Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE.
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 ph...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4734776?pdf=render |
| _version_ | 1818564843812159488 |
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| author | Emiko Kinoshita-Kikuta Eiji Kinoshita Yoko Eguchi Tohru Koike |
| author_facet | Emiko Kinoshita-Kikuta Eiji Kinoshita Yoko Eguchi Tohru Koike |
| author_sort | Emiko Kinoshita-Kikuta |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T01:33:50Z |
| format | Article |
| id | doaj.art-92b61a9335324a6299ff8f07138a76d5 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-14T01:33:50Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-92b61a9335324a6299ff8f07138a76d52022-12-21T23:21:57ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01112e014829410.1371/journal.pone.0148294Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE.Emiko Kinoshita-KikutaEiji KinoshitaYoko EguchiTohru KoikeTripartite 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.http://europepmc.org/articles/PMC4734776?pdf=render |
| spellingShingle | Emiko Kinoshita-Kikuta Eiji Kinoshita Yoko Eguchi Tohru Koike Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE. PLoS ONE |
| title | Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE. |
| title_full | Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE. |
| title_fullStr | Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE. |
| title_full_unstemmed | Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE. |
| title_short | Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE. |
| title_sort | validation of cis and trans modes in multistep phosphotransfer signaling of bacterial tripartite sensor kinases by using phos tag sds page |
| url | http://europepmc.org/articles/PMC4734776?pdf=render |
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