(Received for publication, June 15, 1995; and in revised form, August 4, 1995)
From the
Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Gal4 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD-mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Gal4-p53 TAD also impair the binding activity to TBP severely. Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation-deficient mutant type.
Transcriptional activators have been shown to stimulate in
vitro the assembly of transcriptional preinitiation complexes (1, 2) as well as transcriptional elongation by RNA
polymerase II(3) . This stimulation is thought to depend on
direct or indirect protein-protein interactions between transcriptional
activators and the general transcriptional machinery and/or on
relieving the inhibitory effects of chromatin(4, 5) .
Transcriptional activators can be divided into at least two discrete
functional domains(6) ; a DNA binding/targeting domain is
required to direct the activator to the appropriate DNA sequence
element and then the transcriptional activation domain (TAD) ()can induce the enhanced transcription of target genes.
TADs have been divided into three major classes according to a
predominance of particular amino acid residues: acidic, proline-rich,
or glutamine-rich(7) . Of these classes, the acidic TADs appear
to be unique in that they can apparently function universally in all
eukaryotes tested from yeast to human(8) .
Like other
transcriptional activators, tumor suppressor protein p53 appears to
have a modular domain structure; it contains an NH-terminal
region which functions as a TAD when coupled to a heterologous DNA
binding domain(9, 10) , a central site-specific DNA
binding domain(11, 12) , an oligomerization
domain(13, 14) , and a basic COOH-terminal nuclear
localization domain(15) . The NH
-terminal TAD of
p53 is similar in size, net negative charge, and transactivating
potency to the well defined TAD of herpes simplex virus virion protein
16 (HSV VP16)(16) . This region is also rich in proline
residues which are conserved through evolution(17) . Like VP16
and a number of other transactivators, p53 is thought to be a
transactivator of the acidic type(9, 18) .
Early studies suggested TFIID as the target for various activators(19, 20) . Subsequently, the TATA-binding proteins (TBP) of yeast and human were shown to bind in vitro to the strong TADs of such viral and cellular activators as VP16(21) , E1A(22) , Zta(23) , and p53(18, 24, 25) . It has also been shown that another general transcription factor, TFIIB, interacts with various transactivators such as VP16 (26) , Rel oncogene product(27) , and CTF(28) . Recent report showed that VP16 TAD and p53 TAD can also bind to TFIIH(29) . In addition to general transcription factors, coactivators or adaptors are required for transactivation in the in vitro transcription system. The best characterized proteins among adaptors are the TBP-associated factors (TAFs) of the Drosophila melanogaster and humans (30, 31, 32, 33) . Recently, it was reported that p53 TAD can also interact with two subunits of the TFIID, TAFII40, and TAFII60(34) . Clearly, transcriptional activation appears to be more complicated than originally envisioned (6) and may involve multiple targets that make direct or indirect contacts in different spatial and temporal arrangements with TADs and the transcriptional machinery.
Here, we demonstrate that p53 TAD is a complex activation domain composed of two subdomains, in which positionally conserved hydrophobic residues are critical for activating function. The negatively charged residues and proline residues are also necessary for full activity, but not essential for the activity of p53 TAD. Mutations that severely impair the function of p53 TAD in vivo have been shown to diminish binding activity to TBP in vitro, indicating that the observed in vitro interaction is biologically relevant. Circular dichroism (CD) spectroscopy demonstrates that p53 TAD peptide does not have any detectable secondary structure at physiological condition.
Figure 1: Schematic diagram of chimeric Gal4D-p53 TAD for eukaryotic expression and pGEX-p53 TAD for prokaryotic expression. A series of mutant derivatives were constructed by inserting various TAD derivatives with point mutation into Gal4D and pGEX-KG to generate Gal4D-p53 TAD derivatives and pGEX-p53 TAD derivatives, respectively.
The glutathione S-transferase (GST) fusion plasmids were made by using pGEX-KG which contains a GST gene under the control of tac promoter and a flanked polycloning site(36) . pGEX-p53 TAD was constructed by inserting the 210-bp BamHI-HindIII DNA fragment of pSK-p53 TAD into the BamHI-HindIII site of pGEX-KG (Fig. 1). pGEX-p53 M2, M12, M19, M22, M23, M25, M31, M34, and M1234 were generated by the same method. pGEX-p53 M41 and M241 were made by inserting the BamHI-XhoI DNA fragments of pSK-p53 TAD M41 and M241 into the BamHI-XhoI sites of pGEX-KG, respectively. pGEX-p53(1-40) was generated by inserting the 120-bp BamHI-HindIII DNA fragment of pSK-p53 TAD M41 into the BamHI-HindIII site of pGEX-KG. The reporter plasmid, G5E1bCAT, was described previously(37) .
The p53 TAD is also rich in proline residues (19.2%), which is a characteristic of another class of TAD, such as CTF/NF-1(44) . When M12 and M34 mutants were tested, there were about 39 and 24% reduction in p53 TAD-mediated transactivation, respectively. As expected, the M1234 mutant containing mutations in four Pro residues was shown to be about 71% reduction in the transactivation (Table 1), indicating that there was additive effect with these mutations and that proline residues are also required for the optimal activity of p53 TAD.
Previous studies on the VP16 TAD have suggested that the acidic residues contribute to its activity, but intervening hydrophobic residues are more important than other residues(45) . TADs of a number of transactivators exhibit a conserved pattern of hydrophobic residues (45) . Since p53 TAD also shows the similar pattern of positionally conserved hydrophobic residues (Fig. 2), we generated various mutants in which conserved hydrophobic amino acids were replaced with hydrophilic ones. When these mutants were tested for transactivation activity in BHK-21 and COS-7 cells, the activities of several mutants were significantly impaired (Table 1). Mutations on both residues Leu-22 and Trp-23 reduced p53 TAD-mediated transactivation by about 95%, whereas mutations on Leu-25 and Leu-26 resulted in approximately 88% loss of the activity. Also, single amino acid change on Phe-19 reduced the activity by about 85%. In contrast, mutations on both Val-31 and Leu-32, which are not positionally conserved, did not impair the transactivation function but rather enhance the activity. Therefore, we concluded that the positionally conserved hydrophobic residues, Phe-19, Leu-22, Trp-23, Leu-25, and Leu-26 are critical for transactivation function of p53 TAD. These residues are identical in all sequences of p53 protein from several species(17) . The effect of mutations on Leu-22 and Trp-23 is consistent with a previous report(43) , but those of mutations on Phe-19 and on Leu-25 and Leu-26 do not exactly coincide with their results in which human p53 mutant protein containing the double mutation on Leu-14 and Phe-19 was observed to have a 50% reduction in chloramphenicol acetyltransferase activity compared with wild type p53. In addition, the Leu-25 and Leu-26 double mutant showed either enhanced or reduced activity in Saos-2 cells, depending on p53-responsive elements either from the creatine phosphokinase gene or from the mdm-2 gene(43) .
Figure 2: Comparison of the primary amino acid sequences of different TADs. The amino acid sequences of several TADs are aligned using the bulky hydrophobic residues (boxed) as reported by Cress and Triezenberg(45) . Underlined letters of p53 TAD indicate identity in all sequences of p53 from several species(17) . The residue numbers are given for p53 TAD sequence.
To compare the expression level among different Gal4 fusion proteins, electrophoretic mobility shift assay was performed using a labeled DNA fragment containing five Gal4 binding sites and showed that there was no significant difference among them (data not shown). The difference in the chloramphenicol acetyltransferase activity is, therefore, due to the intrinsic biological activity of different Gal4 fusion proteins, but not by the different level of Gal4 fusion proteins in the transfected cells. Although the transactivating abilities of mutants constructed in the foregoing studies were severely impaired, residual activity still remained, suggesting that p53 TAD is composed of separable subdomains just like VP16 (46) and Epstein-Barr virus Rta transactivator(47) . It was previously shown that the minimal activation domain of p53 lies within the first 42 amino acids of the protein(48) . Since Gal4D-p53(1-40) consistently showed about 30-38% activity of Gal4D-p53 TAD, which contains the residues 1-73, residues 43-73 appear to be necessary for the full p53 TAD-mediated transactivation. To be certain that residues of p53 from 43 to 73 also contain an autonomous TAD, Gal4D-p53 (43-73) was constructed and tested for the transactivating ability. The resulting plasmid showed about 6% activity of Gal4D-p53 TAD (Table 1), indicating that there is an autonomous TAD in this subregion. In the case of VP16, the truncated activation domain possesses approximately 50% of wild type activity, whereas the addition of COOH-terminal subdomain restored the full activity(46) . Gal4D-p53(1-40) M22, which deletes the COOH-terminal subregion from M22 mutant, completely lost the residual activity of M22 mutant (Table 1), demonstrating that the residual activity comes from the separable COOH-terminal subdomain, and that Leu-22 and Trp-23 are absolutely required for the function of minimal activating region (residues 1-40) of p53.
