An Oxidant Responsive Signaling Cascade that Regulates p53-p300 Complex Formation
The biochemical activity of p53 most closely associated with tumour suppression is its function as a sequence-specific DNA binding protein and transcription factor that regulates the expression of gene products implicated in growth control. Two regulatory domains that control p53?s transcription activity include an N-terminal motif that drives the binding of the transcriptional co-activator p300 and a negative regulatory C-terminal domain whose phosphorylation by cyclin A-cdk2 kinase may play a coordinated role in stimulating the transcriptional activity of p53. Further characterization of the N-terminal domain of p53 has led to the identification of a novel phosphorylation site of p53 protein within the mdm2/p300-binding site at Ser20 that is constitutive in cycling cells. Ser20 phosphorylation of p53 can dramatically stabilize the p53-p300 protein complex. These data suggest that the Ser20 kinase regulatory cascade normally maintains p53 protein in a p300-competent binding form that allows the production of an active pool of p53 in an undamaged, proliferating cell. Hypo-phosphorylation of endogenous p53 protein at Ser20 occurs in response to cellular damaging agents that induce oxidant stress and this correlates with a down-regulation of Ser20 kinase activity. Ser20 Kinase activity is inactivated in a cell-free assay by the oxidizing agent N-ethylmaleimide and basal level of Ser20 kinase activity can be elevated by pre-treatment of cells with the anti-oxidant N-acetylcysteine. These data suggest the following oxidant-initiated cascade that can place a significant burden on the p53 pathway: (1) down-regulation of Ser20 Kinase activity by direct chemical oxidation; (2) hypo-phosphorylation of p53 at Ser20; (3) de-stabilized interaction of p53 with p300; and (4) reduced specific activity of p53 as a tumour suppressor protein. The sensitivity of the Ser20 Kinase cascade to oxidant stress identifies a mechanism that may accelerate the rate of inactivation of wild-type p53 protein as a transcription factor in developing cancers where chemical oxidants are known to play a role in disease progression.
Ted Hupp, Gloria Luciani, Ashley Craig and David Dornan
Department of Molecular and Cellular Pathology,