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Department of Biochemistry,1 Department of Medicine, Boston University School of Medicine, 715 Albany St., Boston, Massachusetts 02118,2 Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 021153
Received 7 December 2004/ Returned for modification 13 January 2005/ Accepted 26 April 2005
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ABSTRACT |
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Np63 functions to inhibit p63 and other p53 family members. Mutations in the p63 gene that abolish p63 DNA-binding and transactivation activities cause human diseases, including ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndrome. In this study, we show that mutant p63 proteins with a single amino acid substitution found in EEC syndrome are DNA binding deficient, transactivation inert, and highly stable. We demonstrate that TAp63 protein expression is tightly controlled by its specific DNA-binding and transactivation activities and that p63 is degraded in a proteasome-dependent, MDM2-independent pathway. In addition, the N-terminal transactivation domain of p63 is indispensable for its protein degradation. Furthermore, the wild-type TAp63
can act in trans to promote degradation of mutant TAp63 defective in DNA binding, and the TA domain deletion mutant of TAp63 inhibits transactivation activity and stabilizes the wild-type TAp63 protein. Taken together, these data suggest a feedback loop for p63 regulation, analogous to the p53-MDM2 feedback loop. |
INTRODUCTION |
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N isoforms, produced from an intronic promoter, contain the same DNA-binding and oligomerization domains as the TA isoforms but lack the transactivation domain. The N isoforms also contain a region of 26 amino acids (aa) at the very N-terminal end of the protein (TA2) in which an activation function was recently identified (7). In addition, the N isoforms are capable of forming protein complexes with p53 family proteins to inhibit the function of p53 family members (39). Furthermore, both the TAp63 and Np63 isoforms can undergo alternative splicing to yield three different C-terminal tails (TAp63
, -ß, and - isoforms and Np63, -ß, and - isoforms). Among these isoforms, TAp63 is the most transactivation-active isoform of p63 (39). In the C-terminal extension of the -isoforms, there is a sterile alpha motif implicated in protein-protein interactions and thought to be important for mammalian development (32).
Despite their structural homology, the p53 family members have distinctive biological functions. While p53 is a key gatekeeper for genomic stability by regulating cell cycle, DNA damage repair, and apoptosis, p73 and p63 are critical during development and differentiation. In particular, p63 appears to be essential in epithelial and limb development as demonstrated by the mouse models (33). Several dominant human syndromes involving limb development and ectodermal dysplasia have been mapped to the p63 gene, including ectrodactyly, ectodermal dysplasia and cleft lip/palate (EEC) syndrome; nonsyndromic split hand/foot malformation (SHFM); ankyloblepharon, ectodermal dysplasia, clefting (AEC) syndrome; acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome; and limb-mammary syndrome (LMS) (2, 4). Most mutations in the p63 gene identified in EEC patients so far result in amino acid substitutions that are predicted to abolish the DNA-binding capacity of p63. In contrast to p53, the p63 gene is rarely mutated in cancer (12, 21, 25). However, overproduction of Np63 isoforms has been reported in squamous cell carcinoma (10) and in many other types of epithelial tumors (6, 24, 26, 27).
The p53 protein is usually labile in normal cells but is dramatically stabilized upon a variety of cellular stresses. The key negative regulator of p53 is the MDM2 protein, which functions as an E3 ubiquitin ligase for p53 to promote its protein degradation (9, 15). In addition, MDM2 physically binds to the p53 N-terminal transactivation domain, thereby directly inhibiting p53 transactivation activity (5). Importantly, MDM2 is a bona fide target gene downstream of p53. Thus, activation of p53 up-regulates its own inhibitor (MDM2). This feedback loop ensures that p53 protein levels are maintained at low levels in normal cells.
Here we show that, like p53, the protein stability of the transactivation form of p63, TAp63, is also tightly regulated by its DNA-binding and transactivation activities. The N-terminal TA domain is required for its protein degradation independent of MDM2. Our data suggest that TAp63 is regulated through a feedback mechanism similar to the p53-MDM2 feedback loop.
