Phosphorylation site interdependence of human p53 post-translational modifications in response to stress

Modification-specific antibodies were used to characterize the phosphorylation and acetylation of human p53 in response to genotoxic (UV, IR, and adriamycin) and non-genotoxic (PALA, taxol, nocodazole) stress in cultured human cells at 14 known modification sites. In A549 cells, phosphorylation or acetylation was induced at most sites by the three DNA damage-inducing agents, but significant differences between agents were observed. IR-induced phosphorylation reached a maximum 2 h after treatment and returned to near pretreatment levels by 72 h; UV light and adriamycin induced a less rapid but more robust and prolonged p53 phosphorylation, which reached a maximum between 8 and 24 h, but persisted (UV) even 96 h after treatment. Ser33, Ser37, Ser46, and Ser392 were more efficiently phosphorylated after exposure to UV light than after IR. The non-genotoxic agents PALA, taxol and nocodazole induced p53 accumulation and phosphorylation at Ser6, Ser33, Ser46, and Ser392. Some phosphorylation at Ser15 also was observed. Modifications occurred similarly in the HCT116 human colon carcinoma cell line. Analysis of single site mutant p53s indicated clear interdependences between N-terminal phosphorylation sites, which could be classified in four clusters: Ser6 and Ser9; Ser9, Ser15, Thr18 and Ser20; Ser33 and Ser37; and Ser46. We suggest that p53 phosphorylation is regulated through a double cascade involving both the activation of secondary, effector protein kinases as well as intermolecular phosphorylation site interdependencies that check inappropriate p53 inactivation while allowing for signal amplification and the integration of signals from multiple stress pathways.