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Originally published In Press as doi:10.1074/jbc.M305171200 on September 18, 2003

J. Biol. Chem., Vol. 278, Issue 48, 47853-47861, November 28, 2003
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Prohibitin Induces the Transcriptional Activity of p53 and Is Exported from the Nucleus upon Apoptotic Signaling*

Gina Fusaro{ddagger}§, Piyali Dasgupta{ddagger}||, Shipra Rastogi{ddagger}||, Bharat Joshi{ddagger}, and Srikumar Chellappan{ddagger}**

From the {ddagger}Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612 and the §Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Received for publication, May 16, 2003 , and in revised form, September 12, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prohibitin, a potential tumor suppressor protein, has been shown to inhibit cell proliferation and repress E2F transcriptional activity. Though prohibitin has potent transcriptional functions in the nucleus, a mitochondrial role for prohibitin has also been proposed. Here we show that prohibitin is predominantly nuclear in two breast cancer cell lines where it co-localizes with E2F1 and p53. Upon apoptotic stimulation by camptothecin, prohibitin is exported to perinuclear regions where it localizes to mitochondria. The data presented here also show that prohibitin is capable of physically interacting with p53 in vivo and in vitro. Prohibitin was found to enhance p53-mediated transcriptional activity and cotransfection of an antisense prohibitin construct reduces p53-mediated transcriptional activation. Prohibitin appears to induce p53-mediated transcription by enhancing its recruitment to promoters, as detected by chromatin immunoprecipitation assays. These results suggest that prohibitin is capable of modulating Rb/E2F as well as p53 regulatory pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The E2F family of transcription factors play a major role in cell proliferation, differentiation, and apoptosis. The E2F family members have been shown to upregulate the expression of many genes involved in G1/S transition and DNA synthesis such as cyclin E, Cdc25A, DHFR, and DNA polymerase {alpha} (reviewed in Refs. 1–3). The transcriptionally active E2F family members, E2Fs 1–5, are maintained in an inactive state by members of the Rb1 family while E2F6 lacks a transactivation domain (4). The Rb family proteins have been shown to suppress E2F-mediated transcription by recruiting a variety of transcriptional co-repressors including HDAC1, DNMT, polycomb proteins as well as chromatin remodeling complexes like Brg and Brm (5–9). Of the E2F family members, E2F1 is unique in its ability to induce apoptosis (reviewed in Ref. 10); this is achieved through induction of pro-apoptotic genes, including Apaf-1 and p73 (11, 12). In addition, E2F1 is known to induce p53 activity by inducing the expression of pl4/pl9ARF, which inhibits MDM2-mediated degradation of p53 (13, 14).

Studies from our laboratory showed that the activity of E2F transcription factors could be repressed by a potential tumor suppressor protein, prohibitin. Prohibitin was found to bind to the pocket domain of Rb family members and contact E2F family members through the marked box domain (15, 16). Though prohibitin was originally cloned based on its ability to induce growth arrest in human fibroblasts, its mode of action has remained unclear (17, 18). In addition to our findings on E2F regulation, a protein very similar to prohibitin has been found to regulate estrogen receptor-mediated transcription (19, 20). It has also been proposed that prohibitin localizes to the inner mitochondrial membrane, where it functions in maintaining mitochondrial morphology and inheritance (21, 22).

Attempts to study prohibitin-mediated regulation of E2F1 showed that while there are similarities with Rb in the repression patterns, there are significant differences in the mechanisms involved as well as how the two proteins respond to signals. Thus unlike Rb, prohibitin-mediated repression of E2F1 cannot be reversed by the adenovirus E1A protein, nor by cyclin D or E-associated kinase activity (16). While Rb recruits HDAC1 to repress E2F, prohibitin utilizes both HDAC1 and N-CoR for optimum repression of E2F activity (23). Chromatin-remodeling proteins like Brg and Brm are implicated in the repression mediated by both the proteins (24). We also find that prohibitin, when overexpressed in Ramos B cells, can protect against apoptosis induced by the topoisomerase I inhibitor camptothecin (25). These studies suggested that prohibitin is a regulator of E2F function that aids in decisions between proliferation and apoptosis.

It has been demonstrated that the pro-apoptotic activity of E2F1 is negated by the anti-apoptotic activity of Rb (26). Like Rb, prohibitin has been shown to inhibit apoptosis in at least two different scenarios: in camptothecin-induced apoptosis as well as growth factor withdrawal-induced apoptosis (25, 27). Because of the important points of intersection of the E2F and p53 pathways, and because prohibitin regulates E2F function as well as apoptosis, experiments were designed to determine whether prohibitin and p53 functionally interact. We find that a significant portion of prohibitin is localized in the nucleus of T47D and MCF7 cells, where it co-localizes with both E2F1 and p53. After apoptosis induced by the drug camptothecin, however, these proteins are found mainly in the cytoplasm. We provide evidence that prohibitin can bind p53 and induce p53 transcripitonal activity while an antisense prohibitin construct ablates p53 activity. Overexpression of prohibitin also augmented promoter binding by p53 in vivo, as determined by chromatin immunoprecipitation (ChIP) assays. Conversely, prohibitin inhibited E2F1 binding to a target site in ChIP assays. Based on these results, we propose that prohibitin is a unique regulator of both E2F1 and p53, and may provide a link between proliferatory and apoptotic pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Plasmids, and Transfections—T47D and MCF7 breast carcinoma cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The Ramos B cell lymphoma line was maintained in RPMI 1640 supplemented with 10% fetal bovine serum. Stable cell lines were selected in the presence of 40 µg/ml of G418 (Sigma). MCF7 cells expressing tetracycline-inducible prohibitin were grown in DMEM supplemented with 10% tetracyclinefree fetal bovine serum (Clontech), 25 µg/ml Zeocin (Invitrogen), and 0.25 µg/ml Blasticidin (Invitrogen). Prohibitin expression was induced by incubation of the cells in 1 µg/ml tetracycline for 24 h.