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MATERIALS AND METHODS |
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and U2-OS-Tet-Luciferase cells were maintained in DMEM supplemented with 10% FBS, 2 µg/ml doxycycline (BD Biosciences-Pharmingen), and 200 µg/ml hygromycin (BD Biosciences-Pharmingen).
Plasmid construction and transfection.
pcDNA-myc-TAp63 and pcDNA-myc-Np63 were described previously (39). A BamHI/XhoI or BamHI/XbaI PCR fragment containing TAp63 was subcloned into pcDNA-HA or pTRE-tight (BD Biosciences-Pharmingen) to generate pcDNA-HA-TAp63 or pTRE-tight-TAp63. The pcDNA-myc-TAp63 and pcDNA-HA-TAp63 plasmids were used to generate deletion mutants or point mutants by site-directed mutagenesis according to the manufacturer's instructions (Stratagene). The TA domain swapping mutant of TAp63, HA-p53TA-p63, was generated from pcDNA-HA-TAp63 and pcDNA-HA-p53 (40) using overlapping extension as described previously (11). All constructs were confirmed by DNA sequencing.
U2-OS and HeLa cells were transfected with FuGENE 6 (Roche). Saos-2 cells were transfected with CalPhos transfection kit (BD Biosciences-Pharmingen). MEF were transfected with Lipofectamine 2000 (Invitrogen). To examine TAp63 protein stability, U2-OS or HeLa cells were transfected with 500 ng of pcDNA-TAp63 or cotransfected with 500 ng of pcDNA-TAp63 and 1.0 µg of either pcDNA-TAp63(R304W), pcDNA-TAp63, or pcDNA-TAp63(FWL-A). Sixteen hours posttransfection, cells were treated with cycloheximide (Sigma) at a final concentration of 50 µg/ml. Cells were collected at time intervals. For MG132 treatment, cells transfected for 30 h were treated with MG132 (20 µM) (Sigma) for 5 h. To generate U2-OS-Tet-TAp63 and U2-OS-Tet-Luc cells, U2-OS-Tetoff cells (gift from Qiang Yu, Boston University School of Medicine) were cotransfected with 5 µg of pTRE-tight-TAp63 or pTRE-tight-Luciferase (BD Biosciences-Pharmingen) and 0.5 µg of pTK-Hyg. Twelve hours posttransfection, cells were selected with doxycycline (2 µg/ml) and hygromycin (400 µg/ml) for 4 weeks. Single colonies were selected. All clonal cell lines were tested for induction of TAp63 or luciferase in the absence of doxycycline by Western blot analysis or luciferase activity assay. A stable U2-OS-TetTAp63 cell line (clone 2) was chosen for further analysis for its effective repression and induction by doxycycline administration or withdrawal.
Luciferase reporter assay and Western blot analysis.
For luciferase report assay, Saos-2 cells grown in six-well tissue culture dishes at 80% confluence were transfected with 100 ng of either wild-type or mutant pcDNA-TAp63 in the presence of 2 µg of Bax-Luciferase (Bax-Luc) and 100 ng of pCMV-ß-galactosidase plasmids. To examine the dominant-negative effect of p63 mutants, Saos-2 cells were transfected with 100 ng of pcDNA-myc-TAp63, 400 ng of either pcDNA-HA-TAp63 or pcDNA-HA-TAp63(R304W) in the presence of 2 µg of Bax-Luc and 100 ng of pCMV-ß-galactosidase. Thirty-six hours posttransfection, cells were harvested in 1x reporter lysis buffer (BD Biosciences-Pharmingen) and subjected to ß-galactosidase assay and luciferase activity assay (BD Biosciences-Pharmingen). Luciferase activity was normalized to ß-galactosidase activity and presented as the mean ± standard deviation of three independent experiments performed in triplicate.
