Inhibition of hTAFII32-binding Implicated in the Transcriptional Repression by Central Regions of Mutant p53 Proteins*

Shiu-Feng TungDagger §, Jing-Yuan ChuangDagger parallel , Chin-Tarng Lin**, Ming-Yang LaiDagger Dagger , Cheng-Wen Wu§, and Young-Sun Lin§§§

From the § Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, the  Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 100, Taiwan, the parallel  School of Medical Technology, China Medical College, Taichung 404, Taiwan, the ** Institute of Pathology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, and the Dagger Dagger  Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

We previously identified a movable and regulable inactivation function within the central region (CRts247) of a temperature-sensitive p53 (p53ts) mutant, p53N247I. Here we showed that central regions from several p53ts mutants behaved similarly, i.e. they repressed a neighboring activation domain only when existing in the mutant status. Using chimeric protein GAL4VP16-CRts247 as an example, we demonstrated that de novo protein synthesis was not required for the reactivation of the chimeric protein, indicating that a post-translational mechanism was involved in the control of CRts247 activity. The CRts247-conferred thermo-regulability did not work via a mechanism demanding either an alteration of the subcellular compartmentalization of or the inactivation of DNA-binding activity of the GAL4 chimera. Further, CRts247 did not function in trans, eliminating the possibility that the observed repression was because of the competition for a putative factor(s) by the mutant p53 domain. Rather, CRts247 bestowed temperature-dependent interaction with hTAFII32 to the VP16 activation domain. In a parallel experiment, CRts247 also caused a large reduction in the affinity of hTAFII32 to the p53 activation domain at the nonpermissive temperature. These results strongly suggested that inhibition of hTAFII32 binding could be one of the mechanisms responsible for the transcriptional repression by mutant p53 central regions.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

The tumor suppressor p53 protein is an important negative regulator of cell proliferation (1). Although the half-life of the p53 protein is quite short, in response to DNA damage the protein accumulates and turns on the expression of genes that are involved in growth arrest, DNA repair, and apoptosis (2, 3). Thus, the biological activities of p53 are mainly attributed to its function as a transcription factor. In structure, the human p53 protein consists of 393 amino acids and is organized into three functional domains: an N-terminal region involved in transcriptional activation, a central region mediating specific DNA binding, and a C-terminal region responsible for oligomerization, transcriptional repression, and nonspecific DNA binding (4, 5).

The natural forms of cancers almost universally contain mutations in the p53 gene. Mostly, they are single missense point mutations located within the central region, that is, approximately residues 100-300 of the p53 protein. These mutations not only lead to the inactivation of the tumor suppressor function of p53 but also transform p53 into an oncogene through a dominant negative effect (6). It has also been demonstrated that wild-type and mutant p53 proteins appear to exhibit distinctive phenotypes (7, 8), and the two phenotypes represent distinctive conformations of the p53 polypeptide (7, 9).

Biochemically, mutations in the central region of p53 typically result in the loss of the specific DNA-binding activity of the protein (4). Moreover, we have previously demonstrated that the central region (CRts247)1 encompassing residues 101-318 of p53N247I, a p53ts mutant with a substitution of isoleucine for asparagine at residue 247, functions as a movable and regulable inactivation cassette (10). It represses several activation domains present on the same polypeptide chain at the nonpermissive temperature (37 °C) but not at the permissive temperature (30 °C). These observations suggest that CRts247 has the potential to be used as a desirable tool for the construction of temperature-sensitive derivatives of transcription factors (10). Nevertheless, how the mutant p53 central region gives rise to the repression activity in general and how CRts247 confers thermo-regulability to a neighboring activation domain in particular remain largely unknown. Here we exploited the thermo-regulability of CRts247 to investigate the mechanism(s) responsible for the aforementioned transcriptional repression.

