Abstract
The tumor suppressor protein p53 is a sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. Following exposure to a variety of stresses, p53 becomes post-translationally modified with concomitant increases in activity and stability. To better understand the role of acetylation of Lys-317 in mouse p53, the effect of ionizing radiation (IR) on the thymocytes of p53(K317R) knock-in mice was studied at the global level. Using cleavable ICAT quantitative mass spectrometry, the effect of IR on protein levels in either the wild type or p53(K317R) thymocytes was determined. We found 102 proteins to be significantly affected by IR in the wild type thymocytes, including several whose expression has been shown to be directly regulated by p53. When the effects of IR in the wild type and p53(K317R) samples were compared, 46 proteins were found to be differently affected (p < 0.05). The p53(K317R) mutation has widespread effects on specific protein levels following IR, including the levels of proteins involved in apoptosis, transcription, and translation. Pathway analysis of the differently regulated proteins suggests an increase in p53 activity in the p53(K317R) thymocytes as well as a decrease in tumor necrosis factor alpha signaling. These results suggest that acetylation of Lys-317 modulates the functions of p53 and influences the cross-talk between the DNA damage response and other signaling pathways.
Highlights
The tumor suppressor protein p53 is a sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and domains Topoisomerase (DNA) repair
Proteomics Analysis of the Wild Type and p53K317R Thymocytes—To better understand the effects of p300 and CBP-associated factor (PCAF) acetylation of Lys-317 in mouse p53, wild type and p53K317R knock-in mice were exposed to ionizing radiation (IR)
SSRP1 has been shown to be degraded during apoptosis; it is possible that the lower relative amount of this protein in the mutant versus wild type thymocytes is related to increased apoptosis as a result of the mutation [44]
Summary
Sample Preparation—The generation of knock-in mice containing the p53K317R mutation on both alleles of p53 was described previously [15]. An aliquot containing 500 g of total protein was incubated with the appropriate cleavable ICAT reagent (Applied Biosystems) following the manufacturer’s directions. Each total protein sample was denatured and reduced by incubation in the supplied buffers for 10 min at 100 °C. Non-irradiated and irradiated samples were separately incubated with aliquots of the light and heavy reagents, respectively, for 2 h at 37 °C. The respective non-irradiated and irradiated samples from wild type or p53K317R mice were combined (total of 1 mg of protein) and digested with trypsin for 16 h at 37 °C. Quantitation was performed using the ASAPRatio software, which uses extracted ion chromatograms for quantitation, and the peptides were assigned to proteins using ProteinProphet [20, 21].
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