Treatment with a DNA-damaging agent stabilizes de novo interaction of Rad6 with p53 and p14ARF proteins.
To determine the effects of DNA damage on de novo expression of Rad6, normal MCF10A cells treated with cisplatin (5 μg/ml) or untreated were metabolically labeled with [35
S]methionine and analyzed for Rad6 expression by immunoprecipitation with anti-Rad6 antibody. Control immunoprecipitations were performed with equivalent amounts of normal rabbit IgG. Whereas nonimmune IgG did not precipitate 17-kDa proteins from control or cisplatin-treated lysates of MCF10A cells (Fig. , lanes C and T), immunoprecipitation with Rad6 antibody caused similar levels of de novo-synthesized Rad6 to immunoprecipitate from both control and cisplatin-treated MCF10A cells (Fig. , lanes 1 and 2). Besides immunoprecipitating Rad6, the Rad6 antibody effectively coprecipitated a protein doublet of ~53 kDa from both control and treated cells (Fig. , lanes 1 and 2). Since functional interactions between p53 and several key proteins involved in DNA repair have been reported (7
), we determined the identity of the 53-kDa protein to be p53 by subjecting the proteins immunoprecipitated by Rad6 antibody (Fig. , lanes 1 and 2) and those reimmunoprecipitated from Rad6-immunodepleted supernatants by p53 pAb421 antibody (Fig. , lanes 3 and 4) to Western blot analysis with p53 CM-1 antibody (Fig. , lanes 1′ to 4′). The relative levels of p53 recovered from Rad6 immunoprecipitates (Fig. , lanes 1′ and 2′) versus Rad6-immunodepleted supernatants (Fig. , lanes 3′ and 4′) were determined by comparing total p53 levels detected from corresponding samples without prior immunoprecipitation with Rad6 antibody (Fig. , lanes 5′ and 6′). The data from Fig. show that, whereas in the control cells ca. 80% of p53 was present in Rad6-immunodepleted supernatants, >50% of p53 was found to be associated with Rad6 after exposure to the drug. That a physical interaction, perhaps regulated by DNA-damaging agent, occurs between Rad6 and p53 is further confirmed by coprecipitation of a 14-kDa protein, identified to be p14ARF by Western blot analysis (data not shown and Fig. ), by both Rad6 and p53 antibodies from only cisplatin-treated MCF10A cells (Fig. , lanes 2 and 4).
FIG. 1. De novo interaction between Rad6 and p53. Exponentially growing MCF10A cells were either untreated or treated with 5 μg of cisplatin/ml for 6 h and then incubated in methionine-free DMEM supplemented with 100 μCi of [35S]methionine for (more ...)
FIG. 5. Effect of ADR on Rad6-p53-p14ARF interactions. MCF10A (A and B) or MDA-MB-231 (C, D, and E) cells were treated with ADR for 1 h, and cultures were rinsed and incubated with drug-free medium as described in Materials and Methods. At the indicated periods (more ...) Regulation of Rad6, p53, p14ARF, Hdm2, and Rad18 protein levels by ADR treatment.
