scholarly journals Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper.

1997 ◽  
Vol 17 (10) ◽  
pp. 5699-5706 ◽  
Author(s):  
G W Verhaegh ◽  
M J Richard ◽  
P Hainaut
Keyword(s):  
Dna Binding ◽  
Metal Ions ◽  
Copper Ions ◽  
P53 Protein ◽  
Binding Activity ◽  
Redox Activity ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Wild Type P53

Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Evidence for regulation of NF-κB by poly(ADP-ribose) polymerase

2000 ◽  
Vol 346 (3) ◽  
pp. 641-649 ◽  
Author(s):  
Masanori KAMEOKA ◽  
Katsuya OTA ◽  
Toshifumi TETSUKA ◽  
Yasuharu TANAKA ◽  
Asako ITAYA ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Modification ◽  
Binding Activity ◽  
Wild Type ◽  
Dna Binding Activity ◽  
L1210 Cells ◽  
Nuclear Extracts ◽  
Cell Clones ◽  
Defective Mutant

The DNA-binding activity of NF-ĸB in nuclear extracts of poly(ADP-ribose) polymerase (PARP)-defective mutant L1210 cell clones was markedly increased and was inversely correlated with the PARP content in these cells. The DNA-binding activity of NF-ĸB in a clone with the lowest PARP content (Cl-3527, contained 6% of PARP of wild type cells) was about 35-fold of that of the wild-type cells, whereas the change in the DNA-binding activity of AP-1 and SP-1 in the mutant was relatively small or not so significant. Transfection of a PARP-expressing plasmid to the mutant cells decreased the abnormally high levels of NF-ĸB complexes, especially p50/p65(Rel A) complex, to near the normal level. Moreover, poly(ADP-ribosyl)ation of nuclear extracts in vitro suppressed the ability of NF-ĸB to form a complex with its specific DNA probe by approx. 80%. Further analysis with purified recombinant NF-ĸB proteins revealed that both rp50 and rMBP-p65 (Rel A) proteins, but not rGST-IĸB, could be poly(ADP-ribosyl)ated in vitro and that the modification resulted in a marked decrease in the DNA-binding activity of rMBP-p65, whereas a slight activation was observed in rp50. Poly(ADP-ribosyl)ated p65/NF-ĸB was detected in the cytosol of wild type L1210 cells by immunoblotting with anti-poly(ADP-ribose) and anti-p65 antibodies. Taken together, these results strongly suggest that PARP is involved in the regulation of NF-ĸB through the protein modification.

scholarly journals Inhibition of p53 DNA Binding Function by the MDM2 Protein Acidic Domain

2011 ◽  
Vol 286 (18) ◽  
pp. 16018-16029 ◽  
Author(s):  
Brittany Cross ◽  
Lihong Chen ◽  
Qian Cheng ◽  
Baozong Li ◽  
Zhi-Min Yuan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Wild Type ◽  
Methyl Transferase ◽  
Reading Frame ◽  
Acidic Domain ◽  
Dna Binding Activity ◽  
Binding Function

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. Alternate reading frame binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild type conformation and allows p53-MDM2-SUV39H1 complex to bind DNA. These results provide further evidence for an ubiquitination-independent mechanism of p53 regulation by MDM2 and reveal how MDM2-interacting repressors gain access to p53 target promoters and repress transcription. Furthermore, we show that the MDM2 inhibitor Nutlin cooperates with the proteasome inhibitor Bortezomib by stimulating p53 DNA binding and transcriptional activity, providing a rationale for combination therapy using proteasome and MDM2 inhibitors.

scholarly journals Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.

1997 ◽  
Vol 17 (8) ◽  
pp. 4208-4219 ◽  
Author(s):  
B Viollet ◽  
A Kahn ◽  
M Raymondjean
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Hepatocyte Nuclear Factor 4 ◽  
Kinase A

Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers.

Stimulation of polyomavirus DNA replication by wild-type p53 through the DNA-binding site

1994 ◽  
Vol 14 (4) ◽  
pp. 2651-2663
Author(s):  
T Kanda ◽  
K Segawa ◽  
N Ohuchi ◽  
S Mori ◽  
Y Ito
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Activity ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Wild Type P53 ◽  
Stimulation Of

The tumor suppressor p53 possesses characteristics of a transcription factor; it binds to specific DNA sequences and activates transcription from various promoters. Here we found that murine wild-type p53 stimulated not only transcription but also polyomavirus (Py) DNA replication in a sequence-dependent manner. Oncogenic mutant p53, lacking the DNA-binding activity, showed no stimulation of Py DNA replication. Deletion of the N-terminal acidic transactivation domain of wild-type p53, which completely eliminated the ability to stimulate transcription, only impaired the function to stimulate Py DNA replication. The replication-stimulating activity of wild-type p53 was impaired by the deletion of the C-terminal oligomerization domain as well, without affecting the ability to stimulate transcription. The region responsible for the sequence-specific DNA-binding activity mapped to the central portion of the p53 molecule has a minimal activity. The results indicate that both the N-terminal and the C-terminal regions significantly contribute to the p53-mediated stimulation of Py DNA replication.

scholarly journals Mutants of the tumour suppressor p53 L1 loop as second-site suppressors for restoring DNA binding to oncogenic p53 mutations: structural and biochemical insights

2010 ◽  
Vol 427 (2) ◽  
pp. 225-236 ◽  
Author(s):  
Assia Merabet ◽  
Hellen Houlleberghs ◽  
Kate Maclagan ◽  
Ester Akanho ◽  
Tam T. T. Bui ◽  
...  
Keyword(s):  
Dna Binding ◽  
Double Mutant ◽  
Hot Spot ◽  
Binding Activity ◽  
Tumour Suppressor ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Wild Type P53 ◽  
Dynamics Simulations ◽  
Tumour Suppressor P53

To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 ‘hot spot’ mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53; (iv) the effect of H115N as a second-site suppressor to restore DNA-binding activity is specific to R248Q, but not to R248W; (v) molecular-dynamics simulations indicate that R248Q/H115N has a conformation similar to wild-type p53, which is distinct from that of R248Q. These findings could be exploited in designing strategies for cancer therapy to identify molecules that could mimic the effect of H115N in restoring function to oncogenic p53 mutants.

scholarly journals Stimulation of polyomavirus DNA replication by wild-type p53 through the DNA-binding site.

1994 ◽  
Vol 14 (4) ◽  
pp. 2651-2663 ◽  
Author(s):  
T Kanda ◽  
K Segawa ◽  
N Ohuchi ◽  
S Mori ◽  
Y Ito
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Activity ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Wild Type P53 ◽  
Stimulation Of

The tumor suppressor p53 possesses characteristics of a transcription factor; it binds to specific DNA sequences and activates transcription from various promoters. Here we found that murine wild-type p53 stimulated not only transcription but also polyomavirus (Py) DNA replication in a sequence-dependent manner. Oncogenic mutant p53, lacking the DNA-binding activity, showed no stimulation of Py DNA replication. Deletion of the N-terminal acidic transactivation domain of wild-type p53, which completely eliminated the ability to stimulate transcription, only impaired the function to stimulate Py DNA replication. The replication-stimulating activity of wild-type p53 was impaired by the deletion of the C-terminal oligomerization domain as well, without affecting the ability to stimulate transcription. The region responsible for the sequence-specific DNA-binding activity mapped to the central portion of the p53 molecule has a minimal activity. The results indicate that both the N-terminal and the C-terminal regions significantly contribute to the p53-mediated stimulation of Py DNA replication.

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