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Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE

机译：通过使用磷酸化标签SDS-PAGE验证细菌三方传感器激酶多步磷酸转移信号的顺式和反式模式

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摘要

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.
机译：三方传感器激酶（TSK）在His，Asp和His残基上具有三个磷酸化位点，分别在组氨酸激酶（HK）域，受体域和含组氨酸的磷递质（HPt）域中保守。通过三步磷酸化，TSK将一个磷酸基团从ATP的γ-磷酸基团转移到HK域中的第一个His残基，然后转移到接收域中的Asp残基，最后转移到ATP中的第二个His残基。 HPt域。尽管TSK通常形成同型二聚体，但尚不清楚每个步骤中的磷酸化模式是分子内（顺式）还是分子间（反式）。为了检查这种模式，我们进行了体外互补分析，使用了Ala取代的ATP结合区突变体和重组Escherichia coli的ArcB，EvgS和BarA TSKs的三个磷酸化位点。通过使用Phos-tag SDS-PAGE确定的这些激酶的磷酸化谱表明，ArcB，EvgS和BarA的三步磷酸化转移反应的顺序模式都不同：顺-反-反，顺-顺-cis和trans-trans-trans。包含反式模式与形成同型二聚体的需求是一致的。 EvgS的所有步骤都具有顺式模式这一事实特别有趣。因此，磷酸标签SDS-PAGE提供了一种简单的方法，可以为给定的激酶识别独特而特定的磷酸转移模式，而无需考虑复杂的细胞内元件。

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