Since results from Fig. demonstrated a potential de novo interaction regulated by cisplatin between Rad6, p53, and p14ARF, we examined the regulation of Rad6, Rad18, p53, p14ARF, and Hdm2 steady-state levels in normal MCF10A cells after exposure to ADR, a potent anthracycline and topoisomerase II inhibitor that is one of the most widely used cancer chemotherapeutic drugs. MCF10A cells were either left untreated or treated with predetermined IC50 dose of ADR (0.1 μg/ml) for 1 h; cells were rinsed, and cultures were incubated with fresh drug-free medium. Cell lysates were prepared from untreated samples and at 0-, 2-, 4-, 8-, 24-, 48-, and 72-h recovery periods after exposure to the drug and then analyzed for Rad6, Rad18, p53, p14ARF, and Hdm2 steady-state levels relative to β-actin by Western blot analysis. As shown in Fig. , an increase in Rad6 levels was evident at 8 h of recovery and steadily increased by 24 to 72 h posttreatment to approximately two- to fivefold-higher levels relative to the untreated control lysates. Steady-state levels of Rad18 protein exhibited a similar regulation profile by ADR treatment and reached approximately four- to sixfold-higher levels relative to the untreated control cells by 8 to 72 h of treatment (Fig. ). Both p14ARF and p53 exhibited dramatic and steady increases that were evident immediately after exposure to ADR. Levels of p14ARF were enhanced 5-fold relative to untreated control lysates at 0 h of recovery and steadily increased to ~15-fold by 48 to 72 h posttreatment (Fig. ). The p53 expression pattern after ADR exposure mirrored the p14ARF profile; however, levels of p53 were upregulated and were maintained at ca. 25- to 40-fold-higher levels relative to the controls in the period from 4 to 72 h after recovery from drug exposure. Induction of p53 was accompanied by a simultaneous increase in the appearance of p53 as a doublet band, a finding that may indicate a posttranslational modification of p53 (Fig. ). Although the steady-state levels of Hdm2 exhibited a modest increase after drug treatment, maintenance of higher levels of intact Hdm2 is probably impaired by the simultaneous accumulation of several lower-molecular-weight Mdm2-immunoreactive proteins (Fig. ). These data suggest that ADR significantly enhances Rad6, Rad18, p14ARF, and p53 proteins with only modest effects on Hdm2 levels.
FIG. 2. Effects of ADR on steady-state levels of Rad6, Rad18, p53, p14ARF, and Hdm2 proteins. MCF10A cells were treated with ADR (0.1 μg/ml) for 1 h, and the cultures were washed and replaced with fresh drug-free medium to allow for recovery. Cell lysates (more ...) Intact p53, p14ARF, and Hdm2 proteins are physically complexed with Rad6.
Since our data from Fig. and have demonstrated physical interactions between de novo-synthesized Rad6, p53, and p14ARF in cisplatin-treated cells and similar ADR-mediated inductory effects on Rad6, p14ARF, and p53 proteins, respectively, we investigated whether Rad6 exists in vivo as part of a supramolecular complex with proteins of the p53 pathway in ADR-treated MCF10A cells. MCF10A lysates prepared at 8 h of recovery after ADR treatment were subjected to immunoprecipitation with Rad6 antibody. Lysates prepared at 8 h posttreatment showed that Rad6, p53, p14ARF, Hdm2, and Rad18 are all upregulated (Fig. ). Rad6 immunoprecipitates were washed, boiled, and diluted 10-fold prior to reprecipitation with p53 pAb421, p14ARF, Mdm2, or Rad18 antibodies. The results (Fig. ) indicated the presence of immunoprecipitable Hdm2, p53, and p14ARF in Rad6-immunoprecipitated proteins from ADR-treated MCF10A cells. A similar analysis of control MCF10A lysates failed to reveal detectable amounts of p53, p14ARF, or Hdm2 in Rad6-immunoprecipitated proteins (data not shown). These data suggest the presence of a weak interaction between Rad6 and proteins of the p53 pathway in untreated MCF10A cells and that drug-induced effects on p53, p14ARF, and Hdm2 levels perhaps stabilize their interaction with Rad6. Rad18 was not detectable in Rad6 immunoprecipitates from both control and ADR-treated MCF10A lysates, and the majority of Rad18 was detected in Rad6-immunodepleted supernatants (data not shown). Amino acid residues 141 to 149 at the carboxyl terminus of Rad6 are essential for Rad18 binding (5
); thus, immunoprecipitation with the Rad6 antibody, which recognizes an epitope on amino acids 138 to 152, could occlude Rad6 interaction with Rad18.
FIG. 3. Rad6 interacts with p53, p14ARF, and Hdm2. ADR-treated MCF10A cell extracts (200 μg of protein) prepared at 8 h of recovery were subjected to immunoprecipitation with anti-Rad6 antibody. Immune complexes were pelleted with protein A/G-agarose, (more ...) Rad6-p53 interactions by surface plasmon resonance assay.
