Acetylation of Mouse p53 at Lysine 317 Negatively Regulates p53 Apoptotic Activities after DNA Damage

ABSTRACT Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53K317R mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53K317R MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53K317R thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53K317R thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Download Full-text

Functional Analysis of the Roles of Posttranslational Modifications at the p53 C Terminus in Regulating p53 Stability and Activity

Molecular and Cellular Biology ◽

10.1128/mcb.25.13.5389-5395.2005 ◽

2005 ◽

Vol 25 (13) ◽

pp. 5389-5395 ◽

Cited By ~ 161

Author(s):

Lijin Feng ◽

Tongxiang Lin ◽

Hiroaki Uranishi ◽

Wei Gu ◽

Yang Xu

Keyword(s):

Dna Damage ◽

Cell Line ◽

Posttranslational Modifications ◽

Es Cells ◽

Embryonic Stem ◽

P53 Gene ◽

Missense Mutations ◽

C Terminus ◽

Lysine Residues ◽

P53 Stability

ABSTRACT Posttranslational modification of the tumor suppressor p53 plays important roles in regulating its stability and activity. Six lysine residues at the p53 C terminus can be posttranslationally modified by various mechanisms, including acetylation, ubiquitination, neddylation, methylation, and sumoylation. Previous cell line transfection studies show that ubiquitination of these lysine residues is required for ubiquitin-dependent degradation of p53. In addition, biochemical and cell line studies suggested that p53 acetylation at the C terminus might stabilize p53 and activate its transcriptional activities. To investigate the physiological functional outcome of these C-terminal modifications in regulating p53 stability and activity, we introduced missense mutations (lysine to arginine) at the six lysine residues (K6R) into the endogenous p53 gene in mouse embryonic stem (ES) cells. The K6R mutation prevents all posttranslational modifications at these sites but conserves the structure of p53. In contrast to conclusions of previous studies, analysis of p53 stability in K6R ES cells, mouse embryonic fibroblasts, and thymocytes showed normal p53 stabilization in K6R cells both before and after DNA damage, indicating that ubiquitination of these lysine residues is not required for efficient p53 degradation. However, p53-dependent gene expression was impaired in K6R ES cells and thymocytes in a promoter-specific manner after DNA damage, indicating that the net outcome of the posttranslational modifications at the C terminus is to activate p53 transcriptional activities after DNA damage.

Download Full-text

The Absence of Ser389 Phosphorylation in p53 Affects the Basal Gene Expression Level of Many p53-Dependent Genes and Alters the Biphasic Response to UV Exposure in Mouse Embryonic Fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.01610-07 ◽

2008 ◽

Vol 28 (6) ◽

pp. 1974-1987 ◽

Cited By ~ 25

Author(s):

Wendy Bruins ◽

Oskar Bruning ◽

Martijs J. Jonker ◽

Edwin Zwart ◽

Tessa V. van der Hoeven ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Uv Irradiation ◽

Target Genes ◽

Late Response ◽

Wild Type ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Endogenous Gene ◽

Cellular Processes

ABSTRACT Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53−/− mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53−/−, possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53−/− but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.

Download Full-text

DNA damage, p53 gene expression and p53 protein level in the rat brain aging

Journal of Neurochemistry ◽

10.1046/j.1471-4159.85.s2.18_10.x ◽

2003 ◽

Vol 85 ◽

pp. 26-26 ◽

Cited By ~ 1

Author(s):

J. Dorszewska ◽

Z. Adamczewska-Goncerzewicz

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Rat Brain ◽

Protein Level ◽

Brain Aging ◽

P53 Protein ◽

P53 Gene

Download Full-text

Mutation of Mouse p53 Ser23 and the Response to DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.22.8.2441-2449.2002 ◽

2002 ◽

Vol 22 (8) ◽

pp. 2441-2449 ◽

Cited By ~ 76

Author(s):

Zhiqun Wu ◽

John Earle ◽

Shin'ichi Saito ◽

Carl W. Anderson ◽

Ettore Appella ◽

...

Keyword(s):

Dna Damage ◽

Ionizing Radiation ◽

Radiation Treatment ◽

Es Cells ◽

P53 Protein ◽

Embryonic Stem ◽

P53 Gene ◽

Wild Type ◽

Embryonic Fibroblasts ◽

Phosphorylation Event

ABSTRACT Recent studies have suggested that phosphorylation of human p53 at Ser20 is important for stabilizing p53 in response to DNA damage through disruption of the interaction between MDM2 and p53. To examine the requirement for this DNA damage-induced phosphorylation event in a more physiological setting, we introduced a missense mutation into the endogenous p53 gene of mouse embryonic stem (ES) cells that changes serine 23 (S23), the murine equivalent of human serine 20, to alanine (A). Murine embryonic fibroblasts harboring the p53S23A mutation accumulate p53 as well as p21 and Mdm2 proteins to normal levels after DNA damage. Furthermore, ES cells and thymocytes harboring the p53S23A mutation also accumulate p53 protein to wild-type levels and undergo p53-dependent apoptosis similarly to wild-type cells after DNA damage. Therefore, phosphorylation of murine p53 at Ser23 is not required for p53 responses to DNA damage induced by UV and ionizing radiation treatment.