The MDM2-CAT reporter and pCMV.p53 vectors have been previously described (28), as has the pCMV.MDM2 (29), pCMV.p300, and pGEX2TK.p53 vectors (30). pCDNA3.E2F, pCDNA3.prohibitin, pCR3.1.antisense-prohibitin, pCR3.1.prohibitin-(1–157) and pCR3.1 prohibitin-(116–275) and pGEX2TK.prohibitin are described in Refs. 15 and 16.

Transient transfections of T47D and MCF7 cells were performed by the calcium phosphate method with 2–8 µg of each plasmid DNA as previously described. Transient transfection of 1 x 107 Ramos cells was performed by electroporation using a Bio-Rad Gene Pulser at 400 mV. Each reaction contained 2 µg of {beta}-galactosidase to serve as an internal control for transfection efficiency. Assays for {beta}-galactosidase activity and chloramphenicol acetyltransferase activity were performed 72 h after transfection using standard protocols (31).

Preparation of Nuclear and Cytsolic Extracts—Nuclear and cytosolic fractions were made as described in Ref. 38. Asynchronous MCF-7 cells were cultured to 70% confluence and incubated in the presence or absence of 30 µM camptothecin for 4 h at 37 °C. Briefly, the cells were washed in PBS and resuspended in one packed cell volume of ice-cold nuclear extraction buffer 1 or NE1 (10 mM HEPES, pH 8.0, 1.5 mM MgCl2 10 mM KCl, 1 mM dithiothreitol). The cell suspension was passed through a 22-gauge needle five times. Thereafter the cell suspension was spun at 20,000 x g for 30 s. The supernatant was collected and spun down at 100,000 rpm. This was the cytosolic fraction that was aliquoted and stored at –70 °C. The nuclear pellet was resuspended in two-third-packed cell volume of high salt buffer (20 mM HEPES, pH 8.0, 1.5 mM MgCl2 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and rotated on ice for 30 min. The nuclear debris was spun down at 20,000 x g for 5 min. The supernatant was dialyzed against NE1 for 2 h. The dialysate was aliquoted and stored at –70 °C. The purity of the nuclear fraction was assessed by performing a Western blot for PARP, using 50 µg of nuclear extract and cytosolic fraction. Physical interaction between proteins in vivo was analyzed by immunoprecipitation-Western blot analysis using 200 µg of extract and 2 µg of the indicated antibody, as described before.

In Vitro Binding Assay—GST, GST-p53, and GST-Phb were purified from bacterial cultures and bound to glutathione-Sepharose beads as previously described (15). Beads were then washed three times with PBS, and protein integrity was checked by polyacrylamide gel elecrophoresis and Coomassie Blue staining. [35S]Methionine-labeled lysates of p53 or prohibitin were made using the rabbit reticulocyte translation system according to the manufacturer's directions (Promega). 10 µl of labeled lysates was incubated with an equivalent amount of GST or GST-Phb beads in a buffer containing 20 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40, 50 mM KCI, 500 mM EDTA, and 3 mg/ml bovine serum albumin, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. Samples were incubated for 2 h at 4 °C and then washed in binding buffer six times. Bound proteins were eluted in SDS-PAGE loading buffer and resolved by polyacrylamide gel electrophoresis.

Drug Treatment, Antibodies, and Immunostaining—The prohibitin monoclonal antibody was purchased from NeoMarkers, Inc. (Freemont, CA). The p53 polyclonal, E2F1 polyclonal and cytochrome c polyclonal antibodies were purchased from Santa Cruz Biotechnology. T47D or MCF7 cells were plated onto poly-D-lysine (Sigma) coated 8-well glass chamber slides (10,000 cells per well). Cells were either untreated or treated with 30 µM camptothecin for 4 h (Sigma) unless otherwise indicated. Cells were fixed in 3.5% paraformaldehyde for 25 min, permeabilized in 0.2% Triton X-100/PBS for 5 min, and blocked in 5% normal goat serum in PBS at room temperature for 1 h. Primary antibody incubations were performed overnight with appropriate antibodies at 4 °C. After washing, secondary antibody incubation was performed with goat anti-mouse IgG Alexa Fluor-488 and goat anti-rabbit IgG Alexa Fluor-546 for 30 min at room temperature. DNA was labeled with Hoechst staining at a final concentration of 0.0025 mg/ml. Cells were visualized with a Zeiss LSM 510 (Zeiss, Thornwood, NY) confocal microscope and areas of co-localization were determined using LSM 510 software (Zeiss).

Chromatin Immunoprecipitation Assay—One confluent plate of T47D or MCF7 cells (about 3 x 106 cells per plate) were used for each immunoprecipitation reaction, as described previously (23). The HA monoclonal, p53 monoclonal, and E2F1 monoclonal antibodies were purchased from Santa Cruz Biotechnology. The prohibitin monoclonal antibody was purchased from Neomarkers, Inc. (Freemont, CA). PCR reactions were then performed using 5 µl of the DNA from the immunoprecipitation reactions or 1 µl of DNA from the input reaction as template. PCR cycling conditions were as follows: 94 °C for 2 min; then 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 68 °C for 30 s; followed by 68 °C for 2 min. The sequences of the PCR primers used in the PCR reactions were as follows: MDM2 promoter (forward primer) 5'-AGTGTGAACGCTGCGCGTAGTC-3', MDM2 promoter (reverse primer) 5'-CCCACAGGTCTACCCTCCAATC-3', cdc25A promoter (forward primer) 5'-TCT GCT GGG AGT TTT CAT TGA CCT C-3', cdc25A promoter (reverse primer) 5'-TTG GCG CCA AAC GGA ATC CAC CAA TC-3', fos promoter (forward primer) 5'-TGT TGG CTG CAG CCC GCG AGC AGT TC -3', fos promoter (reverse primer) 5'-GGC GCG TGT CCT AAT CTC GTG AGC AT-3', MT-1G promoter (forward primer) 5'-TGC GCT CAA GGG ACC TTG CA-3', MT-1G promoter (reverse primer) 5'-CTC GAG CCC AAC AGC CA-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subcellular Localization of Prohibitin Changes upon Apoptotic Stimulation—Our earlier studies have shown that prohibitin represses E2F-mediated transcription and associates with E2Fs 1–5 in immunoprecipitation-Western blotting experiments. Since E2F1 is predominantly nuclear and other groups have proposed mitochondrial functions for prohibitin, we decided to examine where in the cell prohibitin binds to E2F1. For this purpose, human breast carcinoma T47D and MCF7 cells were immunostained with a monoclonal antibody against prohibitin, and its presence detected by a secondary antibody labeled with Alexa Fluor-488, a green fluorochrome. Cells were co-stained with a polyclonal antibody against E2F1 and detected by Alexa Fluor-546, a red fluorochrome. Cells were visualized by confocal microscopy. E2F1 localized primarily in the nucleus of healthy cells, as expected (Fig. 1, A and C). Prohibitin also stained in a strikingly nuclear fashion in both cell lines. Discrete yellow or white areas of co-localization could be observed when the images were superimposed (Fig. 1, A and C).



