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Post-translational modification of p53 protein in response to ionizing radiation analyzed by mass spectrometry
J Abraham1, J Kelly, P Thibault
1Ontario Cancer Institute/Princess Margaret Hospital and Department of Medical Biophysics, Toronto, Ontario, M5G 2M9, Canada.
Abstract:
The p53 tumor suppressor protein promotes cell cycle arrest or apoptosis in response to DNA damage and other forms of stress. p53 protein functions as a transcription factor by binding to specific DNA sequences and regulating the transcription of target genes. This activity of p53 is reported to be regulated by phosphorylation and acetylation occuring at various sites on the molecule. Here, we have used a direct and non-radioactive approach involving mass spectrometric analysis of p53 protein to identify sites that are covalently modified in vivo, either constitutively or in response to ionizing radiation. Following partial purification by immuno-affinity chromatography and enzymatic in-gel digestion, the resulting p53 peptides were analyzed by MALDI-TOF and nanoelectrospray mass spectrometry. Mass spectrometry analyses identified four sites at the N terminus that were phosphorylated in response to irradiation, a single constitutive phosphorylation site at serine 315 and several acetylation sites.
Insights
The p53 tumor suppressor protein
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- The p53 protein acts as a tumor suppressor by regulating cell cycle arrest and apoptosis in response to cellular stress.
- p53 functions as a transcription factor, with its activity modulated by post-translational modifications like phosphorylation and acetylation.
- Identifying these modification sites is crucial for understanding p53's regulatory mechanisms.
Purpose of the Study:
- To identify specific sites of covalent modification on the p53 protein in vivo.
- To distinguish between constitutive modifications and those induced by ionizing radiation.
Main Methods:
- Utilized a direct, non-radioactive mass spectrometry approach for p53 analysis.
- Employed immuno-affinity chromatography for partial purification of p53 protein.
- Analyzed enzymatically digested p53 peptides using MALDI-TOF and nanoelectrospray mass spectrometry.
Main Results:
- Identified four N-terminal phosphorylation sites on p53 induced by ionizing radiation.
- Confirmed a constitutive phosphorylation site at serine 315.
- Detected several acetylation sites on the p53 protein.
Conclusions:
- Mass spectrometry is effective for identifying p53 post-translational modifications in vivo.
- Ionizing radiation induces specific N-terminal phosphorylations on p53.
- Characterization of p53 modification sites provides insights into its tumor suppressor functions.