The p53 homolog TAp63α is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By measuring activation kinetics in ovaries and single-site phosphorylation kinetics in vitro with peptides and full-length protein, we show that TAp63α phosphorylation follows a biphasic behavior. Although the first two CK1 phosphorylation events are fast, the third one, which constitutes the decisive step to form the active conformation, is slow. Structure determination of CK1 in complex with differently phosphorylated peptides reveals the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63α substrate interaction in which the product of one phosphorylation step acts as an inhibitor for the following one.

p53 同系物 TAp63α 是卵母细胞基因组完整性的转录关键调节因子。DNA 损伤后，TAp63α 被多步磷酸化激活，涉及激酶 CK1 的多次磷酸化事件，触发从二聚体和非活性构象转变为开放的活性四聚体，从而启动细胞凋亡。通过用肽和全长蛋白测量卵巢中的激活动力学和体外单位点磷酸化动力学，我们表明 TAp63α 磷酸化遵循双相行为。虽然前两个 CK1 磷酸化事件很快，但构成形成活性构象的决定性步骤的第三个磷酸化事件很慢。