| Summary: | 博士 === 國立臺灣大學 === 分子醫學研究所 === 91 === Protein modifications are important mechanisms that regulate various properties of proteins. Therefore, it is important for us to characterize protein modifications in order to understand how protein functions change in response to cellular stimuli. We have previously reported a tandem mass spectrometry-based method, namely SIT analysis, what can be applied to the identification of phosphopeptides as well as exact mapping of the phosphorylated residues within. Here, we employed the same strategy to identify acetylated peptides on p53 in vitro p300-mediated acetylation system. Consistent with the previous finding, lysine residues 370/372/373, 381 and 382 were shown as the acetylated targets of p300 in vitro. Intriguingly, two novel acetylation sites, Lys-292 and Lys-305, were also serendipitously found. Immunoblotting using anti-acetyl Lys-305 specific antibody demonstrated that Lys-305 was acetylated by p300 in vivo as well. We also showed that an alanine or glutamime substitution at Lys-305 (K305A, or K305Q) suppressed its transcriptional activity, while the arginine mutation (K305R) caused an elevation in transcriptional activity. These observations are reminiscent of transcriptional activities of Lys-382 mutants. However, K305A mutant showed cytoplasmic mislocalization as well as suppressed K382 acetylation. Paradoxically, it has stronger affinity toward oligonucleotides containing p21 p53-binding elements. On the other hand, K305R mutant has enhanced transcriptional activity without any effects on DNA binding or subcellular localization. These data showed that, although Lys-305 and Lys-382 mutants share similar changes in transcriptional activity, acetylation at the former position seems to mediate p53 function through nucleocytoplasmic distribution and/or Lys-382 acetylation. Finally, these results showed that SIT analysis can be used for the studies of a variety of modifications seen in various regulated cellular processes.
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