In vivo and in vitro analysis of RegA response regulator mutants of Rhodobacter capsulatus.

In the facultative photosynthetic bacterium Rhodobacter capsulatus, the transcription of genes encoding pigment binding proteins is tightly regulated in response to the oxygen partial pressure by the RegB/ RegA two component system. After a shift from high to low oxygen tension, the response regulator RegA enhances transcription of the puf and puc operon coding for the reaction center, light-harvesting complex I (LHI), and LHII proteins. Various regA mutant strains were analyzed in this study. In a RegA deficient strain, activation of puf and puc transcription is severely impaired which consequently leads to the synthesis of only a few photosynthetic complexes. Strains carrying a mutation in the helix-turn-helix domain of RegA or a mutation of the phosphorylation site, Asp63, show a phenotype like the RegA deficient mutant, although the RegA(D63K) mutant protein showed the same DNA binding behavior as the wild type protein. In contrast, the puf and puc mRNAs still reach about 50-70 % of the wild type level after reduction of oxygen tension in strains which synthesize the C-terminal RegA activator domain only or a hybrid protein composed of the RegA activator and the FixJ receiver domain, while both mutant proteins are impaired in DNA binding. Our data suggest that phosphorylation is not required for DNA binding but rather plays a role for efficient initiation of transcription.

T cell antigen receptor dependent signalling in human lymphocytes: cholera toxin inhibits interleukin-2 receptor expression but not interleukin-2 synthesis by preventing activation of a protein kinase C isotype, PKC-alpha.

Activation and translocation of protein kinases C is a key event in the regulation of T lymphocyte activation, proliferation and function. Stimulation of human peripheral blood lymphocytes with the monoclonal antibody BMA 031 raised against the T cell antigen receptor led to a bimodal activation of protein kinases C. The immediate activation and translocation of the protein kinase C isoform PKC-alpha was followed by activation and translocation of the protein kinase C-beta isoenzyme after 90 min of stimulation. Pretreatment of the cells with cholera toxin for 90 min completely abolished activation of protein kinase C-alpha. In sharp contrast, activation and translocation of protein kinase C-beta was not influenced by the bacterial toxin, suggesting that activation and translocation of different protein kinase C isoenzymes are regulated by distinct mechanisms of transmembrane signalling coupled to the T cell antigen receptor/CD3 complex. The expression of high affinity IL-2 receptors was completely inhibited by cholera toxin, while IL-2 synthesis and secretion were not influenced in BMA 031-stimulated human lymphocytes. Extensive control experiments have shown that the effects of cholera toxin were not mediated by its B subunit, and were independent of elevation of intracellular cAMP concentration, suggesting that cholera toxin interfered with a signalling pathway leading to activation of protein kinase C-alpha, which could be responsible for the inhibition of IL-2 receptor expression. This hypothesis was substantiated by the finding that upon introduction of antibodies against protein kinase C-alpha, IL-2 receptor gene expression was completely suppressed. The results suggest, that protein kinase C-alpha might be the major protein kinase C isoenzyme of a signal transduction cascade regulating IL-2 receptor expression in stimulated human lymphocytes.