    MATERIALS AND METHODS

Plasmid Constructions-- Construction of plasmids pG5E1bCAT, pL6E1bCAT, pSG424, pSGVP, pGAL4VP16-CRwt (previously referred to as pGAL4VP16-p53(101-318)), pGAL4VP16-CRts247 (previously referred to as pGAL4VP16-CR), and pLex-VP was described previously (10). pGAL4VP16-CRts143 and pGAL4VP16-CRts173 were constructed by inserting the corresponding p53 fragment between the EcoRI and BamHI sites of pSGVP. pL6EG5CAT was created by inserting the PstI/XbaI fragment of pG5E1bCAT into the SmaI site of pL6EC-BSK (10). pGEX3X-hTAFII32 was constructed by inserting a DNA fragment encoding residues 1-140 of hTAFII32, which is soluble and sufficient for interaction with VP16 (11-13), between the BamHI and EcoRI sites of pGEX-3X (Amersham Pharmacia Biotech). pH8HMKGAL4VP16-CRwt, pH8HMKGAL4VP16-CRts247, pH8HMKp53-(1-318) and pH8HMKp53N247I-(1-318) were cloned by inserting the DNA fragment encoding GAL4VP16-CRwt, GAL4VP16-CRts247, p53-(1-318), and p53N247I-(1-318) proceeded by a consensus motif for heart muscle kinase (HMK), respectively, between the NdeI and BamHI sites of 8His-pET11d (14). pRK5F-CRts247 was constructed by inserting CRts247 between the EcoRI and BamHI sites of pRK5F (15).

Transient Expression Assays and CAT Protein and RNA Analyses-- Saos-2 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Unless specified, calcium phosphate-mediated DNA transfections were performed as described previously (10, 16). CAT activity was measured and quantified according to the method by Carey et al. (17). For temperature-sensitive assays, the incubation temperature was switched to 30 °C after a 12-h transfection at 37 °C. To block protein synthesis, cycloheximide was added to the medium to a final concentration of 10 µg/ml. RNA preparation and primer extension were performed as described previously (18, 19).

Western Blotting Analysis-- The anti-GAL4 antibody was purchased from Upstate Biotechnology, Inc., New York. The anti-FLAG antibody was purchased from Kodak, New York. Immunoblotting was performed as described previously (10, 16).

Immunostaining-- Cells were plated on acid-washed cover slides and transfected as described above. Cells were fixed for 10 min with 3% formaldehyde in phosphate-buffered saline (PBS) 36 h after transfection. The fixed cells were washed twice with PBS, permeabilized for 10 min in a mixture containing an equal volume of acetone and ethanol at 4 °C, and then washed twice with PBS. For immunofluorescence, slides were first incubated with 10% normal goat serum in PBS at room temperature for 30 min to block nonspecific binding. The slides were then incubated at room temperature for 12 h with the polyclonal anti-GAL4-(1-147) (Upstate Biotechnology, Inc.) diluted (1:1000) in PBS containing 0.1% saponin and 0.05% sodium azide. The slides were washed twice for 10 min with PBS at room temperature and incubated for another 1 h with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody. Finally, the slides were washed extensively with PBS containing 0.02% Tween 20 and mounted on a microscope.

GST Fusion Proteins, His-tagged Proteins, and Pull-down Assay-- Glutathione S-transferase (GST) and GST-hTAFII32 were expressed in and purified from Escherichia coli XA90 strain according to standard protocols (18, 20, 21). The ligand concentration, using bovine serum albumin as a standard, was adjusted to approximately 0.125 mg per ml of resin for GST and GST-hTAFII32. The His-tagged GAL4VP16-CRwt, GAL4VP16-CRts247, p53-(1-318), and p53N247I-(1-318) were expressed in and purified from E. coli JM109(DE3) strain according to the suggestions of the manufacturer (QIAGEN, Inc.). In vitro phosphorylation of the His-tagged proteins was performed as follows: 16 µg of the His-tagged protein were incubated with 8 pmol of [gamma -32P]ATP and 20 units of heart muscle kinase (Sigma) in 60 µl of a buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 100 mM NaCl, and 12 mM MgCl2 at 37 °C for 1 h. The reaction sample was passed through a NucTrap column (Stratagene) to remove free [gamma -32P]ATP. Aliquots (40 µl) of the GST protein beads were incubated for 2 h at either 30 °C or 37 °C as indicated in the figure legends with 2 µg of 32P-labeled GAL4 and p53 derivatives in 500 µl of a buffer (NETN buffer) containing 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.2% Nonidet P-40. After being washed five times with 1 ml of NETN buffer containing either 200 mM NaCl and 0.2% Nonidet P-40 (for derivatives of GAL4VP16) or 500 mM NaCl and 0.01% Nonidet P-40 (for derivatives of p53) prewarmed to the indicated temperature, bound proteins were eluted from beads and analyzed by electrophoresis on a 10% SDS-polyacrylamide gel.

