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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling
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Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling

机译：浓度依赖性的磷酸化分析在体内探测两组分系统的激酶和磷酸酶活性

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摘要

Quantitative analyses of protein concentrations, modifications and activities in their native environments are playing an increasingly vital role in unraveling the general principles underlying signal trans-duction pathways. The prevalent bacterial two-component systems (TCSs) use a central phosphotransfer for signaling; however, in vivo characterization of the kinase and phosphatase activities of TCS proteins is often limited by traditional transcriptional reporter assays and complicated by simultaneous actions of multiple TCS activities. Here, we report a strategy that combines concentration-dependent phosphorylation profiling and mathematical modeling to characterize the cellular activities of the archetype Escherichia coli PhoR/PhoB system. Phosphorylation of the response regulator (RR) PhoB has been found to be dependent on the total concentrations of PhoB/PhoR and saturated at high concentrations. The relationship between RR phosphorylation and total concentrations has been defined by the modeling of the kinase and phosphatase reactions and quantified to derive the biochemical parameters of the PhoR/PhoB system in vivo. In a further test of this approach on a PhoB mutant, PhoB~(F20D), it proved highly effective in exploring the mechanistic differences of TCSs that are not revealed by traditional reporter assays. Measurement of biochemical parameters for PhoB~(F20D) led to the discovery that a weaker interaction between the histidine sensor kinase and RR could result in a higher and nonrobust phosphorylation due to diminished phosphatase activities.%Center for Advanced Biotechnology and Medicine, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854;Center for Advanced Biotechnology and Medicine, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854;
机译：对蛋白质天然环境中蛋白质浓度，修饰和活性的定量分析在阐明信号转导途径的一般原理方面发挥着越来越重要的作用。流行的细菌两组分系统（TCS）使用中央磷酸转移来进行信号传导。但是，TCS蛋白的激酶和磷酸酶活性的体内表征通常受到传统转录报告基因检测的限制，并且由于多种TCS活性的同时作用而变得复杂。在这里，我们报告了一种策略，该策略结合了浓度依赖性磷酸化分析和数学模型来表征原型大肠杆菌PhoR / PhoB系统的细胞活性。已发现响应调节剂（RR）PhoB的磷酸化取决于PhoB / PhoR的总浓度，并在高浓度下饱和。 RR磷酸化和总浓度之间的关系已经通过激酶和磷酸酶反应的建模进行了定义，并进行了量化以得出体内PhoR / PhoB系统的生化参数。在针对PhoB突变体PhoB〜（F20D）的这种方法的进一步测试中，它被证明在探索传统报告基因检测未发现的TCS机理差异方面非常有效。对PhoB〜（F20D）生化参数的测量导致发现，由于磷酸酶活性的降低，组氨酸传感器激酶与RR之间较弱的相互作用可能导致较高的磷酸化，而磷酸化的活性不强。％，高级化学技术和医学中心，生物化学新泽西医科大学牙科与分子生物学-罗伯特伍德·约翰逊医学院，皮斯卡塔维，新泽西州08854；新泽西医科大学牙科与生物医学系先进生物技术与医学中心-罗伯特·伍德·约翰逊医学院，皮斯卡塔维，新泽西州08854；

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  • 作者

    Rong Gao; Ann M. Stock;

  • 作者单位

    Center for Advanced Biotechnology and Medicine, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854;

    Center for Advanced Biotechnology and Medicine, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    in vivo phosphorylation; two-component signal transduction;

    机译：体内磷酸化;两成分信号转导;

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