Site-specific phosphorylation of human p53 protein determined by mass spectrometry.
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 Ser(315) 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 Ser(315), which may be critical molecular events in defining p53 activity.
Merrick
B A
BA
Laboratory of Molecular Carcinogenesis and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Zhou
W
W
Martin
K J
KJ
Jeyarajah
S
S
Parker
C E
CE
Selkirk
J K
JK
Tomer
K B
KB
Borchers
C H
CH
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010748:Phosphopeptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D018411:Spodoptera / Q000235:genetics; D014357:Trypsin; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D019319:Okadaic Acid; D012694:Serine; D014357:Trypsin