    RESULTS

The Central Regions of p53ts Mutants All Possess a Thermo-regulable Inactivation Function-- We have shown that CRts247 represses a neighboring transactivation domain only at the nonpermissive temperature (10), suggesting that the p53 central region, when existing in the mutant conformation, gains an inactivation function. It is not clear, however, whether the gain of function is a general phenomenon for mutant p53 central regions or just a special property of CRts247. To address this question, we analyzed the central regions of other mutants. Individual CRts from the three p53ts mutants identified to date (22) was fused to GAL4VP16, and the transcriptional activity of the resultant fusion protein was measured. As shown in Fig. 1A, fusion of CRwt to GAL4VP16, consistent with previous results (10), exhibited little effect on the transactivation activity of the hybrid protein regardless of the assaying temperature (compare lanes 3 and 9 to lanes 2 and 8). In contrast, all three of the CRts repressed the transactivation activity of the GAL4 fusion protein at 37 °C and not at 30 °C (compare lanes 10-12 to lanes 4-6). The rationale to work with p53ts mutants was as follows. First, p53N247I, p53V143A, and p53V173L all acquire a mutant-specific conformation at the nonpermissive temperature (22, 23). Second, it was aimed to take advantage of their thermo-regulability so that the repression activity could be directly related to their mutant conformation. However, the mutant conformation-specific repression activity was not unique to the three CRts because central regions from other p53 denaturing mutants (23) all repressed the GAL4 fusion protein.2 Nonetheless, the three CRts differed quantitatively. Their ability to restore the activity of the fusion protein at the permissive temperature was repeatedly observed to obey the following order: CRts247 >=  CRts173 >> CRts143 (Fig. 1A, lanes 4-6), suggesting that they were somewhat dissimilar in the extent of recovery to the wild-type conformation at the permissive temperature. An immunoblotting analysis (Fig. 1B) showed that the difference in the transcriptional activity of the fusion proteins could not be explained by variations in their levels of expression. For example, whereas the concentration of GAL4VP16-CRts143 appeared to be equal to, if not higher than, that of other fusion proteins (Fig. 1B, compare lane 4 to lanes 2, 3, and 5), its transactivation activity was the lowest (Fig. 1A, compare lane 5 to lanes 3, 4, and 6). Another example was that, although all three CRts-fusion proteins were synthesized to a level similar to that of GAL4VP16-CRwt at the nonpermissive temperature (Fig. 1B, compare lanes 8-10 to lane 7), CRts-fusion proteins were inactive in stimulating transcription (Fig. 1A, compare lanes 10-12 to lane 9). Thus, it was concluded that all of the CRts possessed a movable and thermo-regulable inactivation function and that, among CRts of the p53ts mutants, CRts247 was optimal in terms of such an activity. In addition, these data suggested that the closer the mutation site was positioned to the N-terminal end of the central region of a p53ts mutant, the less the corresponding CRts functioned as a movable regulatory cassette.


