The Journal of biological chemistry.
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Fig. 1. Components of p53 signaling pathways. p53 accumulates and is modified and activated in response to signals generated by a variety of genotoxic stresses. Several proteins, including ATM, PARP, FAS, BLS, and NBS (see the text for full names), are involved in activation, but the pathways that lead to modification are largely unknown. The RAS-MAP kinase pathway is involved in establishing basal levels of p53 and may also affect function. Some of the cellular functions affected by p53 can be compromised by deregulated expression of Myc, Bcl2, E1B, or E2F. The control of p53 activity includes an autoregulatory loop involving Mdm2. The intact set of p53-dependent pathways helps to maintain genomic integrity by eliminating damaged cells, either by arresting them permanently or through apoptosis. p53 also helps to regulate entry into mitosis, spindle formation, and centrosome integrity, cell cycle checkpoints that are likely to be involved in preventing DNA damage from occurring.
- For example, -irradiation activates p53 to turn on the transcription of p21/Waf1, which binds to and inhibits cyclin-dependent kinases, causing hypophosphorylation of Rb, thus preventing the release of E2F and blocking the G1-S transition (Fig. 1).
- This might be explained by the release of E2F (Fig. 1), which when overexpressed on its own can induce apoptosis (60).
- This observation emphasizes the fact that p53-dependent pathways can be inhibited at any of several different points (Fig. 1).
- p53 signaling pathways connect with tumor suppressors and oncogenes known to influence the cell cycle machinery (Fig. 1).
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Fig. 1. Purification of NBP from HeLa nuclear extracts. (A) Purification procedure for NBP protein from Hela cell nuclear extracts. (B) Silver staining (SS) and western blot (WB) analyses of the fractions from the Mono S and p53RE-specific DNA affinity (W2) columns. Aliquots of 20 µl of active fractions were loaded directly onto a 10% SDS gel, followed by silver staining (left) or western blotting (right) using polyclonal antibodies against p63. MW denotes molecular weight markers as indicated on the left (this is true for all the following figures). (C) EMSA of the active fractions from Mono S FPLC and W2 columns.
- The p53RE-binding and transcriptional activities of the fractions were assayed by electrophoresis gel mobility shift assay (EMSA) and in vitro transcription reactions as described (40,44). p53RE DNA-binding and transcription activities were detected in both the 0.3 and 0.5 M washes of the first column and the flow-through and 0.3 M fraction from the second column (Figure 1A).
- As shown in Figure 1, this p53RE-binding activity has been purified to apparent homogeneity through four columns: phosphocellulose P11, DEAE–Sepharose 52, Mono S and p53RE-specific DNA affinity columns.
- A representative result from the DNA affinity column is shown in Figure 1B.
- Surprisingly, this 50 kDa polypeptide was detected by both polyclonal and monoclonal anti-p63 antibodies (Figure 1B, lanes 2 and 3), but not by an antibody specifically against the unique N-terminus of p40 (data not shown).
- This protein was active in binding to p53RE-containing oligomers to form a DNA–protein complex (Figure 1C) as well as in a transcription reaction in vitro (37; data not shown).
- Because we primarily traced complexes migrating faster than the p53–DNA complex as indicated in Figure 2B, we were able to purify the NBP activity (Figure 1).
- This is why the input Mono S fractions for the affinity column (Figure 1C, lane 2) did not display the non-specific complex detected in the Mono S fractions used in the experiments in Figure 2B (lane 7).
- This strategy was helpful in purifying NBP (Figure 1B, left, lane 3).
- First, the purified NBP was primarily composed of a singular polypeptide with a molecular weight of ~50 kDa, as revealed by silver staining (Figure 1B).
- This polypeptide immunoreacted with anti-p63, but not anti-p53 or anti-p73, antibodies (Figure 1B).
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Fig. 2. Co-purification of NBP with p63. (A) Western blot analysis of the fractions from each step of the purification. Aliquots of 25 µl of each fraction were loaded directly onto a 10% SDS gel, followed by western blotting using antibodies against p73, p53 (Pab421) or p63. S100 denotes cytoplasmic extracts; NE is the nuclear extracts; P11, DE and MS represent the phosphocellulose, DEAE–Sepharose and Mono S columns, respectively. (B) EMSA analysis of the active fractions from each step of purification. An aliquot of 2 µl of each protein sample was used in the DNA-binding assay as described in Materials and Methods. #, a non-specific complex. An aliquot of 50 ng p53 purified from baculovirus-infected SF9 cells was used as a control in lane 2.
