Price B D, Hughes-Davies L, Park S J
Joint Center for Radiation Therapy, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Oncogene. 1995 Jul 6;11(1):73-80.
DNA damage increases p53 protein levels and activates transcription of the p21 gene. The p21 protein binds to and inhibits cdk2 kinase, causing G1 arrest. Here, we have investigated if a p53 fusion protein is a substrate for cdk2 kinase in vitro. Cdk2 kinase was immunoprecipitated from NIH3T3 cells and allowed to phosphorylate a human p53-GST (glutathione-s-transferase) fusion protein. Cdk2 and cyclin E-cdk2 efficiently phosphorylated both wild-type (wt) and mutant p53-GST. Cdk2 immunoprecipitated from cells in Go and early G1 exhibited minimal p53 kinase activity, whereas cells in S-phase displayed high levels of p53 kinase activity. If NIH3T3 cells were X-ray irradiated to induce DNA damage, cdk2 p53 kinase activity was rapidly inhibited within 1 h, but had recovered by 4 h post irradiation. Mutation of serine 315 of p53 to alanine (p53-S315A) abolished phosphorylation by cdk2 kinase. However, wtp53 and p53-S315A were equally effective at activating transcription when cotransfected with a p53 reporter construct. The results demonstrate that ser 315 of p53 is phosphorylated by cdk2 in vitro. However, ser 315 of wtp53 is not required for transcriptional activity in vivo, suggesting that cdk2 phosphorylation of p53 may be involved in regulating other cellular functions of wtp53.
DNA损伤会增加p53蛋白水平并激活p21基因的转录。p21蛋白结合并抑制cdk2激酶，导致G1期停滞。在此，我们研究了p53融合蛋白在体外是否是cdk2激酶的底物。从NIH3T3细胞中免疫沉淀cdk2激酶，并使其磷酸化人p53-GST（谷胱甘肽-S-转移酶）融合蛋白。cdk2和细胞周期蛋白E-cdk2均能有效地磷酸化野生型（wt）和突变型p53-GST。从处于G0期和早期G1期的细胞中免疫沉淀的cdk2表现出最小的p53激酶活性，而处于S期的细胞则显示出高水平的p53激酶活性。如果用X射线照射NIH3T3细胞以诱导DNA损伤，cdk2 p53激酶活性在1小时内迅速受到抑制，但在照射后4小时恢复。将p53的丝氨酸315突变为丙氨酸（p53-S315A）可消除cdk2激酶的磷酸化作用。然而，当与p53报告基因构建体共转染时，wtp53和p53-S315A在激活转录方面同样有效。结果表明，p53的丝氨酸315在体外被cdk2磷酸化。然而，体内转录活性并不需要wtp53的丝氨酸315，这表明p53的cdk2磷酸化可能参与调节wtp53的其他细胞功能。