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Fig. 1.   The central regions of temperature-sensitive p53 mutants possess a thermo-regulable inactivation function. A, transactivation of reporter pG5E1bCAT by GAL4 chimeras at 30 °C (lanes 1-6) and at 37 °C (lanes 7-12). Activator GAL4VP16 was included as a positive control for transactivation (lanes 2 and 8). Diagrams of reporter and activator structure are shown below the autoradiogram. Experiments were repeated three times. An autoradiogram of a typical experiment is shown. The relative CAT activity (± S.D.) was 1 (± 0), 47.5 (± 6.3), 30.3 (± 4.1), 25.6 (± 3.6), 8.9 (± 1.4), 21.3 (± 4.0), 1 (± 0), 64.0 (± 10.6), 41.9 (± 6.5), 1.5 (± 0.2), 1.1 (± 0.1), and 1.2 (± 0.1) for lanes 1-12, respectively. B, expression levels of GAL4VP16-CR derivatives. The chimeras are the same as in panel A. The GAL4 derivative is indicated above each track of the immunoblot. Lane M, molecular mass markers in kilodaltons are indicated on the left. The position of GAL4 derivatives is shown on the right (arrowhead). Approximately 30 µg of proteins (using bovine serum albumin as a standard) from transfected Saos-2 cells were fractionated on a 10% SDS-polyacrylamide electrophoresis gel. GAL4 derivatives expressed at 30 °C (lanes 2-5) and at 37 °C (lanes 7-10) were detected with an anti-GAL4-(1-147) antibody.

De Novo Protein Synthesis Is Not Required for CRts247 Activity-- Next, using GAL4VP16-CRts247 as an example, we set out to address the underlying molecular mechanism(s) responsible for the observation that CRts-chimeric proteins turned into transcriptional activators after the temperature was shifted from 37 to 30 °C (Fig. 1; also see Ref. 10). Conceivably, an increase in the transactivation activity of GAL4VP16-CRts247 at the permissive temperature might be because of the reactivation of the hybrid molecules already present in the cell. Alternatively, de novo synthesis of additional hybrid molecules was the underlying mechanism for the increase of transactivation activity after the temperature shift. To differentiate these possibilities, we examined the effect of cycloheximide, a potent inhibitor of translation, on the transactivation activity of the hybrid protein. Because protein synthesis was shut down in the presence of cycloheximide (data not shown), the transactivation activity of GAL4VP16-CRts247 was determined by quantifying the CAT mRNA rather than the CAT protein. As shown in Fig. 2, at 37 °C and in the presence of GAL4VP16-CRts247, the CAT transcript was barely detectable by primer extension (lane 5). After the assaying temperature was shifted to 30 °C, the level of CAT transcript dramatically increased (lane 3). Importantly, the thermo-regulated increase of CAT transcript was still observed in the presence of cycloheximide (lane 4), demonstrating that de novo protein synthesis was not required for the restoration of the activity of the fusion protein at the permissive temperature; i.e. a post-translational mechanism was involved in the reactivation of GAL4VP16-CRts247.


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Fig. 2.   Cycloheximide fails to prevent the reactivation of GAL4VP16-CRts247. Upper panel, analysis of CAT mRNA by primer extension. Transfection was performed at 37 °C for 36 h. Cycloheximide was added to the medium to a final concentration of 10 µg/ml 1 h before the incubation temperature was shifted to 30 °C. Total RNA from 2 × 106 transfected cells 20 h after the temperature shift was isolated and subjected to the primer extension analysis. The presence (+) or absence (-) of activator, reporter, and cycloheximide is indicated above each track of the autoradiogram. Lower panel, an equivalent amount of total RNA as used for the primer extension was loaded in each lane and was then illustrated by the ethidium bromide staining of the agarose gel.

CRts247 Does Not Interfere with the Subcellular Distribution of the GAL4 Chimera-- Because nuclear localization is a prerequisite for a transcription factor to exert its regulatory role, one potential mechanism responsible for the inactivity of GAL4VP16-CRts247 at the nonpermissive temperature is that the fusion protein is sequestered in the cytoplasm, especially in light of the report that some mutant p53 proteins are found to be distributed abnormally (4). To test this possibility, we performed an immunostaining study to inspect the subcellular distribution of the fusion protein. Fig. 3 shows that GAL4VP16-CRts247 was predominately localized to the nucleus, regardless of the assaying temperature. Thus, the inactivity of GAL4VP16-CRts247 in transactivating gene expression at 37 °C could not be attributed to the exclusion of the protein from the nucleus.


