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Comparative Study
, 20 (8), 1008-16

Analysis of the Oligomeric State and Transactivation Potential of TAp73α

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Comparative Study

Analysis of the Oligomeric State and Transactivation Potential of TAp73α

L M Luh et al. Cell Death Differ.

Abstract

The proteins p73 and p63 are members of the p53 protein family and are involved in important developmental processes. Their high sequence identity with the tumor suppressor p53 has suggested that they act as tumor suppressors as well. While p63 has a crucial role in the maintenance of epithelial stem cells and in the quality control of oocytes without a clear role as a tumor suppressor, p73's tumor suppressor activity is well documented. In a recent study we have shown that the transcriptional activity of TAp63α, the isoform responsible for the quality control in oocytes, is regulated by its oligomeric state. The protein forms an inactive, dimeric and compact conformation in resting oocytes, while the detection of DNA damage leads to the formation of an active, tetrameric and open conformation. p73 shows a high sequence identity to p63, including those domains that are crucial in stabilizing its inactive state, thus suggesting that p73's activity might be regulated by its oligomeric state as well. Here, we have investigated the oligomeric state of TAp73α by size exclusion chromatography and detailed domain interaction mapping, and show that in contrast to p63, TAp73α is a constitutive open tetramer. However, its transactivation potential depends on the cellular background and the promoter context. These results imply that the regulation of p73's transcriptional activity might be more closely related to p53 than to p63.

Figures

Figure 1
Figure 1
Domain structure and sequence alignment of p73 and p63. (a) The domain structure of p53 is compared with the domain structures of several p63- and p73-isoforms used in this study. Corresponding domains have the same color. The degree of sequence identity between the p63 and p73 domains is indicated. (b) Sequence alignment of the TA, the tetramerization (TD) and the TI domains of p63 and p73 (coloring according to domain structure in (a)). Color intensity indicates the degree of homology. Amino acids mutated in this study are highlighted by asterisks. (c) Structure of the TD of p73. The tetramer consists of a dimer of dimers. The dark and light blue monomers form one dimer and the red and yellow monomers the second dimer. In contrast to the OD of p53, the TDs of p63 and of p73 have an additional C-terminal helix (H2) that reaches across the tetramerization interface, depicted by the dashed line
Figure 2
Figure 2
SEC analysis of p73 (ac) and p63 (d and e) isoforms expressed in rabbit reticulocyte lysate. Lysates were applied to a Superose 6 PC 3.2/30 column. For each isoform the western blot analysis of the individual fractions (given in mL of elution volume underneath the bar diagram) and a bar diagram representing the relative intensity of the western blot signals are shown. The sum of the intensity of all individual fractions is set to 100%. (f) Calibration of Superose 6 PC 3.2/30 column using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa). Corresponding molecular weights, the respective elution volumes of calibration proteins and the void volume are indicated
Figure 3
Figure 3
Pull-down experiments with external TA and TI domains confirm the open state of TAp73α. For each pull-down experiment the input signal (IN) and the pull-down signal (PD) of the western blot analysis are shown. Pull-down efficiencies as indicated by the bar diagrams are normalized to either TAp63γ or TAp73β in case of TI-pull-downs or to ΔNα isoforms for the TA-pull-downs. The external TA or TI domains were expressed as GST-fusion proteins and immobilized on glutathione sepharose beads. Panel (a) shows experiments with the external TI domains, while (b) displays experiments with the external TA domains
Figure 4
Figure 4
TAp73α forms heterotetramers with tetrameric p63 isoforms and mutants. Different p63 and p73 isoforms or mutants were coexpressed in Saos-2 cells and subsequently coimmunoprecipitated using either (a) anti-p63 antibodies (H-129) for precipitation and anti-p73 antibodies (ER-15) for detection or (b) vice versa. (c) TAp63γ was detected with a different anti-p63 antibody (4A4) due to the lack of the H-129 antibody epitope (resulting IgG heavy chain background is indicated by the arrowhead). Normal IgG was used for the negative controls. The bar diagrams show the efficiency of each coimmunoprecipitation relative to the respective input signal (lysate). TAp63αMI harbors two point mutations in the TD (M374Q and I378R), which leads to an open, yet dimeric conformation, which is not capable to tetramerize. Coimmunoprecipitation experiments with TAp63αMI were therefore used to prove specific heterooligomerization via the TD
Figure 5
Figure 5
TAp73α is transcriptionally active. The transcriptional activity of different p73 isoforms and mutants were measured on the Bax promoter in transiently transfected SK-N-AS cells and compared with the transcriptional activities of different p63 isoforms and mutants as well as to p53. Bar diagrams indicate fold change of promoter induction compared with empty vector control. Significant differences are indicated with an asterisk and non significant differences are labeled as n.s.
Figure 6
Figure 6
Schematic representation of the proposed activation mechanisms of p53 and the TAα-isoforms of p63 and p73. As the protein level of tetrameric p53 is kept low due to fast degradation, upregulation of p53 target genes requires the stabilization of p53 by phosphorylation and acetylation (simplified, exemplary phosphorylation and acetylation are indicated in blue and green, respectively). TAp63α on the other hand accumulates to high concentrations in an inactive and closed dimeric conformation and becomes activated upon phosphorylation, resulting in the formation of active tetramers. Despite its greater sequence homology to p63, TAp73α can readily form tetramers but like p53 needs further post-translational modifications and coactivators for full transactivation potential

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