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  		FIG. 1.
Prohibitin co-localizes with E2F1 in the nuclei of untreated breast cancer cells, but not in camptothecin-treated cells. A and B, untreated T47D cells (A) or cells treated with 30 µM camptothecin for 4 h (B) were immunostained with an anti-prohibitin monoclonal antibody and a polyclonal anti-E2F1 antibody. Cells were visualized by confocal microscopy. C and D, the same experiment described above was performed in MCF7 cells. Yellow and white foci indicate areas of colocalization.

 
Experiments were designed to examine whether the localization pattern of the proteins was altered upon apoptotic stimulation with camptothecin. T47D and MCF7 cells were treated with 30 µM camptothecin for 4 h and the localization of prohibitin and E2F1 was determined by immunostaining as above. Both prohibitin and E2F1 were found primarily in the cytoplasm in both cell lines after treatment (Fig. 1, B and D). In addition, prohibitin staining appeared to occur in perinuclear regions. Almost no areas of co-localization were observed after camptothecin treatment, suggesting that very little, if any, prohibitin is associated with E2F1 in the cytoplasm.(Fig. 1, B and D). It may be concluded that the previously observed binding and functional interaction between E2F1 and prohibitin are predominantly nuclear events in proliferating cells. In response to camptothecin treatment, prohibitin translocates to the cytoplasmic compartment modulating the apoptotic response.

Camptothecin Treatment Induces Partial Mitochondrial Localization of Prohibitin—Since it was found that camptothecin treatment induced prohibitin to exit from the nucleus to the perinuclear regions, it was next examined whether prohibitin co-localized to mitochondria in this area. Toward this purpose, cells were stimulated with camptothecin and a double immunofluorescence experiment was conducted to see whether prohibitin co-localized with the mitochondrial marker cytochrome c. The staining pattern was visualized by confocal microscopy. It was found that prohibitin staining was predominantly nuclear in untreated cells while cytochrome c appeared punctate throughout the cytoplasm and was absent in the nucleus (Fig. 2, A and C). Superimposing the images did not reveal any areas of co-localization. However, after camptothecin treatment, a significant amount of prohibitin could be observed outside the nucleus, in a similar staining pattern as cytochrome c. Overlay of these images revealed many areas of co-localization throughout the cytoplasm and in the perinuclear region, in both the cell lines tested (Fig. 2, B and D). These results suggested that prohibitin migrates to the mitochondria only when the cells are subjected to stress.



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  		FIG. 2.
Prohibitin co-localizes with cytochrome c after camptothecin treatment. A and B, untreated T47D cells (A) or cells treated with 30 µM camptothecin for 4 h (B) were immunostained with an anti-prohibitin monoclonal antibody and a cytochrome c polyclonal antibody. After addition of goat anti-mouse IgG Alexa Fluor-488 and goat anti-rabbit IgG Alexa Fluor-546 secondary antibodies, cells were visualized by confocal microscopy. Areas of colocalization are seen as yellow or white spots. C and D, the same experiment described above was performed in MCF7 cells.

 
Prohibitin Co-localizes with p53—The preceding experiments suggested that prohibitin underwent a signal-dependent translocation upon apoptotic signaling. Together with our previous results indicating that prohibitin has growth suppressive as well as anti-apoptotic properties, it seemed likely that prohibitin may be interacting with apoptotic proteins as well. Since proteins like DP1 and MDM2 interact with both E2F and p53, experiments were designed to examine whether prohibitin interacts with p53 as well. As a first step, a double immunofluorescence experiment was done on T47D and MCF-7 cells using an anti-prohibitin mouse monoclonal antibody and an anti-p53 rabbit polyconal antibody. Both prohibitin and p53 were predominantly nuclear in both the cell lines; upon superimposing the images, distinct yellow or white areas were observed in the nucleus where prohibitin co-localized with p53 (Fig. 3, A and C). This result suggested a potential physical interaction between these proteins. Though a certain amount of p53 was observed in the cytoplasm, there was minimal colocalization with prohibitin outside the nucleus. It was next examined how the interaction of prohibitin with p53 was affected by camptothecin treatment. Treatment with camptothecin led to prohibitin and p53 exiting the nucleus in both the cell lines tested (Fig. 3, B and D). Camptothecin-induced translocation of p53 to mitochondria has been reported earlier in agreement with our findings (32). Interestingly, both prohibitin and p53 were found to concentrate in perinuclear regions, where they co-localized considerably. This result raised the possibility that prohibitin may be modulating apoptotic processes by affecting p53 function. The co-localization of prohibitin and p53 in T47D cells was examined 2, 4, and 8 h after camptothecin treatment (Fig. 4). It was found that in untreated cells and in the 2 h time point, prohibitin and p53 were found to co-localize in the nucleus (Fig. 4, A and B). After 4 h and 8 h of camptothecin treatment, majority of prohibitin and p53 were in the cytoplasm (Fig. 4, C and D) suggesting that the nuclear exit occurs as the cells progress through the apoptotic process.



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  		FIG. 3.
Prohibitin and p53 co-localize in the nuclei of untreated breast cancer cells, and migrate to the nuclear periphery after camptothecin treatment. A and B, untreated T47D cells (A) or cells treated with 30 µM camptothecin for 4 h (B) were immunostained with an anti-prohibitin monoclonal antibody and an anti-p53 polyclonal antibody and visualized by confocal microscopy. C and D, the same experiment described above was performed in MCF7 cells. Yellow and white foci indicate areas of co-localization.