- As shown in Figure 2A, both p53 and p73 were detectable in the nuclear extracts and fractions from the first column (lanes 2 and 3), but not in the DEAE–Sepharose 52 and Mono S columns (lanes 4 and 5), indicating that p63 was separated from p53 and p73 on the DEAE–Sepharose 52 column.
- In contrast, p63 was detected throughout each step of purification, co-purifying with the p53RE-binding activity (Figure 2B).
- During fractionation we detected several different p53RE–protein complexes in fractions from the phosphocellulose column (data not shown and Figure 2B, lane 5) as well as in DEAE–Sepharose 52 fractions (Figure 2B, lane 6).
- Because we primarily traced complexes migrating faster than the p53–DNA complex as indicated in Figure 2B, we were able to purify the NBP activity (Figure 1).
- This is why the input Mono S fractions for the affinity column (Figure 1C, lane 2) did not display the non-specific complex detected in the Mono S fractions used in the experiments in Figure 2B (lane 7).
- Second, reprobing the active fractions from each step of the purification with these antibodies confirmed that NBP co-purified with p63, but not p53 or p73 (Figure 2A).
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Fig. 3. The NBP–p53RE complex is supershifted by anti-p63 but not anti-p53 antibody. Aliquots of 2 µl of NBP purified through a p53RE DNA affinity column were used in this EMSA experiment. An aliquot of 50 ng p53 was used in lane 2. Poly(dI•dC) (0.5 µg) was used as non-specific competitor in all the reactions and, additionally, 0.2 µg specific DNA competitors as indicated at the top were used in lanes 4–6. Aliquots of 0.5 µg polyclonal anti-p63 or Pab421 antibodies as indicated at the top were used.
- As shown in Figure 3, the oligomers containing wild-type but not mutant p53RE sequence derived from the p21 promoter specifically inhibited formation of the p53RE–NBP complex (lanes 3–5).
- Finally, formation of a p53RE–NBP complex was specifically supershifted by the anti-p63 antibody but not by the anti-p53 antibody Pab421 (Figure 3).
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Fig. 1. The complex signalling pathway of p53. This diagram outlines a range of cellular stresses that can ‘activate’ p53 and lists the modifiers and partners that were discussed at the meeting. It is not a comprehensive list of all stresses, modifiers and outputs involved in the p53 pathway.
- We now know that the role of p53 extends to participation in a diverse array of signalling pathways associated with cellular stress and DNA damage (Figure 1), and recent studies have even raised the possibility that p53 may be involved in pathways that have little to do with cancer.
- However, beyond its role in tumour suppression, and having regard for the many complex signalling pathways with which p53 appears to be involved in (Figure 1), Levine proposed that p53 may be a ‘ubiquitous sensor’ of stress and as such is involved in the prevention of other, possibly aging-related disorders.
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Fig. 1. Diagram showing the domain structure of the p53 protein. The p53 protein is a transcription factor that contains several well-defined domains. At the N-terminus are the transactivation domain and a proline-rich region, which is required for apoptotic function. Within the N-terminus are the interaction sites of p53 with components of the transcriptional machinery as well as ubiquitin ligase Mdm2. The central domain harbors the sequence specific DNA binding region, where most of the tumor associated mutations occur. This central region also contains binding sites for interaction with members of the Bcl2 protein family. The C-terminal region contains the oligomerization domain as well as nuclear localisation and export signals. Several sites within the N-terminal region have been shown to be phosphorylated and the C-terminal region contains numerous sites of modification which influence stability, localization and activity of p53.
- Assembling into a tetramer, p53 shows sequence specific DNA binding activity through its central domain (Figure 1), and activates the expression of genes that contain p53 binding sites in their promoters by virtue of interactions of the N-terminal domain of p53 with the transcriptional machinery.
- Mdm2 binds to the N-terminal region of p53 that also contains the transcriptional activation domain (Figure 1), and this interaction of Mdm2 with p53 can block the binding of other components of the transcriptional machinery and inhibit the ability of p53 to activate transcription.
- The mechanism through which Mdm2 drives nuclear export of p53 is not completely clear, although it is associated with ubiquitination of p53 (42,43) and many involve the unmasking of the nuclear export sequences that are present in the C-terminal region of p53 (Figure 1).
