Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE.
Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE.
复制标题
通过使用PHOS-TAG SDS-PAGE，对细菌三方传感器激酶的多步磷酸转移信号的顺式和反式模式的验证。
DOI：
10.1371/journal.pone.0148294
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Koike T
中科院分区：
文献类型：
--
作者：
Kinoshita-Kikuta E;Kinoshita E;Eguchi Y;Koike T
三方传感器激酶（TSKs）在His、Asp和His残基上有三个磷酸化位点，分别保守于组氨酸激酶（HK）结构域、受体结构域和含组氨酸磷酸化递质（HPt）结构域。TSKs通过三步磷接力，将ATP γ-磷酸基的一个磷酸基传递到HK结构域的第一个His残基，然后传递到接收器结构域的Asp残基，最后传递到HPt结构域的第二个His残基。虽然TSKs通常形成同型二聚体，但尚不清楚每一步的磷酸化模式是分子内（顺式）还是分子间（反式）。为了检验这种模式，我们使用来自大肠杆菌的重组ArcB、EvgS和BarA TSKs的atp结合区域的ala取代突变体和三个磷酸化位点进行了体外互补分析。利用Phos-tag SDS-PAGE测定这些激酶的磷酸化谱显示，ArcB、EvgS和BarA的三步磷酸化转移反应顺序模式不同：分别为顺式-反式-反式、顺式-顺式-顺式和反式-反式。包含反式模式与形成同型二聚体的需要是一致的；《绝地求生》的所有步骤都具有顺式模式，这一点非常有趣。因此，Phos-tag SDS-PAGE提供了一种简单的方法来识别特定激酶的独特和特异性磷酸转移模式，而无需考虑复杂的细胞内因素。
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.