Site-specific phosphorylation of human p53 protein determined by mass spectrometry

B. Alex Merrick, W. Zhou, K. J. Martin, S. Jeyarajah, C. E. Parker, J. K. Selkirk, K. B. Tomer, C. H. Borchers

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30 Citations (Scopus)


Human recombinant p53 (r-p53) protein was studied by mass spectrometry (MS) to determine site-specific posttranslational differences between basal and hyperphosphorylated r-p53. Wild-type p53 was basally expressed after baculovirus infection while a parallel preparation was treated with the phosphatase inhibitor okadaic acid during the terminal stages of expression to create a hyperphosphorylated form of p53 known for its higher DNA binding and transcriptional activation. After immunoaffinity and HPLC purification, MALDI/MS measured a higher molecular mass for r-p53 from okadaic acid treatment relative to control, suggesting a higher phosphorylation state. This was supported by an acidic shift of r-p53 isoforms separated by gel isoelectric focusing. Employing a variety of mass spectrometric analyses combined with separation and affinity techniques, six specific phosphorylation sites of p53 were identified. The MS data indicated that hyperphosphorylated p53 showed a higher degree of phosphorylation than basal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser315 was entirely phosphorylated after okadaic acid treatment, as confirmed biochemically by CDK2 kinase assay and by isoelectric focusing. In summary, MS analysis uniquely revealed increased, site-specific phosphorylations on p53 after phosphatase inhibition, particularly at Ser315, which may be critical molecular events in defining p53 activity.

Original languageEnglish
Pages (from-to)4053-4066
Number of pages14
Issue number13
Publication statusPublished - 3 Apr 2001
Externally publishedYes


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