Requirement of ATM for Rapid p53 Phosphorylation at Ser46 without Ser/Thr-Gln Sequences

Abstract

p53 phosphorylation at Ser46 following DNA damage is important for preferential transactivation of proapoptotic genes. Here, we report that ataxia-telangiectasia mutated (ATM) kinase is responsible for Ser46 phosphorylation of p53 during early-phase response to DNA damage. To elucidate the direct phosphorylation of p53 at Ser46 by ATM, an ATM mutant (ATM-AS) sensitive to ATP analogues was engineered. In vitro kinase assays revealed that p53 was phosphorylated at Ser46 by ATM-AS, even when ATP analogues were used as phosphate donors, although this phosphorylation site is not in an SQ motif, a consensus ATM site. Furthermore, Ser46 phosphorylation by ATM was dependent on the N- and C-terminal domains of p53, unlike Ser15 phosphorylation. Immunofluorescence analyses showed that Ser46-phosphorylated p53 was observed as foci in response to DNA damage and colocalized with γ-H2AX or Ser1981-phosphorylated ATM. These results suggest that ATM phosphorylates a noncanonical serine residue on p53 by mechanisms different from those for the phosphorylation of Ser15.

We are grateful to Michael B. Kastan (St. Jude Children's Research Hospital) for the expression vector for FLAG-tagged ATM wild type. We also thank Kiyotsugu Yoshida (Tokyo Medical and Dental University, Japan) for valuable discussions with respect to DYRK2 and Kyoko Fujinaka (National Cancer Center Research Institute, Japan) for technical assistance.

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (17013088) (to Y.T. and M.E.); SORST, Japan Science and Technology (to Y.T.); and a grant from the Takeda Science Foundation (to M.E.).