 



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  		FIG. 4.
Site of co-localization of prohibitin and p53 in the cell changes upon camptothecin treatment. T47D cells were stimulated with 30 µM camptothecin for 2, 4, or 8 h (B–D) and the localization of prohibitin and p53 detected as in Fig. 3. Yellow and white spots indicate areas of co-localization.

 
Prohibitin and p53 Interact in Vivo—The co-localization of prohibitin with p53 raised the possibility that these two proteins are physically interacting with each other in the cell. This was examined by an immunoprecipitation-Western blot experiment. Cell lysates from MCF-7 cells were immunoprecipitated with antibodies to p53; anti-HA antibody was used as a negative control and antibodies to E2F1 and Rb were positive controls. As shown in Fig. 5A, prohibitin could be detected by Western blotting in p53, E2F1, and Rb immunoprecipitates, but not in the HA immunoprecipitate. We had shown earlier that prohibitin associates with Rb and E2F1; this experiment shows that prohibitin can associate with p53 as well and the interaction can be observed without overexpressing any component. The time course of association with p53 was next examined. Lysates from MCF-7 cells treated with camptothecin for 4, 8, or 16 h were immunoprecipitated with a p53 antibody and the presence of prohibitin in the immunoprecipitates was examined. It was found that prohibitin could be detected in p53 immunoprecipitates of all four samples (Fig. 5B) suggesting that the magnitude of the interaction does not change significantly during the time points tested.



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  		FIG. 5.
Association of prohibitin and p53 in vivo. A, an immunoprecipitation-Western blot analysis showing the association of endogenous prohibitin with p53, Rb, and E2F1. An anti-HA antibody was used as negative control for the immunoprecipitations. B, amount of prohibitin associated with p53 does not change significantly during camptothecin treatment, as seen by immunoprecipitation-Western blots. C, association of prohibitin and p53 can be detected in nuclear extracts of control MCF-7 cells but in cytoplasmic extracts of cells treated with camptothecin for 4 h. This correlates with the subcellular localization of the two proteins. A Western blot for PARP is shown as a nuclear marker. D, time course of apoptosis induction in MCF-7 cells by 30 µM camptothecin as seen by PARP cleavage.

 
It was next examined whether the two proteins interacted with each other in different compartments of the cell upon camptothecin treatment, as seen in the immunofluorescence experiment. Nuclear and cytoplasmic extracts were prepared from control MCF-7 cells or those treated with camptothecin for 4 h and the interaction examined by immunoprecipitation-Western blot experiments. Both prohibitin and p53 were found in the nuclear extract of control cells, but in the cytoplasmic extract of camptothecin-treated cells (Fig. 5C). As shown in Fig. 5C, top panel, prohibitin could be observed in p53 immunoprecipitate from the nuclear extract of control cells, but not in the cytoplasmic extract. In contrast, it could be found only in the immunoprecipitate of the cytoplasmic extract from camptothecin-treated cells, not in nuclear extracts. The extracts were tested for cross-contamination by doing a Western blot for PARP, which is a nuclear protein; as shown in Fig. 5C, bottom panel, PARP could be detected only in the nuclear extracts. This experiment shows that though the magnitude of the interaction did not change significantly, the site of interaction in the cell was altered upon camptothecin treatment, in agreement with the co-localization studies. Attempts were then made to examine whether the changes in the localization of prohibitin and p53 correlated with induction of apoptosis by camptothecin. Toward this purpose, PARP cleavage was examined in lysates from MCF-7 cells treated with 30 µM camptothecin for different periods. This is because several convergent studies have shown that lysate protease activity characterized by PARP cleavage is a good marker for caspase activation. PARP cleavage could be observed as early as 4 h after camptothecin treatment (Fig. 5, C and D) indicating onset of apoptosis; significant amount of PARP cleavage was observed within 8 h. The nuclear exit of prohibitin and p53 thus appears to correlate with the onset of camptothecin-induced apoptosis.

Prohibitin Interacts with p53 in Vitro—The finding that prohibitin co-localized and co-immunoprecipitated with p53 raised the possibility that these two proteins are interacting directly with each other. As an initial step to investigate this association, in vitro binding assays were performed. First, prohibitin protein was synthesized in the presence of [35S]methionine in rabbit reticulocyte lysates and incubated with glutathione S-transferase (GST) beads, or GST-p53 beads; GST-Rb was used as a positive control. The beads were washed extensively and the association of prohibitin was examined by autoradiography following SDS-PAGE. It was found that prohibitin bound to both the GST-p53 and GST-Rb beads efficiently, but there was no binding to the control GST beads (Fig. 6A). The binding was confirmed by performing the experiment in the opposite fashion: p53 protein was labeled with [35S]methionine in a rabbit reticulocyte lysate and incubated with control GST or GST-prohibitin (GST-Phb) beads. It was found that p53 could bind to GST-Phb, but not to GST beads (Fig. 6B), which again suggested that these two proteins can interact in vitro.



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  		FIG. 6.
Prohibitin binds to p53 in vitro. A, prohibitin was synthesized with [35S]methionine in a rabbit reticulocyte lysate and 10 µl of lysate was incubated with GST or GST-p53 beads. GST-Rb was used as a positive control. 2 µl of lysate was loaded in the prohibitin lysate lane. Prohibitin bound to both GST-p53 and GST-Rb, but not to GST beads. B, p53 was synthesized with [35S]methionine in a rabbit reticulocyte lysate, and 10 µl was incubated with GST or GST-Prohibitin (Phb) beads. p53 bound to GST-Phb, but not to GST. 2 µl of lysate was loaded in the p53 lysate lane (C) A T47D whole cell extract was incubated with GST or GST-Phb, and bound proteins were resolved by polyacrylamide gel electrophoresis. The presence of p53 was checked by Western blot analysis. A Western for c-Myc was performed as a negative control. D, the N terminus of prohibitin binds to p53. Deletion constructs corresponding to residues 1–157 of prohibitin or 116–275 were synthesized with [35S]methionine in a rabbit reticulocyte lysate, and 10 µl was incubated with GST or GST-p53. 2 µl of each lysate was loaded in the prohibitin lysate lane. Phb-(1–157) bound to GST-p53, but Phb-(116–275) did not bind.

