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Proc Natl Acad Sci U S A. Mar 3, 2009; 106(9): 3142–3147.
Published online Feb 13, 2009. doi:  10.1073/pnas.0900383106
PMCID: PMC2651247
Biophysics and Computational Biology

Molecular basis of the interactions between the p73 N terminus and p300: Effects on transactivation and modulation by phosphorylation

Abstract

The transcription factor p73 belongs to the p53 family of proteins and can transactivate a number of target genes in common with p53. Here, we characterized the interaction of the p73 N terminus with four domains of the transcriptional coactivator p300 and with the negative regulator Mdm2 by using biophysical and cellular measurements. We found that, like p53, the N terminus of p73 contained two distinct transactivation subdomains, comprising residues 10–30 and residues 46–67. The p73 N terminus bound weakly to the Taz1, Kix, and IBiD domains of p300 but with submicromolar affinity for Taz2, in contrast to previous reports. We found weaker binding of the p73 N terminus to the p300 domains in vitro correlated with a significant decrease in transactivation activity in a cell line for the QS and T14A mutants, and tighter binding of the phosphomimetic T14D in vitro correlated with an increase in vivo. Further, we found that phosphorylation of T14 increased the affinity of the p73 N terminus for Taz2 10-fold. The phosphomimetic p73α T14D caused increased levels of transactivation.

Keywords: p73, transcription

p73 belongs to the p53 family of tumor suppressor proteins. It shares domain organization similar to that of p53 and p63, consisting of a flexible N-terminal transactivation domain, a DNA-binding domain, and an oligomerization domain. Both p73 and p63 may exist in one of several C-terminal splice variants; this feature, combined with the use of two distinct promoters that give rise to transactivation (TA) and dominant-negative (ΔN) variants, means that p73 may be expressed in a wide range of various isoforms. p73 has several functions in common with p53, such as activation of target gene expression and suppression of cell growth. However, p73, along with the other p53 family member, p63, does not function as a classical tumor suppressor and is rarely mutated in human cancers (1). Imbalances in the TAp73/ΔNp73 ratio may be more important in tumorigenesis and response to chemotherapy than mutations (2). ΔNp73 is preferentially degraded in response to DNA damage, allowing accumulation of the proapoptotic TAp73 isoform (3). p73, unlike p53, plays an essential role in normal growth and development, with p73-knockout mice displaying severe developmental defects but no increased susceptibility to spontaneous tumorigenesis (4). Despite its important role, relatively little is known about the various mechanisms by which p73 can induce apoptosis. p73 can induce G1 growth arrest, transactivate genes such as p21, Mdm2, Bax, and 14-3-3σ, and is capable of inducing apoptosis regardless of p53 status (for reviews see refs. 5 and 6). It is not yet clear whether transactivation of a similar group of promoters to those of p53 is sufficient for p73 to induce p53-independent apoptosis.

The transcriptional coactivator p300 is a large multidomain protein that possesses histone acetyltransferase (HAT) ability (7). Together with its homolog, CREB-binding protein (CBP), p300 mediates transcription through binding to transcriptional activators such as JUN, E1A, NF-κB, and to the p53 family (8). Previous studies have illustrated that the interaction between the p73 N terminus (p73Nt) and the zinc finger Taz1 domain of p300 is important for p73 to function as a transactivator (9), whereas the HAT activity of p300 is unimportant for stimulation of p73 function (10). The three members of the p53 family of proteins have distinct roles during embryogenesis and tumorigenesis; it is interesting to examine how much of this variation is the result of differences in the N-terminal domain, which has the lowest levels of similarity between family members. Here, we have utilized a combined biophysical and cell biology approach to quantify the binding of the p73Nt to the Taz1 domain and also to three other p300 domains: Taz2, Kix, and IBiD. We have shown that two distinct regions of the p73Nt are involved in binding to the p300 domains Taz1 and Taz2, whereas only one region is necessary for interaction with the Kix and IBiD domains. In contrast to previous work showing that the p73Nt binds only to the Taz1/CH1 domain of p300 (9), we found from fluorescence anisotropy that the Taz2 domain could bind to the p73Nt and did so with a greater affinity than did Taz1. We used evidence from NMR studies to propose a structural basis for the observed effects. Further, we found that mutations in the N-terminal regions that abrogate the interaction with p300 domains had a significant detrimental impact on the ability of p73α to function as a transactivator and that phosphorylation of T14 may play an important regulatory role.