Download Full-text

Regulation of c-Fos Gene Expression by NF-κB: A p65 Homodimer Binding Site in Mouse Embryonic Fibroblasts but Not Human HEK293 Cells

PLoS ONE ◽

10.1371/journal.pone.0084062 ◽

2013 ◽

Vol 8 (12) ◽

pp. e84062 ◽

Cited By ~ 10

Author(s):

Yu-Cheng Tu ◽

Duen-Yi Huang ◽

Shine-Gwo Shiah ◽

Jang-Shiun Wang ◽

Wan-Wan Lin

Keyword(s):

Gene Expression ◽

Binding Site ◽

Hek293 Cells ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts

Download Full-text

Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.4.c946 ◽

1999 ◽

Vol 276 (4) ◽

pp. C946-C954 ◽

Cited By ~ 46

Author(s):

Li Li ◽

Ji Li ◽

Jaladanki N. Rao ◽

Minglin Li ◽

Barbara L. Bass ◽

...

Keyword(s):

Gene Expression ◽

Growth Inhibition ◽

P53 Protein ◽

Specific Inhibitor ◽

P53 Gene ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Polyamine Biosynthesis ◽

Fetal Bovine ◽

P53 Mrna

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

Download Full-text

AMPKα1 deficiency promotes cellular proliferation and DNA damage via p21 reduction in mouse embryonic fibroblasts

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2014.10.002 ◽

2015 ◽

Vol 1853 (1) ◽

pp. 65-73 ◽

Cited By ~ 13

Author(s):

Hairong Xu ◽

Yanhong Zhou ◽

Kathleen A. Coughlan ◽

Ye Ding ◽

Shaobin Wang ◽

...

Keyword(s):

Dna Damage ◽

Cellular Proliferation ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts

Download Full-text

Enrichment of Cxcl12 promoter with TET2: A possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1

Archives of Biological Sciences ◽

10.2298/abs190404027t ◽

2019 ◽

Vol 71 (3) ◽

pp. 455-462

Author(s):

Anja Tolic ◽

Jovana Rajic ◽

Marija Djordjevic ◽

Milos Djordjevic ◽

Svetlana Dinic ◽

...

Keyword(s):

Gene Expression ◽

Technological Development ◽

Cpg Islands ◽

Gene Induction ◽

Dna Hypomethylation ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Cxcl12 Expression ◽

Knock Out ◽

Parp 1

Previously, we described the link between C-X-C motif chemokine 12 (Cxcl12) gene induction and DNA hypomethylation in the absence of poly(ADP-ribose) polymerase 1 (PARP-1). We have now firmly established that demethylation is the primary cause of gene induction on the basis of Cxcl12 gene upregulation upon treatment with the demethylating agent 5-azacytidine (5-aza). Since the demethylation state of Cxcl12 is favored by PARP-1 absence, we investigated the presence of ten-eleven translocation (TET) proteins on the Cxcl12 promoter in order to corroborate the relationship between the demethylation process and increased gene expression that occurs in the absence of PARP-1. Analysis was performed on the promoter region within CpG islands of Cxcl12 from control mouse embryonic fibroblasts (NIH3T3) and PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-). The lack of PARP-1 increased the abundance of TET2 on the Cxcl12 promoter, suggesting that TET-mediated demethylation provoked by the absence of PARP-1 could account for the observed increased expression of this chemokine. Deciphering the regulation of DNA (de)methylation factors that control Cxcl12 expression may provide an additional therapeutic approach in pharmacological interventions where gene switching on or off based on targeted stimulation or inhibition is necessary. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173020]

Download Full-text

Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate

The Journal of Cell Biology ◽

10.1083/jcb.200512059 ◽

2006 ◽

Vol 173 (4) ◽

pp. 533-544 ◽

Cited By ~ 171

Author(s):

Chad D. Knights ◽

Jason Catania ◽

Simone Di Giovanni ◽

Selen Muratoglu ◽

Ricardo Perez ◽

...

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Biological Activities ◽

Expression Profiles ◽

Expression Patterns ◽

Gene Expression Profiles ◽

P53 Gene ◽

Protein Protein Interactions

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH2-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH2-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the “histone code” hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different “p53 cassettes,” each containing combination patterns of posttranslational modifications and protein–protein interactions.

Download Full-text