Our data from metabolic-labeling and immunoprecipitation experiments (Fig. and ) demonstrated the existence of physical interactions between endogenous Rad6 and p53 proteins. Surface plasmon resonance was used to verify whether there is a direct interaction between p53 and Rad6 proteins or whether this association is dependent on the presence of extraneous cellular factors. As shown in Fig. , Rad6 showed a dose-dependent increase in binding to immobilized GST-p53, suggesting that Rad6-p53 interaction occurred independently of cellular factors. The kon was determined from the association phase of binding of Rad6 to immobilized p53 by using the sensorgrams obtained with different concentrations of the soluble component and was 2.6 × 105 M−1 s−1. Similarly, koff was calculated from the dissociation phase and was 9.4 × 10−3 s−1. These data suggest that Rad6 complexes readily with p53, and the apparent Kd (koff/kon) value 3.6 × 10−8 M is in the range of strong interactions. In order to further determine the specificity of the Rad6-p53 interaction, the effects of various amounts of anti-Rad6 antiserum or the corresponding normal rabbit serum were tested on binding of Rad6 (400 nM) to immobilized GST-p53. As shown in Fig. , coinjection of Rad6 antibody caused a dose-dependent inhibition in the binding of Rad6 to immobilized GST-p53. Whereas the injection of Rad6 antibody at 1:10,000, 1:5,000, 1:2,000, or 1:500 produced 7, 15.5, 25.2, or 44% inhibition of Rad6 binding to GST-p53, respectively (Fig. ), similar coinjection of normal rabbit serum failed to alter Rad6 binding to immobilized GST-p53 (data not shown). Direct injection of Rad6 antibody (1:500) or BSA into immobilized p53 failed to elicit binding, further confirming the specificity of binding reactions (Fig. ).
FIG. 4. Rad6 binds to p53 in vitro. (A) Affinity-purified p53-GST was immobilized on a BIAcore 3000 sensor chip, and various concentrations of Rad6 protein were injected over the chip surface. Curves a to g represent binding curves obtained with 50, 100, 200, (more ...) Normal breast epithelial cells exhibit ADR-mediated stabilization of Rad6 complexed p53.
Since our data from Fig. have shown a direct interaction between Rad6 and proteins of the p53 pathway, we examined the effects of ADR on the stability of molecular complexes formed between Rad6 and p53 in normal MCF10A and metastatic MDA-MB-231 breast cells. MDA-MB-231 and MCF10A cells were chosen since they express mutant (T. Soussi, [http://p53.curie.fr/
]) and wild-type (47
) p53, respectively. MCF10A cell lysates prepared from untreated cells and at 0, 4, 24, and 72 h in the recovery period after ADR treatment were subjected to immunoprecipitation with Rad6 antibody, and immune complexes resolved by SDS-PAGE were assessed by Western blot analysis with antibodies to p53 or p14ARF. Complex formation was tested under both nonreducing and reducing conditions to determine whether variations in size and immunoreactivity to specific proteins are detectable. Consistent with ADR-induced regulation of p14ARF steady-state levels (Fig. ), the results (Fig. ) showed that no Rad6-immunoprecipitable p14ARF was detectable in the control MCF10A cell lysates. However, upon exposure to the drug, Rad6-immunoprecipitable p14ARF was detectable immediately after treatment and was localized to an ~150-kDa band. By 4 h posttreatment, p14ARF immunoreactivity was observed as a broad band spanning ca. 100 to 150 kDa, and by 24 h the majority of p14ARF immunoreactivity was increasingly localized to the 100-kDa band (Fig. ). Analysis of the corresponding immunoprecipitates in the presence of β-mercaptoethanol not only confirmed the presence of p14ARF in Rad6-immunoprecipitable complexes but also corroborated DNA damage-induced effects on p14ARF recruitment and formation of Rad6-p14ARF complexes observed in Fig. .