 
A modified in vitro binding assay was performed to confirm these interactions. GST or GST-Phb beads were incubated with a T47D breast carcinoma cell whole cell extract, and the binding of p53 was assessed by Western blot analysis following SDS-PAGE. p53 associated with GST-Phb beads, but not with GST beads (Fig. 6C, upper panel). To confirm the specificity of the interaction, the blot was stripped and re-probed for c-Myc, which does not bind prohibitin. c-Myc was present in the T47D whole cell extract, but did not bind to GST or GST-Phb beads (Fig. 6C, lower panel). These experiments confirm that p53 and prohibitin can interact in vivo as well as in vitro.

Attempts were next made to determine the region of prohibitin, which associates with p53. Deletion fragments of prohibitin were used for this purpose in in vitro binding assays. In vitro synthesized prohibitin corresponding to residues 1–157 bound efficiently to GST-p53, whereas the region spanning residues 116–275 failed to bind (Fig. 6D). These results indicated that the N terminus of prohibitin contains the region necessary for binding to p53. Experiments were then designed to test the functional relevance of this interaction.

Prohibitin Enhances p53 Transcriptional Activity—Since prohibitin was found to repress E2F1-mediated transcriptional activity via the recruitment of HDAC1 and N-CoR, experiments were next designed to examine whether prohibitin could affect the transcriptional activity of p53 as well (23). Toward this purpose, transient transfection assays were conducted using a MDM2-CAT construct, which had a p53-responsive promoter element from the MDM2 gene fused to a CAT gene (MDM2-CAT). Co-transfection of p53 induced this promoter, as expected; surprisingly, co-transfection of prohibitin enhanced the p53-mediated transcription an additional 3-fold in both T47D as well as MCF7 cells (Fig. 7A). Prohibitin did not activate the promoter in the absence of p53 co-transfection, indicating that the effect is p53-dependent (data not shown). To further confirm these observations, a transient transfection experiment was performed on a Ramos human B cell lymphoma line that stably overexpressed prohibitin. This line as well as the parental Ramos line were transiently transfected with the MDM2-CAT reporter and p53. Similar to the results in T47D and MCF7 cells, the cells that stably overexpressed prohibitin had ~3-fold more p53-mediated transcriptional activity compared with the parental cells (Fig. 7B). These two experiments suggest that prohibitin can upregulate p53-mediated transcription, in contrast to its robust repressive effects on E2F family members.



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  		FIG. 7.
Prohibitin activates p53 transcriptional activity. A, 3 µg of a reporter construct encoding two p53 binding sites from the MDM2 promoter fused to a CAT (MDM2-CAT) gene was transiently transfected into T47D or MCF7 cells. Co-transfection of 3 µg of p53 activates the reporter, and this activation is augmented by cotransfection of 8 µg of prohibitin. Prohibitin does not activate the reporter in the absence of p53 transfection. B, Ramos cells or Ramos cells stably overexpressing prohibitin (Ramos-Phb) were transiently transfected with 3 µg of MDM2-CAT. Co-transfection of 3 µg of p53 stimulates reporter activity, which is enhanced in Ramos-Phb lines. C, prohibitin deletion constructs were tested for their ability to activate p53 in transient transfection experiments in T47D cells. While 8 µg of Phb-(1–157) activated p53 as well as full-length (FL) prohibitin, prohibitin, 8 µg of Phb-(116–275) failed to activate. D, co-transfection of antisense prohibitin represses p53 transcriptional activity. Transient transfection of T47D cells was performed as above. Co-transfection of 4 µg of an antisense prohibitin construct reduced p53 activity, to levels less than that of p53 alone. Transfection of the MDM2-CAT reporter plus the antisense construct alone did not result in CAT conversion, and neither did the vector alone (data not shown). A Western on lysates from transfected cells indicated that while prohibitin levels were reduced by antisense prohibitin, p53 levels were not affected.

 
The effect of the prohibitin deletion constructs on p53 activity was next checked in transient transfection experiments in T47D cells. Full-length prohibitin enhanced p53 activity, and the prohibitin construct corresponding to residues 1–157 was equally effective (Fig. 7C). However, the prohibitin construct that spanned residues 116–275, which could not bind p53, did not enhance its activity. Thus, the region of prohibitin that bound to p53 was also necessary to activate p53, suggesting that prohibitin may need to contact p53 in order to enhance its transcriptional activity.

As a further confirmation of the stimulatory effect of prohibitin on p53 function, an antisense experiment was carried out. T47D cells were co-transfected with MDM2-CAT, p53, and prohibitin. Prohibitin activated p53, as we had previously observed. p53 was then co-transfected with an antisense prohibitin construct, to ablate endogenous prohibitin expression. We found that the antisense prohibitin could effectively ablate p53 transcriptional activity, to levels less than that of p53 transfection alone (Fig. 7D). To ensure that the antisense construct was not interfering with p53 protein expression, a Western blot was performed on transfected cell lysates. We found that while prohibitin levels decreased, p53 levels were not affected (Fig. 7D, bottom panel). This indicated that the observed decrease in p53 activation was not due to loss of p53 protein but is due to the reduction in prohibitin levels.

Prohibitin-mediated Activation of p53 Is Independent of MDM2 and p300 —Attempts were made to determine the mechanism by which prohibitin stimulates p53 transcriptional activity. As a first step, transient transfections were performed in T47D cells to test whether prohibitin was activating p53 by inhibiting MDM2-mediated repression of p53 function. Transfection of MDM2-CAT and p53 led to CAT conversion; this was repressed by MDM2, as expected (Fig. 8A). Prohibitin activated p53 when the two proteins were co-transfected. MDM2 repressed p53 even when prohibitin was co-transfected, which implies that prohibitin is not enhancing p53 activity by overcoming MDM2-mediated repression.



