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Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase.
K Sakaguchi1, S Saito, Y Higashimoto
1NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr(18) but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser(15). Thr(18) was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser(15). Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18).
Insights
Ionizing radiation stabilizes tumor suppressor p53 by disrupting its interaction with Mdm2. A phosphorylation cascade, starting with Ser(15) and then Thr(18), inhibits Mdm2 binding, preventing p53 degradation.
Area of Science:
- Molecular Biology
- Cancer Research
- Protein Biochemistry
Background:
- The p53 tumor suppressor protein plays a critical role in preventing cancer.
- p53 is normally degraded by Mdm2, but is stabilized following DNA damage.
- The precise mechanisms regulating p53 stabilization after ionizing radiation are not fully understood.
Purpose of the Study:
- To investigate the role of N-terminal phosphorylation in the interaction between p53 and Mdm2.
- To elucidate the specific phosphorylation sites and kinases involved in p53 stabilization.
- To determine the sequence of phosphorylation events leading to Mdm2 binding inhibition.
Main Methods:
- In vitro phosphorylation assays using p53 N-terminal peptides and casein kinase 1 (CK1).
- Analysis of p53 phosphorylation in vivo following DNA damage.
- Assessment of Mdm2 binding to p53 peptides with different phosphorylation states.
Main Results:
- Phosphorylation of p53 on Thr(18) disrupts the direct association with Mdm2.
- CK1 phosphorylates Thr(18) only after prior phosphorylation of Ser(15).
- Thr(18) phosphorylation occurs in vivo after DNA damage and requires Ser(15) phosphorylation.
Conclusions:
- p53 stabilization after ionizing radiation involves a sequential phosphorylation cascade.
- DNA damage-induced phosphorylation of p53 Ser(15) enables subsequent Thr(18) phosphorylation.
- This cascade inhibits Mdm2 binding, contributing to p53 accumulation and tumor suppression.