Similar analysis of Rad6-p53 interaction in MCF10A cell extracts from untreated control and 0, 4, 24, and 72 h after ADR treatment revealed the presence of Rad6-immunoprecipitable pAb421-immunoreactive p53 both in control and ADR-treated cells (Fig. ). These data suggest that Rad6-p53 interaction in MCF10A cells, unlike that observed with p14ARF, is not contingent upon exposure to drug. Rad6-immunoprecipitable p53 was found to be present as a broad smear spanning ca. 100 to 150 kDa (Fig. ). Analysis of the corresponding immunoprecipitates under reducing conditions confirmed the presence of p53 in Rad6-p53 (Fig. ).
Although equivalent amounts of antibody and total cellular proteins were included in each immunoprecipitation, it is interesting that the sizes of molecular complexes formed not only reflect a supershift caused by antibody reaction but also the stability of interactions between Rad6, p53, and p14ARF. This is evident from regulation of Rad6-p53 complex formation observed in ADR-treated metastatic MDA-MB-231 breast cancer cells (Fig. ). Analysis of Rad6-p53 interaction in metastatic MDA-MB-231 cell extracts from untreated controls and 0, 4, 24, and 72 h after ADR treatment, revealed the presence of detectable Rad6-immunoprecipitable pAb421-immunoreactive p53 in an ~160-kDa band only in drug-exposed cells (Fig. ). These data suggest that, unlike in normal MCF10A cells, exposure to the DNA-damaging drug is required to enhance and/or stabilize Rad6-p53 interaction in metastatic MDA-MB-231 cells (Fig. ). Interestingly, unlike in MCF10A cells, in which stable complexes of Rad6-p53 were detectable at least up to 72 h after ADR treatment, a 75% decrease in Rad6-immunoprecipitable p53 was observed at 24 h posttreatment, and by 72 h negligible p53 reactivity was seen under nonreducing conditions (Fig. ). The presence of p53 in Rad6-immunoprecipitable complexes was confirmed not only by the immunoreactivity of the complex with p53 antibody but also by derivation under reducing conditions of p53-immunoreactive 53-kDa band in amounts that were proportional to that present in complexes under nonreducing conditions (Fig. ). The reduction in the amounts of Rad6-p53 complexes observed at 24 and 72 h after ADR treatment was not due to inefficient immunoprecipitation by Rad6 antibody, since Western blot analysis of corresponding Rad6 immunoprecipitates showed significant amounts of Rad6 in all samples (Fig. ). In contrast, Western analysis of p53 steady-state levels showed p53 induction in ADR-treated samples at 0, 4, and 24 h and significant reduction at 72 h posttreatment (Fig. ). These data indicate that, although ADR-induced p53 response is associated with an upregulation in interaction between Rad6 and p53 in metastatic MDA-MB-231 cells, prolonged maintenance of Rad6-complexed p53 in metastatic MDA-MB-231 cells is impaired, in contrast to the situation in normal MCF10A cells.
ADR induces Hdm2 degradation via the ubiquitin-proteasome pathway.
Since the results of Fig. showed ADR to exert dramatic inductory effects on Rad6, p53, and p14ARF accumulation and modest stimulatory and/or degradation-inducing effects on Hdm2 in MCF10A cells, we investigated whether ADR-induced decline in Hdm2 occurs via the ubiquitin-proteasome pathway. Lysates of control and ADR-treated MCF10A cells were subjected to immunoprecipitation with Mdm2 antibody, and immune complexes resolved by SDS-PAGE under nonreducing conditions were analyzed by Western blotting with antibodies to ubiquitin or Mdm2. The results (Fig. ) demonstrated the presence of a prominent Mdm2-immunoreactive band at ~172 kDa during early periods of recovery (0 to 4 h) after ADR treatment that was not detectable in control cells (data not shown). At 24 h, a reduction in the signal of the 172-kDa band and the emergence of Mdm2-immunoreactive 110- and 55-kDa bands were observed. By 72 h, the intensities of the 172- and 110-kDa bands decreased >90% and were replaced by a proportional increase in a band at 55 kDa and a smaller band at 40 kDa. When the same blot was stripped and reprobed with antiubiquitin antibody, a similar pattern of immunostaining was observed (Fig. ). Intense ubiquitin-immunoreactive Mdm2-immunoprecipitable bands were observed at ~172 and 150 kDa during 0 to 4 h of recovery, followed by decreases in the 172- and 150-kDa bands at 24 h and subsequent increases in bands at ~110 and 55 kDa at 72 h posttreatment (Fig. ). These data suggest that ADR treatment may facilitate Hdm2 degradation via the ubiquitination pathway.