For Western blot analysis, cells were lysed in EBC buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 0.5% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 2 µg/ml leupeptin, 2 µg/ml aprotinin, 50 mM NaF, and 0.5 mM Na3VO4). Equal amounts of total protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Antibodies used were specific for p63 (4A4; Santa Cruz), hemagglutinin (HA) (Y-11; Santa Cruz), p53 (DO-1; Santa Cruz), Myc (9E10; Santa Cruz), ß-galactosidase (23781; Promega), and actin (C-11; Santa Cruz).
Electrophoresis mobility shift assay (EMSA).
H1299 cells were transfected with 3 µg of p53 or a TAp63 construct. Twenty-four hours posttransfection, nuclear extracts were prepared. Briefly, cells were collected and lysed in hypotonic buffer (10 mM HEPES, pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol [DTT], 0.5 mM PMSF, 0.2% Nonidet P-40), followed by centrifugation at 4,000 rpm for 10 min at 4°C. The pellets were then lysed in hypertonic buffer (20 mM HEPES, pH 7.6, 1.5 mM MgCl2, 420 mM NaCl, 0.5 mM DTT, 0.5 mM PMSF, 25% glycerol), followed by centrifugation at 14,000 rpm for 20 min at 4°C. The reaction mixture (total volume of 25 µl) containing 20 µg nuclear extracts in binding buffer [10 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol, 0.25 µg/µl bovine serum albumin, and 0.1 µg/µl of poly(dI-dC)] was incubated at room temperature for 20 min. The competition assay was performed by the addition of 50-fold excess of unlabeled wild-type oligonucleotide (sc-2579; Santa Cruz Biotechnology) or mutant oligonucleotide (sc-2580; Santa Cruz Biotechnology) into the reaction mixture and incubation for 20 min prior to the addition of 32P-labeled probe. DNA-protein complexes were resolved by electrophoresis on 4% polyacrylamide gels in Tris-glycine buffer and revealed by autoradiography. Thirty nanograms of oligonucleotide containing the p53-binding consensus site (sc-2579; Santa Cruz Biotechnology) was end labeled in the presence of 25 µCi of [32P]ATP (NEG002H; New England Nuclear) and 10 U of T4 polynucleotide kinase (M0201; New England BioLabs) for 30 min at 37°C.
Immunofluorescence staining.
U2-OS cells in six-well plates were cotransfected with either 500 ng of pcDNA vector or pcDNA-TAp63, or 200 ng of pcDNA-TAp63(R304W), pcDNA-TAp63(C306R), pcDNA-TAp63(FWL-A), or pcDNA-TAp63(TA) in the presence of 50 ng of pEG-GFP using FuGENE 6. Twelve hours posttransfection, cells were trypsinized and 5 x 104 cells were plated onto Lab-Tek II chamber slides (154461; Nalge Nunc International). Twenty-four hours later, cells were fixed in 4% paraformaldehyde (in phosphate-buffered saline, pH 7.4), permeabilized with 0.2% Triton X-100, blocked in phosphate-buffered saline containing 1% bovine serum albumin, and then immunostained with p63 antibody (sc-8431; Santa Cruz Biotechnology) followed by a Cy3-conjugated goat anti-mouse immunoglobulin G antibody (115-165-146; Jackson ImmunoResearch Laboratory). The cells were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) (300 nM) prior to mounting using ProLong Gold Antifade (P36930; Molecular Probes). Staining was visualized using the following excitation/emission wavelengths: 358/461 nm for DAPI, 595/615 nm for Cy3, and 494/518 nm for green fluorescent protein (GFP). Fluorescent images were captured on a Zeiss Axiovert 200M microscope with Axiovision v 4.3 program.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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N isoforms, which lack the transactivation domain at the N terminus, whereas the levels of TA isoforms are very low under physiological conditions (10, 20, 38). We asked whether this phenomenon is reminiscent of p53, whose mutants defective in transactivation activity unavoidably become stabilized. Thus, we examined the protein expression levels of transactivation-potent TAp63 and transactivation-inert Np63 in U2-OS or HeLa cells by transient transfection. As shown in Fig. 1A, the protein levels of TAp63 were significantly less than those of Np63 in both cell lines. Treatment with proteasome inhibitor MG132 led to stabilization of endogenous p53 proteins, as expected, and to a dramatic increase of TAp63 protein levels. Np63 protein was also stabilized, albeit to a much lesser extent (Fig. 1B). These data suggest that TAp63 is degraded through a proteasome-dependent pathway and that the differences in the protein levels of ectopically expressed TAp63 and Np63 are likely due to different protein turnover rates. Thus, we performed cycloheximide treatment to determine the protein half-lives of TAp63 and Np63 in U2-OS cells. As shown in Fig. 1C, TAp63 exhibited a much shorter half-life (
90 min) than Np63 did (>5 hours). These data revealed a correlation between the transactivation activity and protein stability of p63 isoforms.