Results

Interaction of the p73Nt with the p300 Domains.

To determine the extent of the p73Nt-binding site, we monitored shifts in the 1H 15N heteronuclear single quantum correlation (HSQC) spectra of p73Nt constructs upon the addition of various p300 domains. Two-dimensional 1H 15N HSQC spectra for free p73 1–67 and p73 bound to the four p300 domains and Mdm2 are shown in Fig. 1 A and B. Residues of the p73Nt that shift upon addition of p300 domains are detailed in Fig. 1C. Some peaks in the bound spectra were not assignable because of line-broadening effects and disappearing peaks.

Fig. 1.
Interactions of the p73 N terminus with p300 domains as determined by NMR spectroscopy. (A) NMR HSQC spectra of free 15N p73 1–67 (black) and in the presence of an excess of Taz1 (blue), Taz2 (red), and Mdm2 (green). (B) HSQC spectra of free ...

Upon addition of the zinc finger domains of p300 (Taz1 and Taz2), we saw changes in chemical shift for residues in two distinct regions of the p73Nt. It clearly consisted of two separate subdomains that may be defined as TAD1 from residues 10–31 and TAD2 from residues 46–67, unlike p53, where TAD1 and TAD2 correspond to residues 1–40 and 41–60, respectively (11). There is a linker between TAD1 and TAD2, broadly corresponding to Q33–G45. In this region, few changes in chemical shift were observed upon addition of p300 domains, highlighting the distinct nature of the subdomains. There were no further changes in chemical shifts on addition of Taz2 to greater concentrations than p73Nt, indicating formation of a 1:1 complex.

TAD1s of p53 and p73 have several regions of conservation (Fig. 1D) whereas the TAD2s vary more. The changes in chemical shifts in the HSQC spectrum show that Taz1, Taz2, and Mdm2 bind to the p73Nt in a fashion similar to the p53Nt, involving both transactivation subdomains. But, in contrast to p53, binding of the p300 domains Kix and IBiD results in few chemical shift changes in TAD2.

The Transactivation Domain of p73 Bound More Weakly to the p300 Domains Than Does p53.

In all cases, the full-length peptide bound the most tightly, as measured by fluorescence anisotropy (Fig. 2 and Table 1). Dissociation constants ranged from 0.89 μM for Taz2 to 25.5 μM for IBiD. There were significant increases in affinity between the shorter peptides and the full-length peptides for the Taz2 domain. This finding is consistent with the NMR data and suggests that both subdomains are required for maximum affinity. Interestingly, this was also the case for Mdm2, which has a Kd value comparable with that of Taz2 for binding to the full-length peptide, unlike with p53. Although NMR data for Taz1 and Mdm2 binding to p73 confirmed that both subdomains were involved in binding, Kd values obtained via fluorescence anisotropy suggest that, by itself, TAD2 contributes weakly to binding. All four domains of p300 studied here interacted with p53 1–57 with binding constants of <10 μM (11). In contrast, the p73Nt displays a greater range of affinities. Taz2 and Mdm2 had the smallest dissociation constants of 0.89 and 0.87 μM, respectively. Kix and IBiD bound more weakly, consistent with their binding to only the first transactivation domain. With the exception of the Taz2 domain, we were unable to determine a Kd value for binding of p73 41–70 to p300 domains and Mdm2 by using fluorescence anisotropy. Although NMR data suggested that the second subdomain plays a minimal role in Kix or IBiD binding to the p73Nt, Kd values were obtained by using only the full-length peptide. In general, although extending the peptides to include the second transactivation domain resulted in a noticeable enhancement of avidity, the effect is less marked than for p53.

Fig. 2.
Fluorescence anisotropy titrations of N-terminal peptides of p73 to the p300 domains Taz1, Taz2, Kix and IBiD, and Mdm2.
Table 1.
Dissociation constants for binding of various p300 domains and Mdm2 to p73 N-terminal peptides as determined by fluorescence anisotropy

The Mutant L18QW19S Bound Weakly to the p300 Domains.

The QS1 (L22Q/W23S) and QS2 (W53Q/F54S) mutants of p53 have impaired transcriptional activity because of their greatly reduced affinity for p300 (1214). Although there is no direct equivalent of the QS2 mutant in the p73Nt, residues L18 and W19 are conserved; the hydrophobic residues V51 F52 or V61 M62 may fulfill a role similar to that of the p53 QS2 mutant. We hypothesized that the p73 QS1 mutant, L18QW19S (henceforth referred to as p73QS), would also abrogate binding to the p300 domains. We were unable to detect any binding of the double-point mutation peptide, L18Q/W19S, to any of the p300 domains by using fluorescent anisotropy (Fig. 2).

p73Nt Binds to Taz2 in a Fashion Similar to That of p53.

The binding site of the p53 N-terminal peptide 14–28 in the murine Taz2 domain from the p300 homolog CBP has been reported (15). Shifts in the HSQC spectra of p73 1–67 (Fig. 1 A and B) upon binding to the p300 domains and Mdm2 were indicative of helix formation by the transactivation domains. Formation of small helical regions is common among transcriptional activators; for example, p53 forms a 13-residue helix upon binding Mdm2 (16), and the c-Jun N terminus also forms helical regions upon binding to Kix (17). It is to be expected that the first transactivation domain of p73 behaves in a similar fashion.

The HSQC spectrum of 15N-labeled Taz2 alone and in complex with an excess of unlabeled p73 1–67 is shown in Fig. 3. Not all peaks in the 2D HSQC of the complex were assignable because of line-broadening effects and disappearing peaks. Reassignment of the bound state was challenging because of the low concentration of labeled protein and exchange broadening, despite attempting triple resonance experiments. The structure of Taz2 from the p300 homolog CBP consists of four helices with three zinc-binding motifs, and the binding surface of a peptide representing residues 14–28 of p53 has been mapped (15). Peaks corresponding to Taz2 residues that shifted significantly upon binding (or disappearing/unassignable peaks) to the p73Nt coincided with those that are reported to shift upon binding of the p53 N-terminal peptide. In particular, residues L69–L72, which showed large shifts upon binding to the p53 (1428) peptide, showed large shifts or were not assignable when in complex with the p73Nt. Given the homology between the p53 (1428) peptide and the equivalent region of the p73Nt, we presume that the first transactivation domain binds to an area on the Taz2 surface consisting primarily of residues L69–L72, which form part of the third helix. The abrogation of binding obtained by substituting nonpolar residues L18W19 for Q18S19 suggests that binding between the Taz2 and p73Nt is primarily driven by the interaction of two hydrophobic surfaces. In addition to shifts described by Wright and coworkers (15), we also observed shifts and disappearing signals in a region corresponding to residues N83-C90, and we propose that the second transactivation domain binds here.

Fig. 3.
HSQC of 15N Taz2 free (red) and in a 1:1.2 complex with unlabeled p73 1–67 (blue). For clarity, not all peaks are labeled.

Interaction Between the TAp73 N Terminus and p300 Was Essential for TAp73 to Function as a Transactivator of Bax.

The Bax protein (Bcl-2-associated protein X) was the first identified proapoptotic member of the Bcl-2 family of proteins (18). p73 has a 2-fold effect on Bax: first, it increases steady-state levels of Bax protein through direct transactivation; and second, it promotes translocation of Bax from the cytosol to the mitochondrial membranes through an indirect pathway via PUMA induction (19) (for a review see ref. 6). We investigated the ability of p73αWT and the double point mutant p73αL18Q/W19S to transactivate the bax promoter in H1299 cells. Because we were unable to find binding of the QS peptide to any p300 domain by using fluorescence anisotropy, we hypothesized that p73αQS would be severely weakened in its ability to transactivate Bax expression. To test this hypothesis, we used a bax-luciferase reporter assay in H1299 cells. The QS mutant does indeed show a marked reduction in bax reporter activity (Fig. 4A); residual transactivation ability may be caused by binding between the transactivation and p300 domains that is too weak to detect by fluorescence anisotropy. To eliminate the possibility of Bax transactivation resulting from an alternative pathway that did not involve the TAp73–p300 interaction, we used siRNA to achieve a 3-fold reduction in p300 levels and then repeated the luciferase assay. In cells where p73αWT was cotransfected with p300 siRNA, we saw a decrease in p300 levels concurrent with a decrease in bax reporter activity (Fig. 4B), demonstrating that the ability of p73α to transactivate Bax expression depended on an interaction with p300; both a weakening of the p300–p73 interaction through mutation and a decrease in the levels of p300 obtained through siRNA knockdown resulted in a decrease in bax transactivation ability.

Fig. 4.
p73Nt effects on bax transactivation. (A) H1299 cells transiently cotransfected with p73α WT, p73α QS, and bax-Luc with or without expression plasmids encoding p300. p300 and pcDNA 3.1(−) were used as controls. The QS mutant displays ...

Phosphorylation of the p73Nt at T14 Increased Its Avidity for the p300 Domains.

Phosphorylation of the p53Nt is known to inhibit the p53–Mdm2 interaction. We examined the binding of a p53 T18P analog, p73 10–40 T14P, to the p300 domains and Mdm2 (Fig. 5 and Table 1). This phosphorylated peptide bound with ≈10-fold greater affinity for the p300 domains Taz1 and Taz2, whereas only a 2-fold increase was seen for Mdm2. The phosphorylation of T14 also increased the affinity of the p73Nt to Kix and IBiD; however, a direct comparison between the phosphorylated and unmodified peptide was not possible because we were unable to obtain Kd values for the unmodified peptide for Kix and IBiD. To examine the potential effects of phosphorylation of T14 in vivo, we repeated the bax-luciferase assays with the mutants TAp73αT14A and TAp73αT14D. We hypothesized that the T14A mutant would show reduced bax transactivation ability whereas the T14D phosphomimetic mutant would show increased bax transactivation. Indeed, this was found to be the case, with the T14D resulting in almost twice the transactivation ability of wild-type p73 (Fig. 6A). We further confirmed this with a dose–response luciferase assay with T14D (Fig. 6B). We expect, therefore, the full T14P phosphorylation to exhibit a greater effect, thus suggesting that phosphorylation of T14 is part of an important mechanism that controls p73 behavior in vivo.

Fig. 5.
Fluorescence anisotropy titrations of the phosphorylated peptide p73 10–40 T14P to the p300 domains Taz1, Taz2, Kix and IBiD, and Mdm2.
Fig. 6.
Effects of T14D phosphomimetic mutant on p73α transactivation capability. (A) H1299 cells were transiently cotransfected with p73α WT, p73α T14D, p73α T14A, and bax-Luc; pcDNA 3.1(−) was used as a negative control. ...

Discussion

p73 is thought to be a key determinant of cellular sensitivity to anticancer therapeutics (20), particularly in tumors lacking p53. p73α is, to a greater extent than p73β, widely induced by chemotherapeutic agents in a range of tumor cell lines (20). p300 has been shown to act as a link between transactivators such as the p53 family and the transcription machinery (for a review see ref. 7). Using combined biophysical and cell-based methods, we have quantified the binding of the p73Nt to various domains of p300 and directly correlated binding strength to cellular activity. In contrast to previous studies that suggested that the p73Nt bound only to the Taz1/CH1 domain of p300 (9), we have found that Taz2 bound to the p73Nt and did so with a higher affinity than did Taz1. Further, the p73Nt could bind to several domains of p300. Binding of the N-terminal regions of p53 family members via the Taz2 domain is concurrent with p300 behaving as a general coactivator for a wide range of transcription factors. Despite previous GST pulldown assays not showing an interaction between the Taz2 domain and p73Nt, it is difficult to imagine an in vivo scenario in which the tight affinity reported here between the two domains is irrelevant. Given the high affinity of the p73Nt for Taz2 and its structurally similar mode of binding to p53, it is clear that Taz2-p53 family N terminus binding is part of the general transactivation activity, and specificity between p53 family-specific target genes is achieved by other means.

Role of p300 Domains.

Although we have quantified binding of the p73Nt to four separate domains of p300, it is not yet clear whether all domains are involved in effecting p73 transcriptional activity. The possibility of four p300 domains binding simultaneously to a p73 tetramer cannot be discounted; however, given the weaker binding of the remaining domains, it is difficult to postulate a biological reason for such a mode of binding.

Binding of p73 to Mdm2.

The E3 ubiquitin ligase Mdm2 is a crucial negative regulator of p53. Overexpression of Mdm2 is associated with transformation of NIH 3T3 and Rat2 cell lines; it is believed that this arises because of its inhibitory effect on p53 (21). Inhibition of p53 occurs via two mechanisms: first by inhibition of p53 transcriptional ability through binding to the p53Nt (22) and second through ubiquitination and subsequent degradation of p53 (23, 24). Mdm2 can alleviate p73-induced apoptosis (25). But, the role of Mdm2 in modulating p73 activity is more limited because the diversity in the C terminus of p73 precludes ubiquitination occurring in a fashion similar to p53. Mdm2 is, therefore, only able to prevent transcriptional activity of p73 by binding to the N terminus. Zeng et al. (9) demonstrated that Mdm2 competes with p300 for binding to the p73Nt but not to p53 and hypothesized that binding between p73Nt and p300 occurred via the Taz1 domain. However, our results clearly show that Taz2 is capable of binding the p73 N-terminal domain and does so with an affinity comparable with that of Mdm2 and in excess of that of Taz1. Our data, therefore, support a p73 control mechanism that involves competition between Mdm2 and p300. But, the high affinity of p73Nt for the Taz2 domain implies that it is Taz2 that is involved in p300–p73 binding. Control of p73 transactivation may, therefore, be achieved through the relative levels of p300 and Mdm2 within the cell in concert with selected posttranslational modifications.

Modification of Binding by Phosphorylation.

p53 behavior is modulated extensively through the use of posttranslational modifications (for a review see refs. 26 and 27. In contrast, posttranslational modifications of p73 have not yet been investigated as comprehensively. Hck and c-Src phosphorylate Y28 (28); JNK may phosphorylate S8 (among other sites not located in the N-terminal region) (29) and catalytic subunit β of protein kinase A has been shown to phosphorylate the N terminus at an as yet unidentified site. Other posttranslational modifications have been reported: acetylation of lysines 321, 327, and 331 by p300 activate the apoptotic functions of p73 (30); phosphorylation of tyrosine 86 by various cyclins results in a decrease in p73 transcriptional activity (31, 32) whereas phosphorylation by c-abl (33, 34) has the opposite effect, resulting in an increase in apoptotis-inducing behavior. Phosphorylation of the N terminus is an obvious candidate for altering p73 transcriptional activity; modifications may increase the affinity of the N terminus for p300 with a subsequent increase in transactivation. Alternatively, Mdm2-mediated modifications of the N terminus may decrease the transactivation ability of TAp73, as is the case for Mdm2-mediated NEDDylation (35). Here, we investigated the impact of phosphorylation of T14, equivalent to the p53 phosphorylation at T18. In p53, T18 is phosphorylated by CK1 subsequent to Ser-15 phosphorylation (36, 37); p73 has no equivalent of S15 to act as a recognition site for CK1, and so phosphorylation in vivo may be achieved by an unidentified kinase. We found that phosphorylation of p73 10–40 at T14 resulted in a 10-fold increase in affinity for the Taz2 domain; if this increase in avidity is extended to the full-length peptide, we would expect the phosphorylated 10–70 region to bind with a Kd of ≈90 nM. Further, the phosphomimetic T14D displays increased transactivation ability in cellular assays; we expect the T14 phosphorylation to have even greater transactivation ability. Given the homology and functional overlap between the p53Nt and p73Nt, we expect these data to be validated by further in vivo work to identify a kinase responsible for T14 phosphorylation. Interestingly, although the T18 phosphorylation of p53 resulted in a decrease in affinity for Mdm2, we found that the equivalent phosphorylation in p73, T14P, results in a 2-fold increase in affinity. Recent work has demonstrated that TAp73α is able to enhance p53 stability by two mechanisms: through antagonizing p53 at the Mdm2 promoter level and by competing for Mdm2 protein binding (38). If phosphorylation of the p73Nt occurs concomitantly with p53 phosphorylation in response to stress factors, p73–Mdm2 binding will be favored at the expense of phosphorylated p53, thereby enhancing p53 stability at a time of cellular stress.

Experimental Procedures

Plasmid Construction.

p73 (residues 1–57 and 1–67) coding sequences were amplified from I.M.A.G.E Clone 40125802 (Geneservice Ltd.). Expression vectors for both N-terminal p73 constructs were made by inserting the coding sequence into the pRSETHisLipoTev vector (39).

Protein Expression and Purification.

Escherichia coli C41 cells containing the expression plasmids for p73 1–57 and 1–67 were grown at 37 °C to log phase in 2× TY medium, at which point 0.3 mM isopropyl 1-thio-β-d-galactopyranoside was added. Expression was conducted at 20 °C for 16 h. The N-terminal domains of p73 were purified by using nickel affinity chromatography. Cells were lysed by using sonication in binding buffer [50 mM potassium phosphate (pH 8.0), 400 mM NaCl, 2 mM 2-mercaptoethanol, and 20 mM imidazole] to which protease inhibitors (Roche Complete EDTA-Free) were added. Lysates were loaded onto a nickel affinity column that was preequilibrated with binding buffer and eluted with elution buffer [50 mM potassium phosphate (pH 8.0), 400 mM NaCl, 2 mM 2-mercaptoethanol, and 250 mM imidazole]. Fusion proteins with TEV protease added were dialyzed overnight into reloading buffer [50 mM potassium phosphate (pH 8.0), 100 mM NaCl, and 2 mM 2-mercaptoethanol]; the TEV protease cleaved the His-lipoyl tag. The unwanted tag was separated from the protein of interest by binding to a nickel affinity column; the protein of interest was collected in the flow-through. p73 1–57 and 1–67 were then subjected to HPLC purification on a C18 column and eluted by using a water:acetonitrile gradient. Fractions containing the protein of interest (as determined by mass spectrometry) were combined, lyophilized, and resuspended in appropriate buffer. p300 domains were expressed and purified as described in ref. 11. 15N- and 15N13C-labeled proteins were expressed in M9 minimal medium with 1.1g/L 15NH4Cl as the sole nitrogen source; medium was supplemented with 4 g/L d-glucose (d-[13C]glucose for double-labeled proteins) and vitamin mix. Isotopically labeled proteins were purified as above.

NMR.

1H 15N heteronuclear single quantum correlation experiments were conducted on a Bruker DRX-600 or 500 spectrometer equipped with CryoProbes. Spectra were collected at 298 K in the following buffer: 50 mM Mes (pH 6.8), 100 mM NaCl, 5 mM DTT. The concentration of labeled p73 was ≈100 μM; p300 domains were added in excess to ensure that all labeled p73 was in a bound state. Backbone assignments were obtained by using standard HNCA, HNCACB, HN(CO)CA, CBCA(CO)NH, and HN(CA)CO triple-resonance experiments. Spectra were assigned by using Sparky (University of California, San Francisco); chemical shift differences were calculated in accordance with the methodology presented by Hajduk et al. (40).

Fluorescence Anisotropy.

Peptides were synthesized as described in ref. 11. Experiments were performed at 25 °C on a PerkinElmer LS55 spectrofluorometer equipped with a Hamilton Microlab M dispenser. Protein awaiting titration was kept at 10 °C during the runs. Excitation and emission wavelengths were 328 and 393 nm, respectively. Typically, 250 μL of protein at various concentrations was titrated into 1 mL of 0.5 μM peptide. Buffer conditions were identical to those used for NMR. Data were fitted to a single binding site model modified to account for linear drift as described in ref. 11 by using the program Kaleidagraph (Synergy Software).

Cell Biology.

The coding sequence for WT TAp73α was obtained via PCR from I.M.A.G.E. Clone 40125802. This was inserted into the pcDNA3.1(+) vector (Clontech). The coding sequence for the QS mutant (L18Q /W19S) was obtained by using site-directed mutagenesis; all constructed plasmids were verified by sequencing. N-terminally HA-tagged p300 in the pCMVβ vector was obtained from Upstate Bioscience (now Millipore).

H1299 cells were grown in RPMI medium 1640 (Invitrogen) supplemented with 10% FCS. Twenty-four hours before transfection, the cells were subcultured from confluence to a 1:6 dilution into 6-well plates. Transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were washed twice with PBS and lysed by using RIPA buffer to which Complete protease inhibitor (Roche) had been added. Western blotting was conducted according to standard protocols by using the following primary antibodies: mouse anti-p73α (ab19941; AbCam) and mouse anti-p300 (ab3164; AbCam).

Luciferase Assays.

H1299 cells were grown and transfected as detailed above. Each well received 0.1 μg of pCMV-Renilla (Promega), 1.0 μg of pGL3-Bax-luciferase (a kind gift from Moshe Oren, Weizmann Institute of Science, Rehovot, Israel), and 0.5 μg of p73αWT or p73αQS with 0.5 μg of pcDNA 3.1(−) (Invitrogen) or 0.5 μg of pCMVβ-p300. Cells were harvested 48 h after transfection, and luciferase assays were performed with the Dual-Luciferase Reporter Assay system (Promega) in accordance with the manufacturer's instructions. Wells were prepared in triplicate, and error bars represent 1 SD.

Transfections with siRNA.

In addition to the p73α, pCMV-Renilla, and Bax-luciferase plasmids, cells were transfected with 100 pmol of p300 SMARTpool siRNA (Dharmacon Research) or control siRNA B (Santa Cruz Biotechnology). The siRNA transfection was repeated 24 h later. After 48 h, the cells were harvested, and luciferase assays were conducted as above. Knockdowns of p300 and protein levels were confirmed via Western blotting.

Acknowledgment.

S.B. is supported by a Medical Research Council Career Development Fellowship.

Footnotes

The authors declare no conflict of interest.