FIG. 6. ADR treatment induces Hdm2 degradation. MCF10A cells were treated with ADR for 1 h, and cultures were rinsed and incubated with drug-free medium. Cell extracts prepared at indicated periods of recovery were subjected to immunoprecipitation with Mdm2 antibody, (more ...)
To obtain further evidence that ADR-induced Hdm2 ubiquitination and degradation occur via the 26S proteasome, MCF10A cells were treated with ADR prior to treatment with MG132, a 26S proteasome inhibitor, and lysates were analyzed for Hdm2 steady-state levels by Western blot analysis with Mdm2 antibody. The results of Fig. show that exposure of ADR-treated cells to MG132 resulted in the accumulation of >25-fold-higher levels of intact Hdm2 by 24 h compared to those in untreated control samples or during early periods of recovery. These data suggest that the ADR-induced degradation of Hdm2 in MCF10A cells occurs at least in part via the ubiquitin-proteasome pathway since the decrease in intact Hdm2 levels induced by ADR (Fig. and B) can be rescued by treatment with a proteasome inhibitor (Fig. ).
To determine whether the stabilization of Rad6-p53 complex formation observed in MCF10A cells parallels a corresponding decay of Hdm2, we examined the distribution of Hdm2 and Rad6 by immunofluorescence microscopy in control and ADR-treated MCF10A cells. Hdm2 was localized in the nucleus and excluded from nucleoli in untreated MCF10A cells (Fig. ). After exposure to ADR, i.e., at 2 h of recovery, Hdm2 levels were significantly elevated and Hdm2 immunoreactivity was localized both to nuclear bodies and nucleoli (Fig. ). Consistent with immunoblotting experiments (Fig. ), a 60% decline in Hdm2 immunoreactivity was observed in the nuclei at 24 h posttreatment, and by 72 h >90% of nuclei exhibited only diffuse staining in the nucleoplasm and substantial staining in the cytoplasm (Fig. ). Immunofluorescence localization of Rad6 in control MCF10A cells revealed diffuse staining in the cytoplasmic and nuclear compartments. However, treatment with ADR induced a preferential redistribution of Rad6 from the cytoplasm to the nucleus that was reflected by detection of elevated levels of Rad6 in the nucleus at least until 72 h posttreatment (Fig. ). These results suggest that ADR exerts opposing effects on the stability of Rad6 and Hdm2 in MCF10A cells.
ADR-stabilized p53 is ubiquitinated and colocalizes with Rad6 in the nucleus.
Since treatment of MCF10A cells with ADR enhances both the steady-state levels of p53 and Rad6 and prolongs the stability of Rad6-p53 complexes, we investigated the effects of ADR on in vivo p53 ubiquitination status. Lysates prepared from MCF10A cells treated with ADR prior to treatment with MG132 were immunoprecipitated with pAb421 antibody. Immune complexes were resolved by SDS-PAGE and subjected to Western blot analysis with antibodies specific to polyubiquitinated protein conjugates (Fig. ), p53 CM-1 (Fig. and C), or ubiquitin-protein conjugates (Fig. ). Analysis of p53 with CM-1 antibody in control and ADR-treated MCF10A cells exposed to MG132 revealed that the majority of p53 immunoprecipitated with the p53 antibody was ubiquitinated since overexposure of the blots was necessary to visualize the presence of normal nonubiquitinated p53 (Fig. and C). Quantitation of relative intensities of nonubiquitinated p53 showed that samples at 8, 24, 48, and 72 h after ADR treatment contained ~8-fold-higher levels of p53 compared to control and earlier periods of post-ADR treatment (Fig. ). Short-time exposure revealed that ca. 15- to 25-fold-higher levels of monoubiquitinated p53 were present in ADR-treated samples at 8, 24, 48, and 72 h posttreatment compared to controls or at 0 and 4 h after ADR treatment (Fig. ). In addition to the 62-kDa p53 immunoreactive band, a prominent p53-immunoreactive band with a molecular size of ~100 kDa was also detected, the latter probably comprising p53 molecules carrying five molecules of ubiquitin (Fig. ). The levels of polyubiquitinated p53 in control and at 0 h after ADR treatment were ~8-fold higher than in samples at 4 and 8 h posttreatment and ~5-fold higher than in samples at 24, 48, and 72 h posttreatment (Fig. ).
