Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [32P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction.A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases.Two-component systems are central elements of the bacterial signaling circuitry [1]. Signal transduction by these systems usually involves autophosphorylation of a histidine kinase on a conserved histidine residue and subsequent transfer of the phosphoryl group to a conserved aspartate on a receiver domain. Until recently, two-component systems had only been found in bacteria. In the past few years, genes coding for histidine kinase homologues and their corresponding receivers have also been discovered in eukaryotic organisms [for a review see 2]. Most of the corresponding eukaryotic gene products are part of a phosphoryl relay, which consists of a hybrid histidine kinase with a kinase and a receiver domain on the same polypeptide, a histidine phosphotransfer protein and a second receiver as part of a response regulator [3]. The function of eukaryotic two-component systems as histidine kinases was questio