References

1. Harms K, Nozell S, Chen X. The common and distinct target genes of the p53 family transcription factors. Cell Mol Life Sci. 2004;61:822–842. [PubMed]
2. Zaika AI, et al. ΔNp73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors. J Exp Med. 2002;196:765–780. [PMC free article] [PubMed]
3. Maisse C, Munarriz E, Barcaroli D, Melino G, De Laurenzi V. DNA damage induces the rapid and selective degradation of the ΔNp73 isoform, allowing apoptosis to occur. Cell Death Differ. 2004;11:685–687. [PubMed]
4. Yang A, et al. p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature. 2000;404:99–103. [PubMed]
5. Melino G, De Laurenzi V, Vousden KH. p73: Friend or foe in tumorigenesis. Nat Rev Cancer. 2002;2:605–615. [PubMed]
6. Ramadan S, et al. p73 induces apoptosis by different mechanisms. Biochem Biophys Res Commun. 2005;331:713–717. [PubMed]
7. Goodman RH, Smolik S. CBP/p300 in cell growth, transformation, and development. Genes Dev. 2000;14:1553–1577. [PubMed]
8. Giles RH, Peters DJ, Breuning MH. Conjunction dysfunction: CBP/p300 in human disease. Trends Genet. 1998;14:178–183. [PubMed]
9. Zeng XY, et al. The N-terminal domain of p73 interacts with the CH1 domain of p300/CREB binding protein and mediates transcriptional activation and apoptosis. Mol Cell Biol. 2000;20:1299–1310. [PMC free article] [PubMed]
10. Zeng X, Lee H, Zhang Q, Lu H. p300 does not require its acetylase activity to stimulate p73 function. J Biol Chem. 2001;276:48–52. [PubMed]
11. Teufel DP, Freund SM, Bycroft M, Fersht AR. Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53. Proc Natl Acad Sci USA. 2007;104:7009–7014. [PMC free article] [PubMed]
12. Zhu J, Zhou W, Jiang J, Chen X. Identification of a novel p53 functional domain that is necessary for mediating apoptosis. J Biol Chem. 1998;273:13030–13036. [PubMed]
13. Liu G, Xia T, Chen X. The activation domains, the proline-rich domain, and the C-terminal basic domain in p53 are necessary for acetylation of histones on the proximal p21 promoter and interaction with p300/CREB-binding protein. J Biol Chem. 2003;278:17557–17565. [PubMed]
14. Zhu J, Zhang S, Jiang J, Chen X. Definition of the p53 functional domains necessary for inducing apoptosis. J Biol Chem. 2000;275:39927–39934. [PubMed]
15. De Guzman RN, Liu HY, Martinez-Yamout M, Dyson HJ, Wright PE. Solution structure of the TAZ2 (CH3) domain of the transcriptional adaptor protein CBP. J Mol Biol. 2000;303:243–253. [PubMed]
16. Kussie PH, et al. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science. 1996;274:948–953. [PubMed]
17. Campbell KM, Lumb KJ. Structurally distinct modes of recognition of the KIX domain of CBP by Jun and CREB. Biochemistry. 2002;41:13956–13964. [PubMed]
18. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–619. [PubMed]
19. Melino G, et al. p73 induces apoptosis via PUMA transactivation and Bax mitochondrial translocation. J Biol Chem. 2004;279:8076–8083. [PubMed]
20. Irwin MS, et al. Chemosensitivity linked to p73 function. Cancer Cell. 2003;3:403–410. [PubMed]
21. Fakharzadeh SS, Trusko SP, George DL. Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. EMBO J. 1991;10:1565–1569. [PMC free article] [PubMed]
22. Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 1992;69:1237–1245. [PubMed]
23. Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by Mdm2. Nature. 1997;387:299–303. [PubMed]
24. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature. 1997;387:296–299. [PubMed]
25. Zeng X, et al. MDM2 suppresses p73 function without promoting p73 degradation. Mol Cell Biol. 1999;19:3257–3266. [PMC free article] [PubMed]
26. Toledo F, Wahl GM. Regulating the p53 pathway: In vitro hypotheses, in vivo veritas. Nat Rev Cancer. 2006;6:909–923. [PubMed]
27. Bode AM, Dong Z. Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer. 2004;4:793–805. [PubMed]
28. Paliwal P, Radha V, Swarup G. Regulation of p73 by Hck through kinase-dependent and -independent mechanisms. BMC Mol Biol. 2007;8:45. [PMC free article] [PubMed]
29. Jones EV, Dickman MJ, Whitmarsh AJ. Regulation of p73-mediated apoptosis by c-Jun N-terminal kinase. Biochem J. 2007;405:617–623. [PMC free article] [PubMed]
30. Costanzo A, et al. DNA damage-dependent acetylation of p73 dictates the selective activation of apoptotic target genes. Mol Cell. 2002;9:175–186. [PubMed]
31. Fulco M, et al. p73 is regulated by phosphorylation at the G2/M transition. J Biol Chem. 2003;278:49196–49202. [PubMed]
32. Gaiddon C, et al. Cyclin-dependent kinases phosphorylate p73 at threonine 86 in a cell cycle-dependent manner and negatively regulate p73. J Biol Chem. 2003;278:27421–27431. [PubMed]
33. Gong JG, et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature. 1999;399:806–809. [PubMed]
34. Agami R, Blandino G, Oren M, Shaul Y. Interaction of c-Abl and p73 α and their collaboration to induce apoptosis. Nature. 1999;399:809–813. [PubMed]
35. Watson IR, Blanch A, Lin DC, Ohh M, Irwin MS. Mdm2-mediated NEDD8 modification of TAp73 regulates its transactivation function. J Biol Chem. 2006;281:34096–34103. [PubMed]
36. Dumaz N, Milne DM, Meek DW. Protein kinase CK1 is a p53-threonine 18 kinase which requires prior phosphorylation of serine 15. FEBS Lett. 1999;463:312–316. [PubMed]
37. Sakaguchi K, et al. Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase: Effect on Mdm2 binding. J Biol Chem. 2000;275:9278–9283. [PubMed]
38. Malaguarnera R, et al. TAp73 α increases p53 tumor suppressor activity in thyroid cancer cells via the inhibition of Mdm2-mediated degradation. Mol Cancer Res. 2008;6:64–77. [PubMed]
39. Weinberg RL, Veprintsev DB, Fersht AR. Cooperative binding of tetrameric p53 to DNA. J Mol Biol. 2004;341:1145–1159. [PubMed]
40. Hajduk PJ, et al. NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein. J Med Chem. 1997;40:3144–3150. [PubMed]
41. Chenna R, et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 2003;31:3497–3500. [PMC free article] [PubMed]
42. Clamp M, Cuff J, Searle SM, Barton GJ. The Jalview Java alignment editor. Bioinformatics. 2004;20:426–427. [PubMed]

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