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 (H(2)O(2)) phosphorylation of p53 was rapidly induced in human fibroblast GM00637, and this phosphorylation occurred on serine 9, serine 15, serine 20, but not on serine 392. In addition, H(2)O(2)-induced phosphorylation of p53 was followed by induction of p21, suggesting functional activation of p53. Induction of phosphorylation of p53 on multiple serine residues by H(2)O(2) was caffeine-sensitive and blocked in ATM(-/-) cells. Polo-like kinase-3 (Plk3) activity was also activated upon H(2)O(2) treatment, and this activation was ATM-dependent. Recombinant His(6)-Plk3 phosphorylated glutathione S-transferase (GST)-p53 fusion protein but not GST alone. When phoshorylated in vitro by His(6)-Plk3, but not by the kinase-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 coimmunoprecipitated with Plk3 in cells treated with H(2)O(2). Furthermore, although H(2)O(2) strongly induced serine 15 phosphorylation of p53, it failed to induce serine 20 phosphorylation in Plk3-dificient Daudi cells. Ectopic expression of a Plk3 dominant negative mutant, Plk3(K52R), in GM00637 cells suppressed H(2)O(2)-induced serine 20 phosphorylation. Taken together, our studies strongly suggest that the oxidative stress-induced activation of p53 is at least in part mediated by Plk3.
Xie
S
S
American Health Foundation, Valhalla, New York 10595, USA.
Wang
Q
Q
Wu
H
H
Cogswell
J
J
Lu
L
L
Jhanwar-Uniyal
M
M
Dai
W
W
D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017382:Reactive Oxygen Species; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D000097868:Polo-like Kinases
D018797:Cell Cycle Proteins; D005982:Glutathione Transferase; D006861:Hydrogen Peroxide; D017346:Protein Serine-Threonine Kinases; D017382:Reactive Oxygen Species; D012694:Serine; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C486256:PLK3 protein, human; D000097868:Polo-like Kinases