasm and may be involved in controlling its oligomerization status. Nevertheless, the exact mechanism by which Cul7 inhibits the activity of p53 remains to be determined.

Cul7 knockout mice exhibit neonatal lethality with reduced size and vascular defects (21), suggesting that Cul7 plays a nonessential role in mammalian development, which would be consistent with a function as a checkpoint component. Our data imply that deregulation of p53 activity might at least in part account for the reduced proliferation of Cul7-deficient mouse embryo fibroblasts (21). Furthermore, p53 may contribute to the neonatal lethality observed after deletion of Cul7 (21). Similar phenotypes have also been observed in mice deficient for other negative regulators of p53, as MDM2 and MDMX (30, 32, 33). In line with a positive role of Cul7 in unperturbed cell proliferation mutational inactivation of Cul7 causes the 3-M syndrome, an autorecessive form of dwarfism characterized by pre- and postnatal growth retardation (27). According to our results, it is possible that the increase in p53 activity, which may result from mutated, nonfunctional Cul7, contributes to the 3-M syndrome.

Negative feed-back loops represent a common mode of p53 regulation (8, 34). Several negative p53 regulators, e.g., MDM2, COP1, and Pirh2, are encoded by target genes of p53 (9–11). Interestingly, Cullin proteins have been recently involved in the negative regulation of the DNA damage response as ATR-mediated phosphorylation marks CHK1 for ubiquitination by SCF ligase complexes containing Cul1 or Cul4a thereby limiting the duration of ATR-CHK1 signaling after genotoxic or replicative stress (35). Here, inhibition of PIKK family members by caffeine prevented the increase in Cul7 mRNA and protein after DNA damage. Therefore, we suggest a model in which PIKK kinases not only activate p53 in response to genotoxic stress but also increase the levels of a negative regulator of p53. In summary, we show that the induction of Cul7 after DNA damage attenuates the activation of p53. Pharmacological modulation of Cul7 function may allow to sensitize cancer cells expressing wild-type p53 to genotoxic agents used in cancer therapy.

Materials and Methods

Cell Lines/Culture and Reagents.

U-2OS osteosarcoma cells, MCF-7 breast cancer, and H1299 small cell lung cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, San Diego, CA) containing 10% FBS and penicillin (100 units/ml)/streptomycin (100 μg/ml). DLD1tTA and HCT116 colon cancer cells and their derivatives were maintained in McCoy's medium (Invitrogen) containing 10% FBS, penicillin (100 IE)/streptomycin (100 μg/ml), and, in case of DLD1tTA cells, 100 ng/ml doxycycline. Doxycycline (Sigma, St. Louis, MO) was dissolved in water (100 μg/ml). Etoposide (Sigma) was dissolved in DMSO (40 mM) and used at a final concentration of 20 μM. Caffeine (Sigma) was used at a final concentration of 5 mM.

Plasmids and siRNAs.

Information and sequences are available in SI Materials and Methods.

Generation of Cell Lines.

MCF-7 breast cancer cells or U-2OS cells were transfected with pRTS-1 or pEMI plasmids by using FuGENE reagents (Roche, Basel, Switzerland). After 48 h, cells were selected in media containing 150 μg/ml hygromycin for 2 weeks. For MCF-7 cells, single cell clones were obtained by limiting dilution, and homogenicity of the derived cell lines was tested by addition of 500 ng/ml doxycycline for 24 h and detection of mRFP by fluorescence microscopy.

DNA Content Analysis by FACS.

MCF-7 cells (5 × 104) were plated into T25 cell culture flasks. For analyses after siRNA transfections, cells were seeded and transfected in 12-well plates. Floating cells and trypsinized cells were collected by centrifugation at 290 × g for 7 min, fixed with ice cold 70% ethanol, and stored overnight on ice. After washing with PBS, 1 ml of FACS solution [PBS/0.1% Triton X-100/60 μg/ml propidium iodide (PI)/0.5 mg/ml DNase free RNase (Roche)] was added per sample and incubated at room temperature for 30 min. DNA content was determined by flow cytometry (FACSCalibur; Becton Dickinson).

Western Blot Analysis.

Cell lysis and immunoblotting were done as described in ref. 36. For immunodetection, membranes were incubated with antibodies specific for Cul7 (mouse hybridoma clone SA12 (37), p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA), p21 (Ab-11; NeoMarkers, Fremont, CA), HA (clone HA.11; Covance, Princeton, NJ), Flag (M2, F-3165; Sigma), P-Chk2 (Thr-68, sc-16297-R; Santa Cruz Biotechnology), α-tubulin (clone DM1A; Sigma), or β-actin (A-2066; Sigma). Signals from HRP (horseradish-peroxidase)-coupled secondary antibodies were generated by enhanced chemiluminescence (PerkinElmer Life Sciences, Boston, MA) and recorded with a CCD camera (440CF imaging system, Eastman Kodak).

