From the Laboratories for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Whitson Street, Bristol BS13NY, United Kingdom.
Endocrine Signaling Group, Royal Veterinary College, Royal College St., London NW10TU, United Kingdom.
J Biol Chem. 2013 Jul 19;288(29):21001-21014. doi: 10.1074/jbc.M113.455345. Epub 2013 Jun 10.
Many extracellular signals act via the Raf/MEK/ERK cascade in which kinetics, cell-cell variability, and sensitivity of the ERK response can all influence cell fate. Here we used automated microscopy to explore the effects of ERK-mediated negative feedback on these attributes in cells expressing endogenous ERK or ERK2-GFP reporters. We studied acute rather than chronic stimulation with either epidermal growth factor (ErbB1 activation) or phorbol 12,13-dibutyrate (PKC activation). In unstimulated cells, ERK-mediated negative feedback reduced the population-average and cell-cell variability of the level of activated ppERK and increased its robustness to changes in ERK expression. In stimulated cells, negative feedback (evident between 5 min and 4 h) also reduced average levels and variability of phosphorylated ERK (ppERK) without altering the "gradedness" or sensitivity of the response. Binning cells according to total ERK expression revealed, strikingly, that maximal ppERK responses initially occur at submaximal ERK levels and that this non-monotonic relationship changes to an increasing, monotonic one within 15 min. These phenomena occur in HeLa cells and MCF7 breast cancer cells and in the presence and absence of ERK-mediated negative feedback. They were best modeled assuming distributive (rather than processive) activation. Thus, we have uncovered a novel, time-dependent change in the relationship between total ERK and ppERK levels that persists without negative feedback. This change makes acute response kinetics dependent on ERK level and provides a "gating" or control mechanism in which the interplay between stimulus duration and the distribution of ERK expression across cells could modulate the proportion of cells that respond to stimulation.
许多细胞外信号通过 Raf/MEK/ERK 级联作用，ERK 反应的动力学、细胞间变异性和敏感性都可以影响细胞命运。在这里，我们使用自动化显微镜来探索 ERK 介导的负反馈对表达内源性 ERK 或 ERK2-GFP 报告基因的细胞中这些属性的影响。我们研究了急性而非慢性刺激，分别使用表皮生长因子（ErbB1 激活）或佛波醇 12,13-二丁酸酯（PKC 激活）。在未受刺激的细胞中，ERK 介导的负反馈降低了激活的 ppERK 的群体平均和细胞间变异性，并增加了其对 ERK 表达变化的鲁棒性。在受刺激的细胞中，负反馈（在 5 分钟至 4 小时之间明显）也降低了磷酸化 ERK（ppERK）的平均水平和变异性，而不改变反应的“分级”或敏感性。根据总 ERK 表达对细胞进行分组，令人惊讶的是，最大的 ppERK 反应最初发生在亚最大 ERK 水平，这种非单调关系在 15 分钟内变为增加的单调关系。这些现象发生在 HeLa 细胞和 MCF7 乳腺癌细胞中，并且存在和不存在 ERK 介导的负反馈。它们最好通过假设分布（而不是连续）激活来建模。因此，我们发现了一种新的、与时间相关的总 ERK 和 ppERK 水平之间关系的变化，这种变化在没有负反馈的情况下持续存在。这种变化使急性反应动力学依赖于 ERK 水平，并提供了一种“门控”或控制机制，其中刺激持续时间和细胞间 ERK 表达分布的相互作用可以调节对刺激做出反应的细胞比例。