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Fig. 3.   CRts247 does not alter the subcellular distribution of the GAL4 chimera at the nonpermissive temperature. Immunostaining of transfected GAL4VP16-CRts247 protein in Saos-2 cells grown at 30 °C (left panel) and at 37 °C (right panel). GAL4VP16-CRts247 was accumulated in the nucleus regardless of the assaying temperature. Magnification, ×100.

CRts247 Does Not Inactivate the DNA-binding Activity of the GAL4 Fusion Protein-- Another possible mechanism for the observed inactivity was that GAL4VP16-CRts247, although present in the nucleus at the nonpermissive temperature, failed to bind its cognate site so that a promoter driven by GAL4 sites was, of course, not activated. To investigate this possibility, we determine the in vivo DNA-binding activity of the GAL4 chimeric proteins using the well established blocking assay (10, 24). Briefly, five copies of GAL4-binding sites were placed immediately downstream of the TATA box of a reporter. In this way, binding of GAL4 derivatives would block, via steric hindrance, assembly of the transcription initiation complex on the promoter and therefore reduce CAT activity. GAL4-(1-147), a derivative providing only GAL4 DNA-binding activity, as well as GAL4VP16-CRwt and GAL4VP16-CRts247, all inhibited transcription from the reporter containing GAL4 binding sites at 37 °C (Fig. 4, compare lanes 3-5 to lane 2). The reduction was specific because transcription of a corresponding reporter lacking GAL4 binding sites was not affected (Fig. 4, compare lanes 8-10 to lane 7). It was concluded that the GAL4 module of the fusion protein was still able to bind its cognate site even at the nonpermissive temperature.


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Fig. 4.   CRts247 does not inactivate the DNA-binding activity of the GAL4 chimera at the nonpermissive temperature. One microgram of the illustrated reporter DNA, 1 µg of the activator DNA, and 13 µg of the specified blocker DNA were used for calcium phosphate-mediated DNA transfection. A GAL4 derivative, GAL4-(1-147), which contains residues 1-147 of yeast GAL4 and possesses only DNA-binding activity, was used as a positive control. Cells were harvested 36 h after transfection. The absence (-) or presence (+) of activator is indicated above each track of the autoradiogram, as is the blocker. The reporter was pL6EG5C (lanes 1-5) or pL6EC (lanes 6-10). Diagrams of the structure of activator, reporters, and blockers are shown below the autoradiogram. Otherwise as in Fig. 1A. The relative CAT activity (± S.D.) was 1 (± 0), 12.3 (± 1.7), 2.2 (± 0.4), 1.7 (± 0.2), 2.1 (± 0.4), 1 (± 0), 6.4 (± 1.0), 5.7 (± 0.9), 5.8 (± 0.7), and 5.8 (± 0.8) for lanes 1-10, respectively.

CRts247 Does Not Function in Trans-- Because neither the nuclear localization (Fig. 3) nor the DNA-binding activity (Fig. 4) of the chimeric protein was altered, only the possibility that the VP16 activation domain of the fusion protein was the target of regulation by CRts247 remained viable. It is possible that CRts247 inhibits the VP16 domain by competing for a putative factor(s) that is required for the transactivation by VP16. In other words, this hypothesis predicts that CRts247 should function in trans as well; i.e. the transactivator and CRts247 co-exist as two individual molecules. Accordingly, experiments were performed to examine whether activator GAL4VP16 was repressed at 37 °C by CRts247 in trans. As shown in Fig. 5A, CRts247 was found to reproducibly repress GAL4VP16 in cis (lane 3). In contrast, the transactivation activity of GAL4VP16 remained constant (Fig. 5A, compare lanes 5-8 to lane 4) in the presence of a broad range of CRts247 (Fig. 5B, lanes 1-5). Moreover, the fact that CRts247 was unable to inhibit activator GAL4VP16 in trans was not because of the possibility that CRts247 itself failed to enter into the nucleus because CRts247, a protein of about 30 kDa, was observed to be homogeneously distributed in both the cytoplasm and the nucleus by immunofluorescence (data not shown). Thus, two conclusions could be drawn from the above study. First, CRts247 did not work via competing for a putative transcription factor(s). Second, and consequently, CRts247 could not be exploited as a regulatory cassette in trans.


