In cells of the yeast Sacchammyces cerevisiae, trehalase activation, repression of CTTl (catalase), SSA3 (Hsp7O) and other STRE-controlled genes, feedback inhibition of CAMP synthesis and to some extent induction of ribosomal protein genes is controlled by the Ras-adenylate cyclase pathway and by the fermentable-growth-medium-induced pathway (FGM pathway). When derepressed cells are shifted from a non-fermentable carbon source to glucose, the Ras-adenylate cyclase pathway is transiently activated while the FGM pathway triggers a more lasting activation of the same targets when the cells become glucose-repressed. Activation of the FGM pathway is not mediated by CAMP but requires catalytic activity of CAMP-dependent protein kinase (cAPK; Tpkl, 2 or 3). This study shows that elimination of Sch9, a protein kinase with homology to the catalytic subunits of cAPK, affects all target systems in derepressed cells in a way consistent with higher activity of cAPK in wiwo. In witm measurements with trehalase and kemptide as substrates confirmed that elimination of Sch9 enhances cAPK activity about two-to threefold, in both the absence and presence of CAMP. In wiwo it similarly affected the basal and final level but not the extent of the glucose-induced responses in derepressed cells. The reduction in growth rate caused by deletion of SCHS is unlikely to be responsible for the increase in cAPK activity since reduction of growth rate generally leads to lower cAPK activity in yeast. On the other hand, deletion of SCHS abolished the responses of the protein kinase A targets in glucoserepressed cells. Re-addition of nitrogen to cells starved for nitrogen in the presence of glucose failed to trigger activation of trehalase, caused strongly reduced and aberrant repression of m l and SSA3, and failed to induce the upshift in RPL25 expression. From these results three conclusions can be drawn: (1) Sch9 either directly or indirectly reduces the activity of protein kinase A; (2) Sch9 is not required for glucose-induced activation of the Rasadenylate cyclase pathway; and (3) Sch9 is required for nitrogen-induced activation of the FGM pathway. The latter indicates that Sch9 might be the target of the FGM pathway rather than cAPK itself.
Addition of a nitrogen-source to glucose-repressed, nitrogen-starved G0 cells of the yeast Saccharomyces cerevisiae in the presence of a fermentable carbon source induces growth and causes within a few minutes a five-fold, protein-synthesis-independent increase in the activity of trehalase. Nitrogen-activated trehalase could be deactivated in vitro by alkaline phosphatase treatment, supporting the idea that the activation is triggered by phosphorylation. Yeast strains containing only one of the three TPK genes (which encode the catalytic subunit of cAMP-dependent protein kinase) showed different degrees of nitrogen-induced trehalase activation. The order of effectiveness was different from that previously reported for glucose-induced activation of trehalase in glucose-depressed yeast cells. Further reduction of TPK-encoded catalytic subunit activity by partially inactivating point mutations in the remaining TPK gene further diminished nitrogen-induced trehalase activation, while deletion of the BCY1 gene (which encodes the regulatory subunit) in the same strains resulted in an increase in the extent of activation. Deletion of the RAS genes in such a tpkw1 bcy1 strain had no effect. These results are consistent with mediation of nitrogen-induced trehalase activation by the free catalytic subunits alone. They support our previous conclusion that cAMP does not act as second messenger in this nitrogen-induced activation process and our suggestion that a novel nitrogen-induced signaling pathway integrates with the cAMP pathway at the level of the free catalytic subunits of protein kinase A. Western blot experiments showed that the differences in the extent of trehalase activation were not due to differences in trehalase expression. On the other hand, we cannot completely exclude that protein kinase A influences the nitrogen-induced activation mechanism itself rather than acting directly on trehalase. However, any such alternative explanation requires the existence of an additional, yet unknown, mechanism for activation of trehalase besides the well-established regulation by protein kinase A.
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