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Fig. 2. Models of p53 function as a BH3-only protein. On the left, two possible roles for p53 as an ‘enabler’ type BH3-only protein. In these models p53 is able to disrupt the interaction between anti-apoptotic proteins (such as BclxL, Bcl2 and Mcl1) and pro-apoptotic proteins. This could directly relieve the inhibition of Bax and Bak (A), or free ‘activator’ type BH3 only proteins (Bid and Bim), which can then activate Bax and Bak (B). On the right, models in which p53 functions as an ‘activator’ type BH3-only protein. Activation of Bax and Bak might involve binding and releasing them from interaction with the anti-apoptotic proteins (C). Alternatively, p53 itself may be held inactive by interaction with the anti-apoptotic proteins. In this case it is possible that other ‘enabler’ BH3-only proteins like PUMA might be able to displace p53 from the anti-apoptotic proteins, thus allowing it to activate Bax/Bak (D). Note that although there is evidence to support each of the models, not all the interactions indicated in the Figure have been confirmed.
- The BH3-only proteins are further divided into two groups; those that bind Bax and Bak directly to activate them (the ‘activators’, such as Bim and Bid) and those that bind the anti-apoptotic family members to release the activators (the ‘enablers’ like Bad and Bik) (29,30) (Figure 2).
- In the first, p53 functions like an ‘enabler’ BH3 domain protein, interacting with the anti-apoptotic proteins and presumably releasing pro-apoptotic BH3 domain proteins to drive apoptosis (Figure 2A and B) (31).
- The second model is based on the recent observation that p53 can function in a manner more analogous to the ‘activator’ BH3-only proteins, by directly activating the apoptotic function of Bax or Bak (Figure 2C and D) (32,33).
- Therefore, when expressed at sufficiently high levels, PUMA may be able to dissociate p53 from the pro-survival proteins and thereby drive the activation of apoptosis (Figure 2D).
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Fig. 1. Effects of Hdmx on p53 stability and Mdm2-mediated p53 degradation. C33A cells were transfected with hdmx (A-C), mdm2 (D-F), or hdmx plus mdm2 (G-I) and stained for Hdmx (A) with antibody 6B1A, Mdm2 (D, G) with antibody 4B2 and p53 (B, E, H) with antibody FL393. DNA is stained with DAPI (C, F, I).
- As shown in Fig. 1 (A-C), the cells expressing Hdmx showed no decreased p53 levels, indicating that Hdmx does not target p53 for degradation, whereas overexpression of Mdm2 clearly leads to a reduction of p53 signal (Fig. 1, D-F).
- Immunofluorescence indicated that now the Mdm2-positive cells did not show decreased levels of p53 (Fig. 1, G-I).
The Journal of biological chemistry.
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Fig. 2. Hdmx stabilize both p53 and Mdm2. A, p53{-}/{-} MEFs were transfected with 100 ng of mutant p53 alone or together with 2 µg of mdm2 in the absence or presence of 2, 4, or 7 µg of full-length hdmx. B, p53{-}/{-} MEFs were transfected with 100 ng of mutant p53 without or with 2, 4, or 7 µg of hdmx. Cell lysates were analyzed by Western blotting with anti-p53 (DO-1), anti-Hdmx (6B1A/11F4D/12G11G), anti-Mdm2 (4B2), and anti-LacZ monoclonal antibodies.
- As shown previously, co-transfection of p53 and mdm2 results in a reduction of p53 protein levels (Fig. 2A, compare lanes 1 and 2).
- Besides increased levels of p53, also increased levels of Mdm2 were observed (Fig. 2A, lanes 3, 4, and 5).
- Fig. 2B shows that hdmx can also stabilize p53 in the absence of transfected mdm2, probably due to the inhibition of the endogenous Mdm2 protein.
- Cell lysates were analyzed by Western blotting with the same antibodies as mentioned in Fig. 2.
- Mdm2 and the different Hdmx forms were detected with same antibodies as mentioned in Fig. 2.
- Hdmx and p53 were detected with same antibodies as mentioned in Fig. 2; Mdm2-N was detected with a mixture of monoclonal antibodies 2A10 and 3G5.
- Hdmx and Hdm2 proteins were detected with the same antibodies as mentioned in Fig. 2. p53 protein was detected with mouse monoclonal antibody DO-1.