Sakaguchi K, Saito S, Higashimoto Y, Roy S, Anderson C W, Appella E
NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2000 Mar 31;275(13):9278-83. doi: 10.1074/jbc.275.13.9278.
The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr(18) but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser(15). Thr(18) was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser(15). Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18).
p53肿瘤抑制蛋白在受到电离辐射后会被稳定化，并在细胞核中积累。人们认为这种稳定化涉及到p53蛋白与Mdm2之间相互作用的破坏，Mdm2会将p53作为降解靶点。在此我们表明，p53 N端肽与Mdm2之间的直接结合会被该肽段上Thr(18)的磷酸化所破坏，但不会被包括Ser(15)和Ser(37)在内的其他N端位点的磷酸化所破坏。Thr(18)在体外被酪蛋白激酶(CK1)磷酸化；这一过程需要Ser(15)先被磷酸化。Thr(18)在体内会因DNA损伤而被磷酸化，且这种磷酸化需要Ser(15)。我们的结果表明，电离辐射后p53的稳定化可能部分源于通过一个磷酸化-磷酸化级联反应抑制Mdm2结合，该级联反应涉及DNA损伤激活的p53 Ser(15)磷酸化，随后是Thr(18)的磷酸化。