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  		FIG. 8.
Prohibitin cannot overcome MDM2-mediated repression of p53 nor enhance p300-mediated activation of p53. A, transient transfections were performed in T47D cells. Transfection of 3 µg of MDM2-CAT plus 3 µg of p53 leads to CAT conversion, which was repressed by MDM2 (8 or 10 µg). Transfection of the reporter plus p53 and 8 µgof prohibitin leads to activation, but MDM2 could repress p53 in the presence of prohibitin. B, prohibitin does not cooperate with p300 to activate p53 activity. T47D cells were transfected with 3 µg each of MDM-CAT and p53, plus increasing amount of p300 (1–3 µg), which activated p53 in a dose-dependent manner. Addition of 8 µg of prohibitin with p300 did not lead to any further activation; rather a decrease in p53 activity was observed. Transfection of prohibitin and p53 without p300 led to activation of p53.

 
Next, we tested whether p300 contributes to prohibitin-mediated activation of p53 function. p300 acetylates the C terminus of p53, leading to transcriptional activation of p53 function (30, 33). Transient transfection of MDM-CAT and p53 led to CAT conversion, which was activated by p300 in a dose-dependent manner (Fig. 8B). Transfection of p300 and prohibitin did not lead to any further stimulation, suggesting that prohibitin does not co-operate with p300 to stimulate p53 activity, at least in a transient transfection experiment.

Prohibitin Enhances p53 Binding to Target Sites—The recruitment of transcription factors to promoter regions by regulatory proteins is one mechanism by which transcriptional function is modulated. We wished to determine whether prohibitin was found on p53 or E2F target sites in vivo, and whether it altered the binding ability of these proteins. ChIP assays were conducted for this purpose using MCF7 cells expressing a tetracycline-inducible prohibitin construct. Protein-DNA complexes were isolated and immunoprecipitated with antibodies for p53, prohibitin or HA as a negative control. PCR was performed for a 200-base pair region of the endogenous MDM2 promoter. A PCR product of the correct size was observed when the input (total) DNA was used for PCR, as expected (Fig. 9A). We find that in the unstimulated MCF7 cells, there was a certain amount of p53 on the MDM2 promoter, but not prohibitin (Fig. 9A). Induction of prohibitin with tetracycline for 24 h led to a marked increase in p53 binding; prohibitin also could be found in association with the promoter as seen by the presence of a PCR product in the prohibitin IP lane (Fig. 9A). It is interesting that prohibitin was also found associated with the promoter; this is the first time that prohibitin has been found to be associated with a promoter in vivo. The control lane where an anti-HA antibody was used did not yield a PCR product. To check the specificity of this result, a PCR was performed for an unrelated promoter sequence from the metallothionine-1G gene, which is not known to be regulated by p53 or prohibitin. None of the immunoprecipitated samples yielded a PCR product for this sequence, suggesting that the association of prohibitin and p53 with the MDM2 promoter is a specific event (Fig. 9A). We infer from these results that prohibitin can associate with a p53 target site, most likely through its binding to p53. These results also suggest that prohibitin promotes the binding of p53 to its recognition sequence, which leads to the increase in p53-mediated transcriptional activity.



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  		FIG. 9.
Prohibitin overexpression promotes p53 association with a target promoter in vivo but represses E2F association in ChIP assays. A, MCF7 cells expressing a tetracycline-inducible prohibitin construct were used for ChIP assays in the absence or presence of tetracycline induction for 24 h. Cells were treated with formaldehyde, sonicated, and ChIP lysates were incubated with antibodies for p53, prohibitin, or HA as a negative control. After isolation of bound DNA, PCR was performed for a 200-base pair region of the endogenous MDM2 promoter. While there was no significant binding of p53 in the absence of prohibitin induction, binding could be observed after addition of tetracycline. Prohibitin was also detected on this promoter after induction. Input indicates a PCR performed on DNA without any immunoprecipitation. A PCR for the metallothionein-1G promoter was performed as a negative control. B, Western blot to confirm the induction of prohibitin after 24 h of tetracycline induction. Induction of prohibitin does not affect endogenous p53 levels. C, ChIP assays were performed as in A, with antibodies against E2F1, Phb, or HA as a negative control. After isolation of DNA, PCR was performed for a 200-bp E2F1 responsive region of the cdc25A promoter. Binding of E2F1 was evident in non-induced cells, and this binding was absent after induction of prohibitin. PCR for the c-fos promoter was performed as a negative control.

 
To ensure that the enhanced p53 binding was not due to increased p53 levels, a Western blot was performed before and after tetracycline induction of MCF7 cells for 24 h. While prohibitin levels were induced at this time point, p53 levels were not altered (Fig. 9B). Thus the increase in p53 promoter binding in the ChIP assay was due to the induction of prohibitin levels and not due to increased p53 levels.

Since prohibitin represses E2F1-mediated transcriptional activity, experiments were designed to determine whether prohibitin affected the binding of E2F1 to its recognition sequence in vivo as well. ChIP assays were conducted in MCF7 cells in the absence or presence of prohibitin induction with antibodies against E2F1, prohibitin, or HA as a negative control. After isolation of DNA, PCR was performed for a 200-base pair region of the cdc25A promoter containing an E2F binding site. It was found that in the untreated MCF7 cells, E2F1 was associated with the promoter (Fig. 9C) but the amount of E2F1 bound to the promoter was significantly decreased after tetracycline induction of prohibitin. There was very little prohibitin bound to this promoter in these cells. A PCR for c-fos again confirmed the specificity of this result. These results suggest that one mechanism by which prohibitin can repress E2F1 activity is by reducing the association of E2F1 with a target site. Collectively, these results suggest that prohibitin can enhance promoter binding by p53, a transcription factor it activates, but reduces the promoter binding by E2F1, a transcription factor it represses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Though prohibitin was originally cloned based on its ability to induce a G1/S arrest, not much is known about its cellular functions or mode of action. The subcellular localization of prohibitin has been disputed as well; we had found prohibitin to be ubiquitously distributed in the cell including the nucleus (23), while another report states it to be mitochondrial (34). Nevertheless, the actual data presented in that study showed significant amount of prohibitin in the nucleus as well. Studies from other groups have suggested a mitochondrial function for prohibitin while we consistently find a nuclear transcriptional role (15, 22, 35). Another function attributed to prohibitin is its ability to inhibit apoptosis; we had shown that prohibitin inhibits camptothecin-induced apoptosis, while other groups have shown it can inhibit growth factor-induced apoptosis (25, 27). The studies presented in this paper address many of these issues and indicate that the subcellular localization of prohibitin is affected by apoptotic signals and that prohibitin is a regulator of p53 function.

