Site-Specific Phosphorylation of Human p53 Protein Determined by Mass Spectrometry

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• 作者：B. Alex Merrick ; Wei Zhou ; Karla J. Martin ; Shanthini Jeyarajah ; Carol E. Parker ; James K. Selkirk ; Kenneth B. Tomer ; and Christoph H. Borchers
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 3, 2001
• 年：2001
• 卷：40
• 期：13
• 页码：4053 - 4066
• 全文大小：230K
• 年卷期：v.40,no.13(April 3, 2001)
• ISSN：1520-4995

文摘

Human recombinant p53 (r-p53) protein was studied by mass spectrometry (MS) to determinesite-specific posttranslational differences between basal and hyperphosphorylated r-p53. Wild-type p53was basally expressed after baculovirus infection while a parallel preparation was treated with thephosphatase inhibitor okadaic acid during the terminal stages of expression to create a hyperphosphorylatedform of p53 known for its higher DNA binding and transcriptional activation. After immunoaffinity andHPLC purification, MALDI/MS measured a higher molecular mass for r-p53 from okadaic acid treatmentrelative to control, suggesting a higher phosphorylation state. This was supported by an acidic shift ofr-p53 isoforms separated by gel isoelectric focusing. Employing a variety of mass spectrometric analysescombined 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 thanbasal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser³¹⁵was entirely phosphorylated after okadaic acid treatment, as confirmed biochemically by CDK2 kinaseassay and by isoelectric focusing. In summary, MS analysis uniquely revealed increased, site-specificphosphorylations on p53 after phosphatase inhibition, particularly at Ser³¹⁵, which may be critical molecularevents in defining p53 activity.