FIG. 7. ADR enhances monoubiquitination of p53. Control and ADR-treated cultures were pretreated with MG132, and extracts prepared at the indicated time points were subjected to immunoprecipitation with p53 pAb421 antibody. Immune complexes were subjected to (more ...)
Reprobing the blots with polyubiquitinated protein-specific FK1 antibody further substantiated that the 100-kDa p53-immunoreactive band indeed represented polyubiquitinated p53 and was most prominent in control samples and in samples at 0 h post-ADR treatment (Fig. ). It is interesting that although nonubiquitinated p53 was not detected with polyubiquitin-specific antibody, faint bands corresponding to 62, 70, 80, and 90 kDa were detected in most samples. It is not clear whether these bands represent nonspecific immunoreactivity to p53 molecules with limited ubiquitination or multimonoubiquitination. Densitometric analysis of the FK1-reactive 100-kDa band indicated its presence at ~10-fold-higher levels in control samples and in samples at 0 h post-ADR treatment compared to samples at 4, 8, 24, 48, and 72 h posttreatment. Immunoblotting with FK2, an antibody that recognizes all ubiquitin-protein conjugates, showed the presence of >50-fold-higher levels of monoubiquitinated p53 in ADR-treated samples at 8, 24, 48, and 72 h posttreatment compared to those in control samples or in samples at 0 and 4 h post-ADR treatment (Fig. ). The relative levels of unubiquitinated p53, monoubiquitinated p53, and polyubiquitinated p53 detected with p53 CM-1 and FK1 antibodies, respectively, are graphically summarized in Fig. . These data indicate that p53 is polyubiquitinated in control samples and during the initial periods of ADR treatment. However, the drug-induced response is accompanied by a decrease in polyubiquitinated p53 that is coupled with a dramatic and concomitant increase in the levels of monoubiquitinated p53.
In order to determine whether the increase or decrease in monoubiquitinated p53 versus polyubiquitinated forms, respectively, reflected an increase in deubiquitinating enzyme activity in ADR-treated cells, we measured the ubiquitin-hydrolyzing activity in control and ADR-treated cells at 0, 4, 8, 24, 48, and 72 h of recovery according to the assay described by Dang et al. (13
). The results in Fig. show that there was no significant difference in Ub-AMC-hydrolyzing activity between control and ADR-treated samples that can account for the high levels of polyubiquitinated p53 or monoubiquitinated p53 in control and ADR-treated samples, respectively. Inclusion of ubiquitin hydrolase inhibitor, ubiquitin aldehyde, abolished the Ub-AMC-hydrolyzing capacity of the extracts by >90%, thus confirming the specificity of ubiquitin hydrolase (Fig. ). These data suggest that alterations in the ratio of monoubiquitinated p53 to its polyubiquitinated forms is not a result of an increase in deubiquitinating activity but rather is due to alterations in Hdm2 E3 ligase activity that is required for the polyubiquitination of p53.