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are essential for its protein degradation.
Mutations in the p63 gene have been linked to several human genetic diseases, including EEC syndrome and SHFM (33). In EEC syndrome and SHFM, the germ line mutations in the p63 gene are frequently located in the central DNA-binding domain (4, 34, 36). Strikingly, these missense mutations correspond very well with the somatic mutational hot spots in the p53 gene, which inactivate p53 DNA binding and p53 growth suppression function. We generated three point mutations in the DNA-binding domains of TAp63, TAp63(R204W), TAp63(R304W), and TAp63(C306R). These mutations are found in EEC syndrome and are predicted to abolish the DNA-binding activity of p63 (4). Indeed, all three mutant proteins exhibited no DNA-binding activity (see Fig. 5A) and, as expected, no detectable transcriptional activity (Fig. 2A), yet they were expressed at much higher levels than wild-type TAp63 (Fig. 2B). Inhibition of proteasomes led to a dramatic increase in protein stability of wild-type TAp63 but not the p63 mutant proteins defective in DNA binding (Fig. 2B). Indeed, the mutant proteins exhibited significantly prolonged protein half-lives (Fig. 2C and data not shown). Thus, these data indicate that the specific DNA-binding activity of TAp63 is essential for its protein degradation.
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and its protein degradation, we generated an N-terminal deletion mutant of TAp63, TAp63, which lacks the entire TA domain (aa 1 to 69). Although TAp63 possessed a strong DNA-binding activity (see Fig. 5A) and localized in nuclei (see Fig. 5C), it exhibited no transactivation activity (Fig. 3A) and was highly stable (Fig. 3B and C). Therefore, these data indicate that the transcriptional activity of TAp63 is critical for its protein instability.
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(FWL-A) in which these three amino acid residues (F16, W20, and L23) are replaced by alanine, retained strong DNA-binding activity (Fig. 5A) and partial transactivation activity in a dose-dependent manner (Fig. 4B and 5B), yet the protein was highly stable (Fig. 4C). These data suggest either that the partial transactivation activity is insufficient to promote p63 protein degradation or that the FWL motif is critical for p63 protein turnover. To address this question, we generated a HA-tagged chimeric protein HA-p53TA-p63 in which the TA domain (aa 1 to 64) of TAp63 is replaced with the TA domain (aa 1 to 45) of p53. As shown in Fig. 6, p53TA-p63 was fully competent in transactivation (Fig. 6A). Strikingly, it was stable in Saos-2 cells (Fig. 6B), indicating that the transactivation activity alone is not sufficient for protein degradation. However, in contrast to remarkable protein stability in Saos-2 cells, p53TA-p63 protein was rapidly turned over in U2-OS cells (Fig. 6B). Since U2-OS cells contain wild-type p53 and express much higher levels of MDM2 in comparison to the p53 null Saos-2 cells (Fig. 6C), it is likely that degradation of the p53TA-p63 chimeric protein is dependent on MDM2, which binds to the TA domain of p53. Of note, the FWL motif is important for p53 interaction with MDM2 (16). Thus, the conserved FWL motif in the TA domain of TAp63 may serve as a binding site for MDM2, which might in turn promote p63 degradation. However, the ectopically expressed TAp63 exhibited similar protein half-lives in wild-type MEF and p53–/– MDM2–/– MEF (Fig. 6D), indicating that degradation of TAp63 does not require MDM2. Thus, the FWL motif in the TA domain of TAp63 is critical for its protein degradation in an MDM2-independent manner.
