RESUMO
Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and "activity of bc1 complex" kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase - X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.
Assuntos
Synechocystis , Synechocystis/genética , Synechocystis/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fosforilação , Serina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
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.