Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53

Cai, Bi-He; Chen, Jang-Yi; Lu, Mei-Hua; Chang, Li-Tze; Lin, Hwang-Chi; Chang, Yuan-Hsiou; Chao, Chung-Faye

doi:10.1093/nar/gkp033

Cited by 23 publications

(24 citation statements)

References 24 publications

(45 reference statements)

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“…To verify this, we first performed electrophoretic mobility shift assays (EMSA) using MCF-10-2A nuclear extracts and a radioactively labeled probe that corresponds to the p53 consensus sequence (GTAGCA-TTAGCCCAGACATGTCC) in the 14-3-3s gene promoter (Hermeking et al, 1997;Cai et al, 2009). MCF-10-2A cells were first used for these experiments because they express endogenous plakoglobin and wild-type p53 (Li et al, 2005;Lam et al, 2009).…”

Section: Plakoglobin and P53 Interact In Both The Cytoplasm And Nucleusmentioning

confidence: 99%

“…Briefly, a double-stranded nucleotide corresponding to the p53 consensus sequence in the promoter of the 14-3-3s (SFN) gene (Hermeking et al, 1997;Cai et al, 2009) was radioactively labeled with use of 32 P-ATP (adenosine 59-triphosphate; Perkin Elmer). Nuclear extracts (5 mg) were incubated with oligonucleotide probes (15,000 cpm) on ice for 10 minutes in EMSA reaction buffer [50 mM HEPES pH 7.9, 250 mM KCl, 25 mM MgCl 2 , 5 mM EDTA, 5% glycerol and 1 mg poly (dI-dC) (Sigma)].…”

Section: Electrophoretic Mobility Shift Assaymentioning

confidence: 99%

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Plakoglobin interacts with the transcription factor p53 and regulates the expression of 14-3-3σ

Aktary

Kulak

Mackey

et al. 2013

Journal of Cell Science

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SummaryPlakoglobin (c-catenin), a constituent of the adherens junction and desmosomes, has signaling capabilities typically associated with tumor/metastasis suppression through mechanisms that remain undefined. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We detected several p53-target genes whose levels were altered upon plakoglobin expression. In this study, we identified the p53 regulated tumor suppressor 14-3-3s as a direct plakoglobin-p53 target gene. Coimmunoprecipitation experiments revealed that plakoglobin and p53 interact, and chromatin immunoprecipitation and electrophoretic mobility shift assays revealed that plakoglobin and p53 associate with the 14-3-3s promoter. Furthermore, luciferase reporter assays showed that p53 transcriptional activity is increased in the presence of plakoglobin. Finally, knockdown of plakoglobin in MCF-7 cells followed by luciferase assays confirmed that p53 transcriptional activity is enhanced in the presence of plakoglobin. Our data suggest that plakoglobin regulates gene expression in conjunction with p53 and that plakoglobin may regulate p53 transcriptional activity, which may account, in part, for the tumor/metastasis suppressor activity of plakoglobin.

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Section: Plakoglobin and P53 Interact In Both The Cytoplasm And Nucleusmentioning

confidence: 99%

Section: Electrophoretic Mobility Shift Assaymentioning

confidence: 99%

Plakoglobin interacts with the transcription factor p53 and regulates the expression of 14-3-3σ

Aktary

Kulak

Mackey

et al. 2013

Journal of Cell Science

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“…The exact natures of wt and mut homo-tetramers, as well as the dynamics of tetramer formation are only poorly understood. The equilibrium constant for the formation of the tetramers, approximately 20 nM, is such that there is an equilibrium between dimers and tetramers in the cell, and dimer-tetramerization phenomena may be important in the regulation of the activity of p53 (10)(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

