Reactive oxygen species-induced phosphorylation of p53 on serine 20 is mediated in part by polo-like kinase-3

Upon exposure of cells to hydrogen peroxide (H2O2) phosphorylation of p53 w as rapidly induced in human fibroblast GM00637, and this phosphorylation oc curred on serine 9, serine 15, serine 20, but not on serine 392. In additio n, H2O2-induced phosphorylation of p53 was followed by induction of p21, su ggesting functional activation of p53. Induction of phosphorylation of p53 on multiple serine residues by H2O2 was caffeine-sensitive and blocked in A TM(-/-) cells. Polo-like kinase-3 (Plk3) activity was also activated upon H 2O2 treatment, and this activation was ATM-dependent. Recombinant His(6)-Pl k3 phosphorylated glutathione S-transferase (GST)-p53 fusion protein but no t GST alone. When phoshorylated in vitro by His(6)-Plk3, but not by the kin ase-defective mutant His6-Plk3(K52R), GST-p53 was recognized by an antibody specifically to serine 20-phosphorylated p53, indicating that serine 20 is an in vitro target of Plk3. Also serine 20-phosphorylated p53 was coimmuno precipitated with Plk3 in cells treated with H2O2. Furthermore, although H2 O2 strongly induced serine 15 phosphorylation of p53, it failed to induce s erine 20 phosphorylation in Plk3-difficient Daudi cells. Ectopic expression of a Plk3 dominant negative mutant, Plk3(K52R), in GM00637 cells suppresse d H2O2-induced serine 20 phosphorylation. Taken together, our studies stron gly suggest that the oxidative stress-induced activation of p53 is at least in part mediated by Plk3.