Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli
Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli
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DOI：
10.1074/jbc.m410104200
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发表时间：
2005-01-14
影响因子：
4.8
通讯作者：
Ishihama, A
中科院分区：
文献类型：
--
作者：
Yamamoto, K;Hirao, K;Ishihama, A
细菌拥有一个信号转导系统，称为双组分系统，用于适应外部刺激。每个双组分系统由一个传感蛋白-组氨酸激酶(HK)和一个反应调节蛋白(RR)组成，共同通过组氨酸-天冬氨酸磷酸酯转导形成信号转导通路。目前已发现在大肠杆菌中存在30种传感器HKS，包括尚未鉴定的HKS(Baes、Bass、CREC、CUSS、HydH、RstB、YedV和YfhK)，以及34种RRs(Baer、Basr、CREB、CUSR、HydG、Rsta、YedW、YfhA、YgeK和YhjB)。我们已经纯化了27个传感器HKS的羧基末端催化结构域和所有34个RR的全长蛋白，使其表面均一。体外检测25个HKS的自身磷酸化。不同HKS的自身磷酸化速率不同，而磷酸化水平一般与磷酸化速率相关。然而，尽管反应速度很快，ArcB、HydH、NarQ和NtrB的磷酸化水平很低，而磷酸化速度较慢的物种BASS、CHEA和CREC的磷酸化水平很高。通过使用磷酸化的HKS，我们在体外检测了RRS的所有可能的结合的反式磷酸化。首次检测到HKS对Baes-Baer、Bass-Basr、CREC-CREB、CASS-CUSR、HydH-HydG、RstB-Rsta、YedV-YedW和YfhK-YfhA八对假定同源RRs的反式磷酸化。所有的反式磷酸化都在不到1/2分钟内发生，但磷酸化RRs的稳定性不同，这表明参与了去磷酸化调控。除了同源对之间的反式磷酸化外，我们还检测到大约3%的非同源HK-RR对之间的反式磷酸化，这增加了信号转导中的串扰发生在两个组分系统之间的可能性。
Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Self-phosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.