To determine the cellular localization of Rad6 and p53 and to confirm whether the Rad6 complexed p53 is indeed ubiquitinated, we probed control and ADR-treated (at 72 h of recovery) MCF10A cells with antibodies to Rad6, p53, or ubiquitin. Whereas negligible immunoreactivity to p53 was observed in untreated cells (Fig. ), exposure to ADR caused a dramatic appearance of pAb421-immunoreactive p53 in the nucleoplasm and nucleoli of MCF10A cells (Fig. ). Similarly, treatment with ADR induced preferential accumulation of Rad6 in the nuclei of MCF10A cells (Fig. and Fig. ) compared to untreated cells that displayed diffuse Rad6 staining in the cytoplasm and nucleus (Fig. and Fig. ). Double immunofluorescence labeling and image-merging experiments demonstrated the colocalization of Rad6 with p53 (Fig. ), p53 with ubiquitin (Fig. ) and Rad6 with ubiquitin (Fig. ) in the nucleoplasm and nucleoli of ADR-treated cells when Hdm2 was undetectable (Fig. ). These data not only confirm the results from coprecipitation studies but also provide further evidence for ADR-induced effects on p53 ubiquitination, colocalization of p53 with Rad6, and the stability of Rad6-p53 complexes.
We next examined the effects of ADR on cell cycle progression in MCF10A and MDA-MB-231 cells since both cell lines show Rad6-p53 complex formation but with distinctly different stabilities. Treatment of MCF10A cells with 0.1 μg of ADR/ml induced G2/M cell cycle arrest by 24 h in ~90% of cells that persisted at least up to 72 h of treatment, whereas similar analysis of untreated MCF10A cells at corresponding time points revealed normal cell cycle progression (Fig. ). Similar flow cytometric analysis of MDA-MB-231 cells revealed that 51% of the cells arrested in G2/M at 24 h, and at 72 h a majority of the cells (58%) were found to arrest in G0/G1 compared to only 28% in the G2/M phase (Fig. ).
ADR treatment induces differential effects on cell cycle arrest in MCF10A and MDA-MB-231 cells. Cell cycle progression was analyzed in control or ADR-treated MCF10A and MDA-MB-231 cells at 24, 48, and 72 h by using a FACScan.
Demonstration in vitro of Rad6- and Mdm2-mediated effects on p53 ubiquitination.
Since our data showed that (i) a significant amount of p53 is associated with Rad6 in ADR-exposed MCF10A cells, (ii) ADR induces physical colocalization of Rad6 with p53 in MCF10A nuclei, (iii) a substantial amount of the p53 present in the nuclei of ADR treated MCF10A cells is ubiquitinated under conditions when Hdm2 is undetectable, and (iv) the majority of the posttranslationally stabilized p53 in ADR-treated MCF10A cells is indeed monoubiquitinated, we tested the effects of Rad6 singly or in combination with Mdm2 on p53 ubiquitination in a cell-free system. Recombinant Rad6 was incubated with GST-p53 in the presence or absence of GST-Mdm2 as indicated (Fig. ). Under nonreducing conditions, E1-Ub thioesters are detectable both in the presence and in the absence of p53, and E1 per se has no effect on p53 ubiquitination (Fig. ). Interestingly, although thioester intermediates of E1-Rad6 are readily detectable in reactions lacking p53 (data not shown), E1-Rad6 thioester intermediates were not observed in reactions containing p53. These data suggest that inclusion of a suitable ubiquitination substrate such as p53 may promote rapid transfer of activated ubiquitin to the substrate. The molecular sizes of ubiquitinated p53 correspond with the presence of one or two ubiquitin molecules (Fig. ). The effects of Rad6 on p53 ubiquitination are specific since assay mixtures not containing Rad6 are unable to mediate transfer of ubiquitin to p53. Inclusion of GST-Mdm2 into reactions containing E1 and Rad6 induced polyubiquitination of p53, as confirmed by its immunoreactivity to ubiquitin (Fig. ), polyubiquitin (Fig. ), and p53 CM-1 (Fig. ) antibodies. These data suggest that, whereas Rad6 mediates restrictive ubiquitination of p53, Mdm2 functions by extending ubiquitin chains.
FIG. 9. Effects of Rad6 and Mdm2 on p53 ubiquitination. GST-p53 was incubated with ATP, ubiquitin, and E1 in the absence or presence of Rad6 or in the presence of both Rad6 and GST-Mdm2 as indicated. Reactions were terminated after 2 h at 30°C and then (more ...)