Profiling of protein thiophosphorylation by Phos-tag affinity electrophoresis: evaluation of adenosine 5'-O-(3-thiotriphosphate) as a phosphoryl donor in protein kinase reactions.

Adenosine 5'-O-(3-thiotriphosphate) (ATPγS) has been widely used as a phosphoryl donor to trace protein kinase activities. However, the question remains whether particular kinases accept ATPγS as readily as they accept natural ATP. We investigated the characteristics of several kinase reactions in the presence of ATPγS by using Phos-tag affinity electrophoresis. The Phos-tag gel permitted quantitative analysis of thiophosphorylated proteins produced by kinase reactions in vitro and it identified differences in the efficiencies of utilization of ATPγS and ATP in these reactions. Using the method, we evaluated the utility of ATPγS as a phosphoryl donor in studies on bacterial two-component systems. Histidine kinases accepted ATPγS as readily as they accepted ATP in autophosphorylation reactions. However, downstream phosphotransfer reactions with ATPγS were markedly slower than the corresponding reactions with ATP. In an analysis of the sluggish thiophosphate transfer, we found that detergent-denatured thiophosphorylated histidine kinases gradually hydrolyzed at the P-N bond, even at neutral pH, during incubation for 24 h, whereas the native form of the thiophosphorylated enzymes were much more stable. Profiling of protein thiophosphorylation by using Phos-tag affinity electrophoresis might provide new insights into the characteristics of various types of kinase reactions with ATPγS.

Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.

Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected].