Stimulation of p53 DNA binding by c-Abl requires the p53 C terminus and tetramerization

The carboxyl terminus of p53 is a target of a variety of signals for regulation of p53 DNA binding. Growth suppressor c-Abl interacts with p53 in response to DNA damage and overexpression of c-Abl leads to G(1) growth arrest in a p53-dependent manner. Here, we show that c-Abl binds directly to the carboxyl-terminal regulatory domain of p53 and that this interaction requires tetramerization of p53. Importantly, we demonstrate that c-Abl stimulates the DNA-binding activity of wild-type p53 but not of a carboxyl-terminally truncated p53 (p53Delta363C). A deletion mutant of c-Abl that does not bind to p53 is also incapable of activating p53 DNA binding. These data suggest that the binding to the p53 carboxyl terminus is necessary for c-Abl stimulation. To investigate the mechanism for this activation, we have also shown that c-Abl stabilizes the p53-DNA complex. These results led us to hypothesize that the interaction of c-Abl with the C terminus of p53 may stabilize the p53 tetrameric conformation, resulting in a more stable p53-DNA complex. Interestingly, the stimulation of p53 DNA-binding by c-Abl does not require its tyrosine kinase activity, indicating a kinase-independent function for c-Abl. Together, these results suggest a detailed mechanism by which c-Abl activates p53 DNA-binding via the carboxyl-terminal regulatory domain and tetramerization.     

14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers

Activation of the tumour suppressor p53 on DNA damage involves post-translational modification by phosphorylation and acetylation. Phosphorylation of certain residues is critical for p53 stabilization and plays an important role in DNA-binding activity. The 14-3-3 family of proteins activates the DNA-binding affinity of p53 upon stress by binding to a site in its intrinsically disordered C-terminal domain containing a phosphorylated serine at 378. We have screened various p53 C-terminal phosphorylated peptides for binding to two different isoforms of 14-3-3, epsilon and gamma. We found that phosphorylation at either S366 or T387 caused even tighter binding to 14-3-3. We made by semi-synthesis a tetrameric construct comprised of the tetramerization plus C-terminal domains of p53 that was phosphorylated on S366, S378 and T387. It bound 10 times tighter than did the monomeric counterpart to dimeric 14-3-3. We showed indirectly from binding curves and directly from fluorescence-detection analytical ultracentrifugation that 14-3-3 enhanced the binding of sequence-specific DNA to p53 by causing p53 dimers to form tetramers at lower concentrations. If the in vitro data extrapolate to in vivo, then it is an attractive hypothesis that p53 activity may be subject to control by accessory proteins lowering its tetramer-dimer dissociation constant from its normal value of 120-150 nM.     

New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins

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Scintillation proximity assay for DNA binding by human p53

Many DNA binding proteins are known to regulate gene expression. When that binding is altered, a disease state can result. A common method for measuring DNA binding, namely electrophoretic mobility shift assay (EMSA) is often used but it is not amenable to rapid screening of many samples. As an alternative method, we have developed a DNA binding assay for the tumor suppressor protein p53 in a 96-well microtiter plate format using scintillation proximity assay (SPA) beads. We have shown this assay to be sensitive (as little as 0.5 ng p53 can be detected), quick (assay completed in as little as 15 min), and easily quantitated using a microtiter plate scintillation counter We also used the assay to analyze the kinetics of the DNA binding to p53. The specificity of this p53 DNA binding SPA was confirmed using competition by oligonucleotides either from the same gene or from mutated versions of this sequence. Thus, SPA is a good alternative to gel shift assays for DNA binding and may be useful for the analysis of multiple tumor cell samples or for high-throughput screens for compounds affecting DNA binding by proteins of interest.     

Cooperative binding of tetrameric p53 to DNA

We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of approximately 3 microM for its dimer-tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.     

Discrimination of DNA binding sites by mutant p53 proteins.

Critical determinants of DNA recognition by p53 have been identified by a molecular genetic approach. The wild-type human p53 fragment containing amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA binding assays, and full-length human p53 was used for transactivation assays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically altered forms of a known consensus DNA site. This refinement indicates that p53 binds with high affinity to two repeats of PuGPuCA.TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A/T).(T/A) GPyPyPy. These results were further confirmed by transactivation assays of yeast by using full-length human p53 and systematically altered DNA sites. Dimers of the pentamer AGGCA oriented either head-to-head or tail-to-tail bound efficiently, but transactivation was facilitated only through head-to-head dimers. To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutions were made at several positions within the DNA binding domain of p53, and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gln or Arg, and Arg-283 to Gln bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentamer (PuGPuCA), respectively. Thus, we implicate these residues in amino acid-base pair contacts. Interestingly, mutant Cys-277 to Gln bound a consensus site as two and four monomers, as opposed to the wild-type p53 fragment, which invariably binds this site as four monomers.     

Dissimilar DNA binding by p53 in normal and tumor-derived cells

Comment on: Krassimira Botcheva, et al. Cell Cycle 2011; 10:4237-49.     

p53 stimulates human topoisomerase I activity by modulating its DNA binding

The tumor suppressor protein p53 and the human DNA topoisomerase I (htopoI) interact with each other, which leads to a stimulation of the catalytic activity of htopoI. Moreover, p53 stimulates the topoisomerase I-induced recombination repair (TIRR) reaction. However, little was known about how p53 stimulates this topoisomerase I activity. Here we demonstrate that monomeric p53 is sufficient for the stimulation of the topoisomerase I-catalyzed relaxation activity, but the tetrameric form of p53 is required for the stimulation of TIRR. We also show that p53 stimulates topoisomerase I activity by increasing the dissociation of htopoI from DNA. Since htopoI forms a closed ring structure around the DNA, our results suggest that p53 induces a conformational change within htopoI that results in an opening of the clamp, and thereby releases htopoI from DNA.     

DNA mismatch repair-dependent activation of c-Abl/p73alpha/GADD45alpha-mediated apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G(2) cell cycle checkpoint arrest and apoptosis in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of "mutator" cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G(2) arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G(2) arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G(2) arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73alpha and GADD45alpha protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73alpha/GADD45alpha signaling in cell death responses. STI571 (Gleevec, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73alpha, and GADD45alpha knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73alpha knockdown blocked MMR-dependent apoptosis, but not G(2) arrest, thereby uncoupling G(2) arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73alpha/GADD45alpha retrograde signaling.     

Calpains mediate p53 activation and neuronal death evoked by DNA damage

DNA damage is an initiator of neuronal death implicated in neuropathological conditions such as stroke. Previous evidence has shown that apoptotic death of embryonic cortical neurons treated with the DNA damaging agent camptothecin is dependent upon the tumor suppressor p53, an upstream death mediator, and more distal death effectors such as caspases. We show here that the calcium-regulated cysteine proteases, calpains, are activated during DNA damage induced by camptothecin treatment. Moreover, calpain deficiency, calpastatin expression, or pharmacological calpain inhibitors prevent the death of embryonic cortical neurons, indicating the important role of calpain in DNA damage-induced death. Calpain inhibition also significantly reduced and delayed the induction of p53. Consistent with the actions of calpains upstream of p53 and the proximal nature of p53 death signaling, calpain inhibition inhibited cytochrome c release and DEVD-AFC cleavage activity. Taken together, our results indicate that calpains are a key mediator of p53 induction and consequent caspase-dependent neuronal death due to DNA damage.