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can act in trans to promote degradation of the p63 DNA-binding mutant, but not the p63 TA domain mutants.
Since both the transcriptional activity and the FWL motif of TAp63 are crucial for its protein instability, it is very possible that there may be a feedback control mechanism for regulation of p63 protein levels, analogous to the p53-MDM2 feedback loop. This hypothesis would predict that TAp63 transactivates expression of an unidentified target gene whose product interacts with the FWL motif in the TA domain to promote TAp63 protein degradation via a proteasome-dependent pathway. Accordingly, the wild-type TAp63 should be able to act in trans to promote the degradation of mutant p63 protein defective in DNA binding, but not the mutant p63 lacking the TA domain or the mutant p63 with a defective FWL motif. Indeed, in cotransfection experiments, the protein half-life of HA-tagged TAp63(R304W) was decreased by 50% in the presence of Myc-tagged wild-type TAp63, while the half-life of HA-tagged TAp63 or TAp63(FWL-A) was not affected by the presence of wild-type TAp63 (Fig. 7). A similar phenomenon was observed using an inducible U2-OS-Tet-TAp63 stable cell line. Upon withdrawal of doxycycline, expression of TAp63 was markedly induced (Fig. 8C). Induction of TAp63, but not induction of luciferase in the control cell line, led to a significant decrease in the protein half-life (2 h) of HA-TAp63(R304W) (Fig. 8A and B).
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Np63 isoforms are predominantly expressed in cancer cells and in epithelial progenitor cells (10, 20, 38). We therefore investigated the influence of transactivation-inert p63 mutant TAp63 on the wild-type TAp63 protein stability. As shown in Fig. 9, coexpression of TAp63 led to a marked suppression of transactivation activity and significant stabilization of TAp63 protein. Interestingly, coexpression of DNA-binding-defective TAp63(R304W) exhibited a much lower inhibitory effect on the TAp63 transactivation activity and no significant effect on the stability of TAp63 protein (Fig. 9). Thus, nontransactivation TAp63 can effectively function in a dominant-negative fashion in suppression of transactivation activity of wild-type TAp63 and modulate the stability of transactivation p63 isoforms.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, is tightly regulated likely through a feedback mechanism, analogous to the p53-Mdm2 feedback loop (Fig. 10). We show that both DNA-binding and transactivation activities are essential for TAp63 instability and, furthermore, that the unique features of the TAp63 TA domain play an important role in protein degradation. Moreover, we show that the wild-type TAp63 can act in trans to induce the degradation of the p63 DNA-binding mutant and that the p63 deletion mutant lacking the TA domain can stabilize the TAp63 protein through its dominant-negative effect.
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proteins are highly stable, thus supporting the notion that specific DNA-binding activity, while being a prerequisite for TAp63-mediated transcription, is critical for its protein degradation. Alternatively, it is possible that interaction of TAp63 with DNA is required for its protein degradation in a manner similar to some transcription factors, such as estrogen receptor, Myc, and VP16, whose DNA-binding activity is critical for protein degradation (13, 28, 29, 35). However, our data reveal that wild-type TAp63 can promote the degradation of a DNA-binding-defective mutant, suggesting that TAp63 interaction with DNA is not a prerequisite for its protein degradation. Notably, high levels of wild-type p53 or MDM2 have been shown to promote the degradation of mutant p53 proteins defective in DNA binding (1, 22).
