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 bacul ovirus infection while a parallel preparation was treated with the phosphat ase inhibitor okadaic acid during the terminal stages of expression to crea te a hyperphosphorylated form of p53 known for its higher DNA binding and t ranscriptional activation. After immunoaffinity and HPLC purification, MALD I/MS measured a higher molecular mass for r-p53 from okadaic acid treatment relative to control, suggesting a higher phosphorylation state. This was s upported 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 p 53 were identified. The MS data indicated that hyperphosphorylated p53 show ed a higher degree of phosphorylation than basal p53 at specific amino- and carboxy-terminal sites. In particular, ESI-MS demonstrated that Ser(315) w as entirely phosphorylated after okadaic acid treatment, as confirmed bioch emically by CDK2 kinase assay and by isoelectric focusing. In summary, MS a nalysis uniquely revealed increased, site-specific phosphorylations on p53 after phosphatase inhibition, particularly at Ser(315), which may be critic al molecular events in defining p53 activity.