Stress responses of the unicellular cyanobacterium Synechocystis are fulfilled via a number of regulatory systems, namely, two-component systems as well as through negative supercoiling of the genome DNA. We have studied an involvement of serine/threonine protein kinases (STPK) in the cyanobacterium Synechocystis cold stress response. A search for the STPK mutants allowed us to determine four protein kinases, SpkB, SpkD, SpkE and SpkG, which could regulate transcription under the low temperature. According to a proteome analysis, SpkE significantly affects the protein pattern in Synechocystis. Functional activity of the recombinant SpkE was confirmed in in vitro phosphorylation assay with a use of a set of potential protein kinase substrates. It have been demonstrated that the basic proteins are preferable substrates for the recombinant protein kinase SpkE.