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Fig. 5.   CRts247 does not inhibit GAL4VP16 in trans at the nonpermissive temperature. A, transactivation of reporter pG5E1bCAT by GAL4VP16 in the absence (lane 4) or presence of increasing amounts of CRts247 (lanes 5-8) at 37 °C. Fusion proteins GAL4VP16-CRwt and GAL4VP16-CRts247 were included as controls (lanes 2 and 3). Diagrams of reporter and activator structure are shown below the autoradiogram. The activator is indicated above each track of the autoradiogram. For the expression of CRts247, lanes 5-8 were co-transfected with 1, 2, 5, and 10 µg, respectively, of pRK5F-CRts247, a plasmid expressing FLAG-tagged CRts247; otherwise, as in Fig. 1A. The relative CAT activity (± S.D.) was 1 (± 0), 31.3 (± 5.6), 1.1 (± 0.2), 32.3 (± 6.4), 30.9 (± 4.9), 31.7 (± 5.2), 31.0 (± 5.5), and 30.8 (± 5.9) for lanes 1-8, respectively. B, expression levels of CRts247. The transfection conditions for lanes 1-5 were the same as in lanes 4-8 of panel A. Lane M, molecular mass markers in kilodaltons are indicated on the left. The position of CRts247 is shown on the right (arrowhead). Approximately 30 µg of proteins (using bovine serum albumin as a standard) from transfected Saos-2 cells were fractionated on a 12% SDS-PAGE gel. CRts247 was detected with an anti-FLAG antibody.

CRts247 Confers Temperature-dependent Binding of hTAFII32 to Activation Domains Present on the Same Polypeptide Chain-- Because CRts247 only functioned in cis (Figs. 1 and 5; also see Ref. 10), i.e. it repressed a transactivation domain present on the same polypeptide chain only at the nonpermissive temperature (37 °C) and not at the permissive temperature (30 °C), an alternative mechanism was proposed to explain how CRts247 functions. Perhaps, a spreading of the temperature-dependent structural transition from CRts247 into the neighboring region may account for the distinct behavior of CRts247. In other words, the conformation of a neighboring activation domain could be influenced by CRts247 so that the affinity of the activation domain to its target(s) is greatly reduced. In this regard, it was worthy to note that the activation domains of VP16 and p53 share the ability both to be regulated by CRts247 (Ref. 10) and to interact with a common co-activator, hTAFII32 (11, 25). Thus, it was compelling to question whether CRts247 inhibits the VP16 and p53 activation domains via interference of their interaction with hTAFII32 at the nonpermissive temperature. To test this idea, a GST pull-down assay was performed. In brief, hTAFII32 was expressed and purified from bacteria as a GST fusion protein, GST-hTAFII32. Binding of 32P-labeled GAL4 derivatives to hTAFII32 was measured by their retention on glutathione beads preloaded with the GST fusion protein. As expected, fusion of CRwt to the VP16 activation domain showed little effect on the affinity of the chimeric protein toward hTAFII32, regardless of the assaying temperature (Fig. 6A, compare lane 2 to lane 4). In contrast, binding of the CRts247-fusion protein to hTAFII32 became temperature-dependent: GAL4VP16CRts247 interacted with hTAFII32 at the permissive temperature (Fig. 6A, lane 8) but not at the nonpermissive one (Fig. 6A, lane 6). Besides, neither of the hybrid activators was retained by control GST beads (Fig. 6A, lanes 1 and 5), indicating that the observed binding was specific for hTAFII32. Also, in consistence with previous reports (11, 25), the VP16 domain was required for the observed binding of GAL4VP16-CRts247 to hTAFII32 because GAL4-CRts247, a derivative with the VP16 domain removed, was not retained by the column at 30 °C (data not shown). In a parallel experiment, wild-type p53-(1-318) was also retained by the beads regardless of the assaying temperature (Fig. 6B, lanes 2 and 4). However, the analogous region from p53N247I bound to the beads much better at the permissive temperature than at the nonpermissive one (Fig. 6B, compare lane 8 to lane 6). For the sake of simplicity, the C-terminal truncated p53 proteins were used rather than the full-length ones to exclude the bacterial heat shock 70-kDa protein, which has been shown to interact with the C terminus of p53 (26, 27), from complicating the interpretation of the experiment. Using the C-terminal truncated proteins also excluded the repression domain of the C terminus of p53 (5, 16), further simplifying the experiment. Taken together, the observation that CRts247 conferred temperature-sensitive binding of hTAFII32 to the VP16 and p53 activation domains not only was consistent with the conformation-spreading hypothesis but also strongly suggested that the temperature-dependent interaction of these domains with hTAFII32 was one of the underlying mechanisms responsible for the activity of CRts247 in vivo.


