Tissue repair, immune defence and cancer progression rely on a vital cellular decision between quiescence and proliferation^1,2. Mammalian cells proliferate by triggering a positive feedback mechanism^3,4. The transcription factor E2F activates cyclin-dependent kinase 2 (CDK2), which in turn phosphorylates and inactivates the E2F inhibitor protein retinoblastoma (Rb). This action further increases E2F activity to express genes needed for proliferation. Given that positive feedback can inadvertently amplify small signals, understanding how cells keep this positive feedback in check remains a puzzle. Here we measured E2F and CDK2 signal changes in single cells and found that the positive feedback mechanism engages only late in G1 phase. Cells spend variable and often extended times in a reversible state of intermediate E2F activity before committing to proliferate. This intermediate E2F activity is proportional to the amount of phosphorylation of a conserved T373 residue in Rb that is mediated by CDK2 or CDK4/CDK6. Such T373-phosphorylated Rb remains bound on chromatin but dissociates from it once Rb is hyperphosphorylated at many sites, which fully activates E2F. The preferential initial phosphorylation of T373 can be explained by its relatively slower rate of dephosphorylation. Together, our study identifies a primed state of intermediate E2F activation whereby cells sense external and internal signals and decide whether to reverse and exit to quiescence or trigger the positive feedback mechanism that initiates cell proliferation.

组织修复、免疫防御和癌症进展依赖于静止和增殖之间的重要细胞决定 ^1,2 。哺乳动物细胞通过触发正反馈机制进行增殖 ^3,4 。转录因子 E2F 激活细胞周期蛋白依赖性激酶 2 (CDK2)，进而磷酸化 E2F 抑制剂蛋白视网膜母细胞瘤 (Rb) 并使其失活。这一作用进一步增加了 E2F 活性以表达增殖所需的基因。鉴于正反馈会无意中放大小信号，了解细胞如何控制这种正反馈仍然是一个谜。在这里，我们测量了单细胞中 E2F 和 CDK2 信号的变化，发现正反馈机制仅在 G1 期后期起作用。在进行增殖之前，细胞会在中间 E2F 活性的可逆状态下花费可变且通常较长的时间。这种中间 E2F 活性与由 CDK2 或 CDK4/CDK6 介导的 Rb 中保守 T373 残基的磷酸化量成正比。这种 T373 磷酸化的 Rb 仍然结合在染色质上，但一旦 Rb 在许多位点过度磷酸化，就会从染色质上解离，从而完全激活 E2F。 T373 的优先初始磷酸化可以通过其相对较慢的去磷酸化速率来解释。总之，我们的研究确定了中间 E2F 激活的启动状态，细胞在此状态下感知外部和内部信号，并决定是否逆转并退出静止状态或触发启动细胞增殖的正反馈机制。