Quantitative Kinetic Analyses of Shutting Off a Two-Component System.
Quantitative Kinetic Analyses of Shutting Off a Two-Component System.
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DOI：
10.1128/mbio.00412-17
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发表时间：
2017-05-16
期刊：
影响因子：
6.4
通讯作者：
Stock AM
中科院分区：
文献类型：
--
作者：
Gao R;Stock AM
细胞依靠信号系统的精确控制来适应环境的扰动。刺激去除后的系统失活与信号通路的激活同样重要。双组分系统（TCS）是主要的细菌信号转导机制之一。在许多TCSs中，组氨酸激酶（HK）的磷酸酶活性被认为在关闭该通路和将系统重置到刺激前状态中发挥重要作用。两个基本的挑战是了解系统失活的动态行为和定量评估在自然细胞条件下磷酸酶活性的作用。在这里，我们报告了一项动力学分析，利用转录报告基因测定和体内磷酸化分析，对关闭原型大肠杆菌PhoR-PhoB TCS途径的反应进行了分析。在去除刺激后，PhoB反应调节因子（RR）的快速去磷酸化关闭了这一通路，而PhoB调节的基因产物通过生长稀释逐渐恢复到刺激前的水平。我们开发了一种结合实验和建模的方法，利用多个磷酸酶减少突变体的动力学数据来评估磷酸酶活性的体内动力学参数。这使得在体内PhoR磷酸酶活性的估计比在体外分析PhoR细胞质区域的磷酸酶活性强得多。我们定量模拟了抑制TCSs非特异性磷酸化需要多大程度的磷酸酶活性，并发现PhoR的强磷酸酶活性是抑制交叉磷酸化所必需的。tcs的活化已被广泛研究；然而，关闭TCS通路的动力学并没有很好地表征。我们对PhoR-PhoB系统的关闭反应进行了全面分析，揭示了磷酸酶活性对关闭动力学的影响。这使得定量框架的发展不仅可以表征自然细胞环境中磷酸酶的活性，还可以了解磷酸酶活性的特定强度来抑制非特异性磷酸化的要求。我们的模型表明，磷酸酶率与非特异性磷酸化率的比值与TCS表达水平和RR与HK的比值相关，这可能是不同TCS中酶水平和活性差异很大的原因。
Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo, which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro. We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression. Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.