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Fig. 6.   CRts247 confers temperature-dependent binding of hTAFII32 to a neighboring activation domain. A, GAL4VP16-CRts247 binds to the GST-hTAFII32 bead only at the permissive temperature. Left panel, binding of GAL4VP16-CRwt at 37 °C (lanes 1-2) and at 30 °C (lanes 3-4). Right panel, binding of GAL4VP16-CRts247 at 37 °C (lanes 5-6) and at 30 °C (lanes 7-8). The GST protein bead used is indicated above each track of the autoradiogram. Input, one tenth of the input protein directly loaded onto the gel. Molecular mass markers in kilodaltons are shown on the left. B, interaction of p53N247I-(1-318) with the GST-hTAFII32 bead is also temperature-dependent. Left panel, binding of p53-(1-318) at 37 °C (lanes 1-2) and at 30 °C (lanes 3-4). Right panel, binding of p53N247I-(1-318) at 37 °C (lanes 5-6) and at 30 °C (lanes 7-8). The GST protein bead used is indicated above each track of the autoradiogram. Input, one-twentieth of the input protein directly loaded onto the gel. Molecular mass markers in kilodaltons are shown on the left.


    DISCUSSION

It has been shown that the central region of p53N247I gains a thermo-regulable repression activity toward several transcriptional activation domains (10). In this report, we demonstrate that the central regions of p53ts mutants all behave similarly (Fig. 1). The thermo-regulability of CRts247 requires neither new protein synthesis (Fig. 2), nor an alteration of the subcellular localization of the chimeric protein (Fig. 3). In addition, CRts247 does not inactivate the DNA-binding activity of the chimeric protein (Fig. 4). Further, CRts247 does not work via the competition for a putative transcription factor(s) that is required for the activity of a neighboring activation domain (Fig. 5). Rather, a temperature-dependent binding of hTAFII32 to the activation domains of chimeric proteins may account for the thermo-regulability of CRts247-fusion proteins (Fig. 6). However, both the VP16 and the p53 activation domain can bind several transcription factors (4, 28, 29). It is thus conceivable that, to achieve the observed repression, CRts247 may also interfere with the interaction of VP16 and p53 activation domains with transcription factors other than hTAFII32. This possibility is strengthened by the study that the activation domain of adenovirus E1a protein is likewise subjected to the thermo-regulation by a juxtaposing CRts247 (10). To our knowledge, there is no evidence supporting that the E1a activation domain augments gene expression by interacting with hTAFII32. Taken together, these observations strongly argue that the interference of hTAFII32 binding to neighboring activation domains is probably not the only mechanism responsible for the repression function of CRts247.

