HIGH-THROUGHPUT AUTOPHOSPHORYLATION ASSAY FOR BACTERIAL PROTEIN HISTIDINE KINASES

Protein histidine kinases play a major role in bacteria as sensor comp onents in the so-called ''two-component'' systems involved in signal t ransduction, We describe a high throughput assay for these kinases usi ng CheA, the bacterial chemotaxis-regulating kinase from Escherichia c oli, The purpose of the assay was to monitor ATP-dependent autophospho rylation of the kinase resulting in the phosphorylation of the conserv ed histidine residue, Unlike most eukaryotic protein kinases, ''two-co mponent'' kinases are not known to phosphorylate histones and small pe ptide substrates, This limits the level of phosphorylation and consequ ently the signal generated in these assays. We, therefore, established the desirable reaction conditions first using the conventional method involving the radiolabeling of CheA by [gamma-P-32]ATP followed by ge l electrophoresis-based analysis. Next, we converted the assay to a hi gh throughput format in which CheA, autophosphorylated and radiolabele d with [gamma-P-33]ATP, was trapped in a filter via anionic or hydroph obic interaction using diethyl aminoethyl or nitrocellulose-based 96-w ell filter plates, respectively, Free [gamma-P-33]ATP was removed by w ashing the wells with high salt buffers, The dried plates were then an alyzed for radioactivity associated with the wells by scintillation co unting, Finally, we performed and validated the assay in a partially a utomated format using nitrocellulose-based filter plates.