Ultraslow oligomerization equilibria of p53 and its implications

Natan

Hirschberg

Morgner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The tumor suppressor p53 is in equilibrium at cellular concentrations between dimers and tetramers. Oncogenic mutant p53 (mut) exerts a dominant-negative effect on co-expression of p53 wildtype (wt) and mut alleles in cancer cells. It is believed that wt and mut form hetero-tetramers of attenuated activity, via their tetramerization domains. Using electrospray mass spectrometry on isotopically labeled samples, we measured directly the composition and rates of formation of p53 complexes in the presence and absence of response element DNA. The dissociation of tetramers was unexpectedly very slow (t 1/2 ‫؍‬ 40 min) at 37°C, matched by slow association of dimers, which is approximately four times longer than the half-life of spontaneous denaturation of wt p53. On mixing wt tetramers with the oncogenic contact mutant R273H of low DNA affinity, we observed the same slow formation of only wt 4, wt2mut2, and mut4, in the ratio 1:2:1, on a cellular time scale. On mixing wt and mut with response element DNAs P21 and BAX, we observed only the complexes wt 4.DNA, wt2mut2.DNA, and mut 4.DNA, with relative dissociation constants 1:4:71 and 1:13:85, respectively, accounting for the dominant-negative effect by weakened affinity. p53 dimers assemble rapidly to tetramers on binding to response element DNA, initiated by the p53 DNA binding domains. The slow oligomerization of free p53, competing with spontaneous denaturation, has implications for the possible regulation of p53 by binding proteins and DNA that affect tetramerization kinetics as well as equilibria.mass spectrometry ͉ protein-protein interactions ͉ slow association ͉ tetramerization domain T he tumor suppressor p53 is a transcription factor that is inactivated by mutation in some 50% of human cancers (1, 2). p53 consists of an unstructured N-terminal domain (residues 1-94) which points away from the rest of the protein (3), a folded core domain (residues 94-292), a linker to the tetramerization domain (residues 325-355) and an unstructured C-terminal domain . Nearly all of the mutations reside in its DNA-binding domain. Mutations can be either 'contact,' which remove residues that interact with DNA and lower affinity, or 'structural,' which destabilize the protein, sometimes with concomitant conformational changes (4). The transcriptionally active form of p53 is a homo-tetramer, linked via its tetramerization domains. Since most cancer mutations are located outside the tetramerization domain, in the core domain, the mutant (mut) proteins retain wild-type (wt) ability to form tetramers. Consequently the formation of hetero-tetramer complexes between wt and mut p53 is observed both in vivo and in vitro (5). It is thought that formation of hybrids between the wt and the mut protein is the cause of the trans-dominant effect of the mut over the wt in heterozygous cells (dominant-negative effect), either following somatic mutation or from birth as in the case of Li-Fraumeni syndrome (5, 6). In such events, the chances of initiating cancer are increased (7), and the p53 het...

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“…Human diseases such as Nijmegen breakage syndrome due to mutations in the NBS1 gene result in defects in resection of double strand breaks (3). NBS1 functions as part of the MRN complex whose functions are not restricted to HR but are also involved NHEJ (4). NBS is a rare human autosomal recessive disorder caused by hypomorphic mutations.…”

Section: Introductionmentioning

confidence: 99%

Haploinsufficiency of the Nijmegen breakage syndrome 1 gene increases mammary tumor latency and metastasis

Wan

Crowe

2012

Int J Oncol

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Abstract. Human diseases such as Nijmegen breakage syndrome due to mutations in the NBS1 gene result in defects in resection of double strand breaks. NBS1 functions as part of the MRN complex which functions in homologous recombination and non-homologous end joining. NBS is a rare human autosomal recessive disorder caused by hypomorphic mutations. At the cellular level, NBS is characterized by radiosensitivity, chromosomal breakage and defective cell cycle checkpoints. NBS1 null mutations result in early embryonic lethality in mice, but NBS1 hypomorphic mutants are viable. Cells from these mice are defective in S phase and G2/M checkpoints. In humans, NBS1 polymorphisms have been associated with increased risk of breast cancer. MRN expression was reduced in the majority of breast tumors, and low expression of MRN correlated with increased histologic grade and estrogen receptor negativity. While these studies have shown NBS1 to be important in clinical outcomes of patients with breast cancer, mammary tumors are rare in the NBS1 haploinsufficient mouse. To better understand the role of NBS1 in mammary tumorigenesis, we examined the NBS1+/-;MMTV-neu mouse model. Mammary tumor latency was markedly increased in NBS1+/-;neu mice compared to NBS1+/+;neu control animals. This effect was due to increased apoptosis in early NBS1+/-;neu mammary tumors. However, NBS1+/-;neu mammary tumors were highly metastatic and demonstrated clear differences in gene expression profiles compared to control tumors. We concluded that NBS1 haploinsufficiency results in increased mammary tumor latency and metastasis.

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Functional four-base A/T gap core sequence CATTAG of P53 response elements specifically bound tetrameric P53 differently than two-base A/T gap core sequence CATG bound both dimeric and tetrameric P53

Cited by 23 publications

References 24 publications

Plakoglobin interacts with the transcription factor p53 and regulates the expression of 14-3-3σ

Plakoglobin interacts with the transcription factor p53 and regulates the expression of 14-3-3σ

Ultraslow oligomerization equilibria of p53 and its implications

Haploinsufficiency of the Nijmegen breakage syndrome 1 gene increases mammary tumor latency and metastasis

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