The N-terminal domain of TAp63 is indispensable for its protein degradation. The TAp63 mutant, which lacks the entire transactivation domain and thus is completely inert in transactivation, is highly stable. Replacement of the TA domain of TAp63 with the p53 TA domain in the chimeric protein p53TA-p63 results in increased protein stability in cells expressing low levels of MDM2, despite its high transactivation activity, indicating that transcription activity is not sufficient for p63 protein degradation. In addition, the TAp63(FWL-A) mutant bearing the point mutations in the F16-W20-L23 motif also leads to protein stability. Of note, not only are both TAp63 and TAp63(FWL-A) highly stable but both are also resistant to the degradation induced by wild-type TAp63. These data indicate that the TA domain is required for the degradation of TAp63 protein, probably by serving as an interaction domain for a p63 transcription target protein that can induce the degradation of TAp63. Interestingly, a recent study reported that the F16-W20-L23 motif functions as a protein-protein interaction site with which the transactivation-inhibitory domain on the C terminus of TAp63 is able to interact, resulting in stabilization of the TAp63 protein (31). Importantly, these three hydrophobic amino acids (F19-W23-L26) within the p53 N terminus are critical for the MDM2-mediated degradation of p53 protein (16). Despite the poor homology between the p53 family members, those three amino acids are well conserved in p53, p63, and p73; this may mean that MDM2 might also be able to bind the FWL motif and promote p63 protein degradation. However, our data indicate that MDM2 is not required for p63 degradation, as evidenced by the observation that p63 protein can be degraded equally well in p53–/– MDM2–/– MEF and wild-type MEF. These data are consistent with the observations indicating that overexpression of MDM2 does not lead to p63 protein instability (3, 18).
The notion that p63 is regulated by a feedback loop is further supported by the observation that the TAp63 mutant can significantly inhibit the transactivation activity of the wild-type TAp63 and lead to its protein stabilization. It is possible that excess expression of TAp63 may occupy the promoter regions to which the transactivation p63 normally binds and thereby block TAp63-mediated transcription, which may in turn suppress the expression of proteins involved in p63 protein degradation (Fig. 10). Interestingly, a recent study showed that the transactivation activity of p73 is also critical for p73 protein degradation (37). It is plausible that cells may have developed similar feedback regulation mechanisms during evolution in regulation of p53 family protein expression despite their distinct biological functions.
It has been established that certain unstable transcription factors contain the degron sequences in the transactivation domains so that transcription can be coupled to proteolysis (17, 23, 30). Our data demonstrated that TAp63 degradation is also tightly coupled to its transcriptional activity, which may partially explain the low levels of expression of transactivation-active p63 isoforms in most tissues and cell lines. Recent studies have demonstrated that among the p63 proteins TAp63 isoforms are the first to be expressed during embryogenesis and are required for commitment to an epithelial stratification program. Np63 is the predominant isoform expressed in the basal cells of many epithelial tissues (14, 20). Since TAp63 isoforms seem to inhibit terminal differentiation, their activities must be counterbalanced by various mechanisms, such as transcription-coupled degradation of TAp63 and expression of Np63 in neutralizing TAp63, to allow cells to respond to signals required for the maturation of embryonic epidermis. It is possible that uncontrolled expression of transactivation-potent p63 isoforms leads to adverse effects on cells, since TAp63 has been shown as an inducer for apoptosis (8, 39).
Interestingly, the p63 mutations in the DNA-binding domain observed in several dominant human syndromes are highly stable. It is conceivable that these mutations may regulate the function of p53 family proteins to disrupt normal cell proliferation and development.
Although our data indicate the TAp63 protein stability is tightly controlled by its transcriptional activity, the precise molecular mechanism, however, remains to be elucidated.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant GM-70017 (to Z.-X.J.X.) from the National Institute of General Medical Sciences.
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FOOTNOTES |
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