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Phosphorylation site interdependence of human p53 post-translational modifications in response to stress
Shin'ichi Saito1, Hiroshi Yamaguchi, Yuichiro Higashimoto
1Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
Modification-specific antibodies were used to characterize the phosphorylation and acetylation of human p53 in response to genotoxic (UV, IR, and adriamycin) and non-genotoxic (PALA, taxol, nocodazole) stress in cultured human cells at 14 known modification sites. In A549 cells, phosphorylation or acetylation was induced at most sites by the three DNA damage-inducing agents, but significant differences between agents were observed. IR-induced phosphorylation reached a maximum 2 h after treatment and returned to near pretreatment levels by 72 h; UV light and adriamycin induced a less rapid but more robust and prolonged p53 phosphorylation, which reached a maximum between 8 and 24 h, but persisted (UV) even 96 h after treatment. Ser33, Ser37, Ser46, and Ser392 were more efficiently phosphorylated after exposure to UV light than after IR. The non-genotoxic agents PALA, taxol and nocodazole induced p53 accumulation and phosphorylation at Ser6, Ser33, Ser46, and Ser392. Some phosphorylation at Ser15 also was observed. Modifications occurred similarly in the HCT116 human colon carcinoma cell line. Analysis of single site mutant p53s indicated clear interdependences between N-terminal phosphorylation sites, which could be classified in four clusters: Ser6 and Ser9; Ser9, Ser15, Thr18 and Ser20; Ser33 and Ser37; and Ser46. We suggest that p53 phosphorylation is regulated through a double cascade involving both the activation of secondary, effector protein kinases as well as intermolecular phosphorylation site interdependencies that check inappropriate p53 inactivation while allowing for signal amplification and the integration of signals from multiple stress pathways.
Insights
Human p53 protein modifications, including phosphorylation and acetylation, were analyzed under various stress conditions. Results reveal distinct modification patterns and interdependencies between sites, suggesting complex regulatory pathways for p53 signaling.
Area of Science:
- Molecular Biology
- Cellular Stress Response
- Protein Post-translational Modifications
Background:
- The tumor suppressor protein p53 plays a critical role in cellular responses to stress.
- Understanding p53 modification dynamics is crucial for deciphering its regulatory mechanisms.
- Genotoxic and non-genotoxic stresses induce complex p53 post-translational modifications.
Purpose of the Study:
- To comprehensively characterize p53 phosphorylation and acetylation at 14 known sites.
- To investigate the differential effects of genotoxic and non-genotoxic stresses on p53 modification.
- To elucidate interdependencies among p53 N-terminal phosphorylation sites.
Main Methods:
- Utilized modification-specific antibodies to analyze p53 in cultured human cells (A549 and HCT116).
- Applied genotoxic stresses (UV, IR, adriamycin) and non-genotoxic stresses (PALA, taxol, nocodazole).
- Analyzed single-site mutant p53s to determine phosphorylation site interdependencies.
Main Results:
- Genotoxic agents induced distinct p53 phosphorylation patterns, with UV and adriamycin causing more prolonged modifications than IR.
- Non-genotoxic agents induced p53 accumulation and phosphorylation at specific N-terminal sites (Ser6, Ser33, Ser46, Ser392).
- Identified four clusters of interdependent N-terminal phosphorylation sites, indicating complex regulatory networks.
Conclusions:
- p53 phosphorylation is regulated by a dual cascade involving effector kinases and intermolecular site interactions.
- These interdependencies ensure appropriate p53 inactivation while allowing signal amplification and integration.
- The findings provide insights into the intricate signaling pathways governing p53 function under stress.