engleski

Can bacterial Hanks-type kinases constitute signal transduction cascades?

Signal transduction in bacteria most often includes phosphorylation of histidine and aspartate residues, within relatively simple two-component systems. Some bacteria also possess serine/threonine-specific protein kinases, which belong to a large superfamily of Hanks-type kinases, together with the homologous eukaryotic enzymes. Aim of this research was to find out whether bacterial Hanks-type kinases constitute a phosphorylation cascade. Signal transduction through protein kinase cascades enables amplification of the signal and integration of different signalling pathways. This type of signal transduction is well studied and an important phenomenon in eukaryotes, where it regulates various cellular processes, but it hasn’t been associated with bacteria. A model Gram-positive bacterium Bacillus subtilis possesses a few Hanks-type kinases, two of them being membrane proteins. To determine whether these kinases phosphorylate each other, it was necessary to abolish their autophosphorylation activity. This was accomplished by site-directed mutagenesis of the active site. After expression and purification of recombinant proteins, in vitro phosphorylation assays with 32P-γ-ATP were performed. Wild-type and mutant forms of different kinases were combined. It was determined that both membrane-linked kinases, PrkC and YabT, phosphorylate a citosolic kinase YbdM. Also, intermolecular kinase activity of PrkC was confirmed. Structural resemblance of PrkC to eukaryotic receptor kinases and its ability to transphosphorylate, make this membrane kinase a good candidate for the first step of the newly characterized signal transduction cascade in bacteria.

Bacterial Hanks-type kinases; protein kinase cascade; protein phosphorylation; signal transduction

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