Our finding that prohibitin localizes mainly to the nucleus in breast carcinoma cells and to the cytoplasm in camptothecin-stimulated cells suggest that prohibitin undergoes export from the nucleus to mitochondrial fractions upon receiving apoptotic or stress signals. Co-localization with cyctochrome c occurs only upon apoptotic signaling in both the cell lines examined, though considerable amount of cytochrome c and prohibitin staining can be seen in unstimulated cells. The translocation of prohibitin from the nucleus to the cytoplasm seems to have different effects on its interaction with transcription factors: camptothecin treatment seems to abolish its interaction with E2F1 in the nucleus, and there is negligible co-localization in the non-nuclear regions. In contrast, camptothecin treatment did not appear to disrupt its binding to p53; only the site of co-localization is affected. Recent studies have proposed mitochondrial functions for p53 in promoting apoptosis (36, 37). It is not clear how prohibitin affects p53 functions in the mitochondria, though it has distinct effects on p53-mediated transcription in the nucleus.

The positive effects of prohibitin on p53-mediated transcriptional activation were unexpected, given that it strongly represses E2F1-mediated transcription. We had earlier shown that prohibitin-mediated repression of E2F1 involved co-repressors; the experiments presented here show that prohibitin might be reducing the binding of E2F1 to its target sequences as well. Repression could be due to a combination of reduced DNA binding and recruitment of co-repressors. In the case of inducing p53 activity, we found that prohibitin was associated with the MDM2 promoter, and that prohibitin could promote p53 association with this sequence. Prohibitin did not cooperate with p300 to stimulate p53 activity or suppress MDM2-mediated repression. Rather, prohibitin reduced the stimulatory effects of p300 on p53. Perhaps the association of prohibitin with HDAC1 abrogates the positive effect. Furthermore, we found no evidence that prohibitin could alter the histone acetylation status of a p53-responsive promoter region (data not shown). It is likely that prohibitin enhances the binding of p53 to the target sites either directly, or through the mediation of other proteins.

Our previous studies indicated that cells overexpressing prohibitin were protected from cell death induced by camptothecin. While camptothecin treatment enhanced endogenous E2F activity, this activation was reduced in cells overexpressing prohibitin (25). In contrast, we did not find any major change in p53 activity after camptothecin treatment in the presence or absence of prohibitin overexpression (data not shown). The fact that p53 remains functional while E2F1 is repressed when excess prohibitin is present might affect the balance between proliferation and apoptosis. It is also possible that the association of prohibitin with p53 in the mitochondrial fraction might modulate non-transcriptional functions of p53. Thus prohibitin might be able to tilt the balance in favor of survival by ablating proliferative signals from E2F1 while modulating the function of p53.

The results described in this study raise the possibility that prohibitin is another link between Rb/E2F and p53 regulatory pathways. Its ability to translocate to different subcellular domains in response to specific signals as well as its ability to differentially regulate the transcriptional activity of E2F1 and p53 places it in a unique situation where it could integrate proliferatory and apoptotic signals. It is quite possible that the levels and activity of prohibitin could help determine the fate of cells while facing proliferatory as well as apoptotic signals.


   FOOTNOTES
 
* This study was supported by Grant CA77301 from the NCI, National Institutes of Health (to S. P. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

A recipient of Department of Defense Student Fellowship DAMD 17-01-1-0215 for breast cancer research. Back

|| These authors contributed equally to this work. Back

** To whom correspondence should be addressed. Tel.: 813-903-6892; Fax: 813-632-1328; E-mail: Chellasp{at}moffitt.usf.edu.

1 The abbreviations used are: Rb, retinoblastoma; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; HA, hemagglutinin; PARP, poly(ADP-ribose) polymerase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Wei Gu for the gift of the GST-p53 and p300 expression vectors, and Dr. Jiandong Chen for the gifts of the MDM2-CAT plasmid, p53 and MDM2 expression vectors. We also thank Ed Seijo and the Moffitt Analytical Microscopy Core Facility for help with co-localization experiments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dyson, N. (1998) Genes Dev. 12, 2245–2262[Free Full Text]
  2. Nevins, J. R. (2001) Hum. Mol. Genet. 10, 699–703[Abstract/Free Full Text]
  3. Stevaux, O., and Dyson, N. J. (2002) Curr. Opin. Cell Biol. 14, 684–691[CrossRef][Medline] [Order article via Infotrieve]
  4. Kaelin, W. G., Jr. (1999) Bioessays 21, 950–958[CrossRef][Medline] [Order article via Infotrieve]
  5. Brehm, A., Miska, E. A., McCance, D. J., Reid, J. L., Bannister, A. J., and Kouzarides, T. (1998) Nature 391, 597–601[CrossRef][Medline] [Order article via Infotrieve]
  6. Dahiya, A., Wong, S., Gonzalo, S., Gavin, M., and Dean, D. C. (2001) Mol. Cell. 8, 557–569[CrossRef][Medline] [Order article via Infotrieve]
  7. Magnaghi-Jaulin, L., Groisman, R., Naguibneva, I., Robin, P., Lorain, S., Le Villain, J. P., Troalen, F., Trouche, D., and Harel-Bellan, A. (1998) Nature 391, 601–605[CrossRef][Medline] [Order article via Infotrieve]
  8. Robertson, K. D., Ait-Si-Ali, S., Yokochi, T., Wade, P. A., Jones, P. L., and Wolffe, A. P. (2000) Nat. Genet. 25, 338–342[CrossRef][Medline] [Order article via Infotrieve]
  9. Trouche, D., Le Chalony, C., Muchardt, C., Yaniv, M., and Kouzarides, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11268–11273[Abstract/Free Full Text]
  10. Phillips, A. C., and Vousden, K. H. (2001) Apoptosis 6, 173–182[CrossRef][Medline] [Order article via Infotrieve]
  11. Irwin, M., Marin, M. C., Phillips, A. C., Seelan, R. S., Smith, D. I., Liu, W., Flores, E. R., Tsai, K. Y., Jacks, T., Vousden, K. H., and Kaelin, W. G., Jr. (2000) Nature 407, 645–648[CrossRef][Medline] [Order article via Infotrieve]
  12. Moroni, M. C., Hickman, E. S., Denchi, E. L., Caprara, G., Colli, E., Cecconi, F., Muller, H., and Helin, K. (2001) Nat. Cell Biol. 3, 552–558[CrossRef][Medline] [Order article via Infotrieve]
  13. Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L., and Vousden, K. H. (1998) Nature 395, 124–125[CrossRef][Medline] [Order article via Infotrieve]
  14. Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin, L., Potes, J., Chen, K., Orlow, I., Lee, H. W., Cordon-Cardo, C., and DePinho, R. A. (1998) Cell 92, 713–723[CrossRef][Medline] [Order article via Infotrieve]
  15. Wang, S., Nath, N., Adlam, M., and Chellappan, S. (1999) Oncogene 18, 3501–3510[CrossRef][Medline] [Order article via Infotrieve]
  16. Wang, S., Nath, N., Fusaro, G., and Chellappan, S. (1999) Mol. Cell. Biol. 19, 7447–7460[Abstract/Free Full Text]
  17. Nuell, M. J., Stewart, D. A., Walker, L., Friedman, V., Wood, C. M., Owens, G. A., Smith, J. R., Schneider, E. L., Dell' Orco, R., Lumpkin, C. K., Danner, D. B., and McClung, J. K. (1991) Mol. Cell. Biol. 11, 1372–1381[Abstract/Free Full Text]
  18. Jupe, E. R., Liu, X. T., Kiehlbauch, J. L., McClung, J. K., and Dell'Orco, R. T. (1995) Exp. Cell Res. 218, 577–580[CrossRef][Medline] [Order article via Infotrieve]
  19. Delage-Mourroux, R., Martini, P. G., Choi, I., Kraichely, D. M., Hoeksema, J., and Katzenellenbogen, B. S. (2000) J. Biol. Chem. 275, 35848–35856[Abstract/Free Full Text]
  20. Montano, M. M., Ekena, K., Delage-Mourroux, R., Chang, W., Martini, P., and Katzenellenbogen, B. S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6947–6952[Abstract/Free Full Text]
  21. Nijtmans, L. G., de Jong, L., Artal Sanz, M., Coates, P. J., Berden, J. A., Back, J. W., Muijsers, A. O., van der Spek, H., and Grivell, L. A. (2000) EMBO J. 19, 2444–2451[CrossRef][Medline] [Order article via Infotrieve]
  22. Steglich, G., Neupert, W., and Langer, T. (1999) Mol. Cell. Biol. 19, 3435–3442[Abstract/Free Full Text]
  23. Wang, S., Fusaro, G., Padmanabhan, J., and Chellappan, S. P. (2002) Oncogene 21, 8388–8396[CrossRef][Medline] [Order article via Infotrieve]
  24. Wang, S., Zhang, B., and Faller, D. V. (2002) EMBO J. 21, 3019–3028[CrossRef][Medline] [Order article via Infotrieve]
  25. Fusaro, G., Wang, S., and Chellappan, S. (2002) Oncogene 21, 4539–4548[CrossRef][Medline] [Order article via Infotrieve]
  26. Hseih, J.-K., Fredersdorf, S., Kouzarides, T., Martin, K., and Lu, X. (1997) Genes Dev. 11, 1840–1852[Abstract/Free Full Text]
  27. Vander Heiden, M. G., Choy, J. S., VanderWeele, D. J., Brace, J. L., Harris, M. H., Bauer, D. E., Prange, B., Kron, S. J., Thompson, C. B., and Rudin, C. M. (2002) J. Biol. Chem. 277, 44870–44876[Abstract/Free Full Text]
  28. Wu, X., Bayle, J. H., Olson, D., and Levine, A. J. (1993) Genes Dev. 7, 1126–1132[Abstract/Free Full Text]
  29. Chen, J., Wu, X., Lin, J., and Levine, A. J. (1996) Mol. Cell. Biol. 16, 2445–2452[Abstract]
  30. Gu, W., and Roeder, R. G. (1997) Cell 90, 595–606[CrossRef][Medline] [Order article via Infotrieve]
  31. Sambrook, J., Fritsch, E., and T. Maniatis. (1989) in Molecular Cloning, 2 Ed. (Nolan, C., ed) Vol. III, pp. 16.59–16.62, Cold Spring Laboratory Press, Cold Spring Harbor, NY
  32. Marchenko, N. D., Zaika, A., and Moll, U. M. (2000) J. Biol. Chem. 275, 16202–16212[Abstract/Free Full Text]
  33. Liu, L., Scolnick, D. M., Trievel, R. C., Zhang, H. B., Marmorstein, R., Halazonetis, T. D., and Berger, S. L. (1999) Mol. Cell. Biol. 19, 1202–1209[Abstract/Free Full Text]
  34. Coates, P. J., Nenutil, R., McGregor, A., Picksley, S. M., Crouch, D. H., Hall, P. A., and Wright, E. G. (2001) Exp. Cell Res. 265, 262–273[CrossRef][Medline] [Order article via Infotrieve]
  35. Berger, K. H., and Yaffe, M. P. (1998) Mol. Cell. Biol. 18, 4043–4052[Abstract/Free Full Text]
  36. Schuler, M., Bossy-Wetzel, E., Goldstein, J. C., Fitzgerald, P., and Green, D. R. (2000) J. Biol. Chem. 275, 7337–7342[Abstract/Free Full Text]
  37. Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P., and Moll, U. M. (2003) Mol. Cell. 11, 577–590[CrossRef][Medline] [Order article via Infotrieve]
  38. Baldwin, A. S. (1996) Annu. Rev. Immunol. 14, 649–683[CrossRef][Medline] [Order article via Infotrieve]

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