Coimmunoprecipitation.

For immunoprecipitation (IP) of exogenous proteins, H1299, MCF-7, and HEK293 cells were transfected by using a calcium phosphate method. Cells were lysed at 4°C in lysis buffer [25 mM Tris·HCl, pH 8.0/150 mM NaCl/10 mM MgCl2/0.5% Nonidet P-40/2 mM sodium orthovanadate/1 mM DTT/1 mM NaF/50 units/ml DNase I (Roche)/complete mini protease inhibitor mixture (EDTA free, Roche)/0.25% Phosphatase Inhibitor Mixture I (Sigma)]. Cleared lysates were rotated at 4°C with the respective antibodies for 3 h, and 25 μl of Protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) was added for an additional 2 h. After washing four times with 25 mM Tris·HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, and 0.5% Nonidet P-40, proteins were separated by SDS/PAGE and subjected to Western blot analysis.

In Vivo Ubiquitination.

MCF-7 and H1299 cells were transfected by using a calcium phosphate method with VSV-tagged p53, His-tagged ubiquitin [pMT107 (38)] and the respective E3 ligase or empty vector control. MG132 (Axxora, San Diego, CA), if used, was added at a final concentration of 20 μM 4 h before harvest. Cells were harvested 36 h after transfection by scraping into lysis buffer (6 M guanidine-HCl/0.1 M NaHPO4/0.1 M Tris/5 mM N-ethylmaleimide, pH 8.0) and sonicated to shear the DNA. Lysates were incubated for 8 h at 4°C with 40 μl of Ni-NTA-agarose beads (Qiagen, Hilden, Germany), after which they were washed twice with wash buffer A (8 M urea/10 mM Tris/0.1 M NaHPO4/0.05% Tween 20/5 mM N-ethylmaleimide, pH 8.0), followed by two washes in wash buffer B (8 M urea/10 mM Tris/0.1 M NaHPO4/0.05% Tween 20/5 mM N-ethylmaleimide, pH 6.4), and one final wash with PBS. Ni-NTA-agarose bound proteins were eluted by boiling 10 min in 2× Laemmli buffer plus 250 mM imidazole, separated by SDS/PAGE and subjected to Western blot analysis.

Microscopy.

mRFP and phase contrast images of living cells were obtained on an inverted Axiovert 200M microscope (Zeiss, Oberkochem, Germany) by using Metamorph software (Universal Imaging).

Supplementary Material

Supporting Information
pnas_0609467104_index.html (12.1KB, html)

Acknowledgments

We thank Stefan Müller (MPI of Biochemistry, Martinsried, Germany), Gunter Meister (MPI of Biochemistry, Martinsried, Germany), Zhen-Qiang Pan (Columbia University, New York, NY), James DeCaprio (Dana–Farber Cancer Institute, Boston, MA), Bert Vogelstein (The Johns Hopkins University, Baltimore, MD), and Georg Bornkamm (Institute for Clinical Molecular Biology and Tumor Genetics, GSF, Munich, Germany) for generously providing reagents. The H.H. laboratory is supported by the Max-Planck-Society.

Abbreviations

ATM

ataxia telangiectasia mutated

Cul7

Cullin 7

PARC

PARkin-like, cytoplasmic, p53-binding protein

PIKK

phosphatidylinositol 3-kinase-related kinase

ATR

ATM- and Rad3-related

IP

immunoprecipitation

DOX

doxycycline.

Footnotes

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/0609467104/DC1.

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Supplementary Materials

Supporting Information
pnas_0609467104_index.html (12.1KB, html)
pnas_0609467104_1.pdf (93.9KB, pdf)
pnas_0609467104_2.pdf (55.1KB, pdf)
pnas_0609467104_3.pdf (143.8KB, pdf)
pnas_0609467104_09467Fig9A.JPG (81.9KB, JPG)
pnas_0609467104_09467Fig9B.JPG (174KB, JPG)
pnas_0609467104_4.pdf (84.1KB, pdf)
pnas_0609467104_09467Fig10C.JPG (181.4KB, JPG)
pnas_0609467104_09467Fig10D.JPG (67.3KB, JPG)
pnas_0609467104_5.pdf (164.1KB, pdf)
pnas_0609467104_09467Fig11C.JPG (58.7KB, JPG)

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