p53 is the most frequently mutated gene found in human cancer. Mutant p53 is believed to adopt a conformation that is different from that of wild-type p53 (7, 9). Because of their increased stability, mutant p53 proteins accumulate to a high concentration in cancer cells, and in many cases, they are found to be localized to the nucleus (30, 31). The strength of p53 activation domain (32-34) is comparable with that of VP16, the strongest one known to date. Theoretically, it should cause a problem described as transcriptional squelching (35) in cancer cells harboring a mutant p53 if the activation domain of such a mutant p53 is still active. In light of this, the finding that p53 mutants usually lose not only the DNA-binding but also the transactivation activities (this work and see Ref.10) seems biologically relevant. However, it is not clear how p53 mutants inactivate their transactivation domain. Using temperature-sensitive p53 mutants as a model, we demonstrate that the central region, when trapped in the mutant conformation, gains a repression function by preventing, at least, hTAFII32 from interacting with the p53 activation domain. In support of this hypothesis, destruction of the intactness of the central region of wild-type p53 by deletion, which is thought to cause a conformational change by disrupting the compact packing of the region (36) so that a repression domain approximately encompassing amino acids 101-170 is activated (16), also renders the resultant p53 fragments with a repression activity (16, 37). In contrast, the central regions of p53 mutants that retain the wild-type conformation, such as p53R248W, p53R273H, and p53R280K (23), behaved like that of wild-type p53 and could not repress the p53 and VP16 activation domains (22).2

The ligand-binding domain (LBD) of steroid receptors can also function as a movable and regulable inactivation cassette (38). There exist, however, differences between CRts247 and LBD. First of all, the activity of CRts247 is regulated by temperature (Ref. 10 and this work), in contrast to the case of LBD which is controlled by its cognate steroid hormone (38). Second, whereas it is presumed that steric hindrance via the interaction of heat shock protein 90 (HSP90) with the unliganded LBD is mainly responsible for the inactivation of fusion proteins (39), several lines of evidence indicate that CRts247 does not require the interaction with a putative bulky protein to function as an autonomous regulatory cassette. First, although HSP70 and HSC70 are known to bind to p53, they do not bind to the central region (4). In fact, we could not detect any interaction between CRts247 and HSP70, HSC70, or HSP90 by various biochemical and genetic approaches (data not shown). Second and more importantly, recombinant hTAFII32 and CRts247-fusion proteins, expressed in and purified from bacteria, directly interact with each other in a temperature-dependent manner (Fig. 6). Accordingly, our experimental results do not favor the bulky protein model to explain how CRts247 functions. Instead, available data are consistent with the conformation-spreading hypothesis to explain the molecular mechanism by which CRts247 represses the neighboring transactivation domain present on the same polypeptide chain. Nevertheless, because the partial digestion patterns of GAL4VP16-CRts247 by several proteases, including V8 protease, endoproteinase Arg-C, alpha -chymotrypsin, m-calpain, and alkaline protease, in the presence or absence of hTAFII32 at either 30 °C or 37 °C were not informative (data not shown), future studies are required to directly test the hypothesis.

    ACKNOWLEDGEMENT

We acknowledge Dr. J. Y. Chen for p53N247I, p53V143A, and p53V173L clones.

    FOOTNOTES

* This work was supported by grants from the Academia Sinica and the National Science Council of Taiwan (to C. T. L., M. Y. L., C. W. W., and Y. S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to the study.

§§ To whom correspondence should be addressed. Tel.: 886-2-26523074; Fax: 886-2-27829142; E-mail: bmyslin{at}ccvax.sinica.edu.tw.

2 S.-F. Tung, J.-Y. Chuang, C.-T. Lin, M.-Y. Lai, C.-W. Wu, and Y.-S. Lin, unpublished results.

    ABBREVIATIONS

The abbreviations used are: CRts, temperature-sensitive mutant of the central region; CRwt, wild-type mutant of the central region; PBS, phosphate-buffered saline; GST, glutathione S-transferase; LBD, ligand-binding domain.

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