Figure 3: Direct correlation between the binding activity of p53 TAD to TBP and p53 TAD-mediated transactivation. A, SDS-PAGE analysis of purified GST-p53 TAD fusion protein and its mutant derivatives. 1 µg each of samples were subjected to 10% SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue. B, the p53 TAD and mutant derivatives were assayed for the ability to bind in vitro translated human TBP in a GST pull-down assay as described under ``Materials and Methods.'' C, the relationship between transactivation and TBP binding activities of p53 TAD and mutant derivatives. The autoradiogram corresponding to two independent GST pull-down experiments were analyzed and quantitated by a photoimaging system. Each signal was plotted with the relative chloramphenicol acetyltransferase activity shown in Table 1. The signal measured for p53 TAD was defined as 100% arbitrarily.
Figure 4: The CD spectra of purified p53 TAD (A) and the M22 mutant (B) obtained at pH 7.0 with several different concentrations of TFE. a, 0%; b, 10%; c, 20%; d, 30%; e, 50%.
The determination of the critical amino acid residues and protein structures involved in mediating the biological activity of TADs would represent an important step toward understanding the mechanism of transcriptional activation. Since p53 is an important tumor suppressor protein and contains a distinct TAD, including acidic residues (23.3%) and proline residues (19.2%), we have chosen p53 TAD to study the molecular mechanism of transcriptional activation. Due to its high content of acidic and proline residues, p53 TAD may fall into the category of a combination of acidic and proline-rich domain as in the cases of Jun and Fos(51, 52) . Our mutational analyses showed that the negatively charged residues and proline residues of p53 TAD are necessary for full activity but not essential for the transactivation ability. Several reports recently suggested that acidic residues are not essential for transactivation function, but hydrophobic and bulky aromatic residues may be more important in defining the transactivation domain. Importance of hydrophobic residues was also observed in p53 TAD, since our results revealed that the conserved hydrophobic residues (Phe-19, Leu-22, Trp-23, Leu-25, and Leu-26) are critical for transactivation ability ( Fig. 2and Table 1).
Interestingly, p53 TAD is composed of two separate
functional subdomains. The COOH-terminal subdomain (amino acids
43-73) has weaker transactivation ability than minimal activating
region (amino acids 1-40) in BHK-21 and COS-7 cells when linked
to Gal4 DNA-binding domain (Table 1). This COOH-terminal
subdomain contains a similar level of acidic residues and proline
residues (acidic: 25.8%, proline: 22.6%). The requirement of
COOH-terminal subdomain in cis for optimal transactivation
ability of p53 TAD suggests the possibility that the subdomain may be
required for stabilizing the interaction between p53 TAD and the target
molecules. Alternatively, the subdomain may directly contact with
different cellular factor(s). It has been shown that the full-length
VP16 TAD, but not NH-terminal subdomain, interacts with
TAFII40(53) , whereas NH
-terminal subregion can
interact with TBP (54) and TFIIB (26) . Many
characteristics of p53 TAD are shared with those of VP16 TAD, including
(i) essential bulky hydrophobic residues, (ii) an overall negative
charge, (iii) the lack of secondary structure in
solution(55, 56) , (iv) separable two subdomains, and
(v) in vitro interaction with yeast and human TBP. These
findings suggest that p53 TAD has the same mechanism of action as does
the VP16 TAD. We have observed that overexpression of p53 TAD can
efficiently inhibit the function of VP16 TAD and vice versa in an in vivo squelching experiment. (
)Also apparently
shared with VP16 TAD is that transcriptionally compromised mutants of
p53 TAD have reduced binding ability to TBP(54) . In addition,
there are accumulative evidences that several nonfunctional TADs have
reduced binding abilities to TBP, suggesting that there is the
biological relevance of these
interactions(22, 57, 58) .
Two hypotheses
for the role of critical hydrophobic residues are that these residues
are necessary for either maintaining the structure of the activation
domain or the direct interaction with TBP. Based on the results of CD
spectroscopy, there is no significant structural difference between
wild type p53 TAD and M22 mutant. This suggests that Leu-22 and Trp-23
may be directly interacting residues with TBP and may not be involved
in structural determination. In contrast to highly ordered DNA binding
modules, activation domains may be not so highly ordered on their own,
but appear to become structured only upon interactions with target
molecules. This hypothesis is supported by the finding that the
biological function of TADs does not require a well defined amino acid
sequence. Based on this sequence flexibility, it was suggested that
acidic regions are unstructured negative noodles which become
structured upon interaction with some part of the transcription
apparatus(59) . This ``induced fit'' model seems to
be further supported by our results and previous data on VP16 TAD (55, 56) showing the apparent lack of any detectable
-helical or other structure. Tight interactions between TADs and
target molecules seem to be dependent on hydrophobic interactions that
are formed by the induced fit. However, it is necessary to perform the
structural analysis in a complex form of p53 TAD and TBP for a complete
definition of the induced structure.