Studies on activation and phosphorylation of protein kinase A during the transition from respiratory to fermentative metabolism in S.cerevisiae

MORENO, S AND PORTELA, S.

Rio de Janeiro, Brasil

Congreso; 11th International Congress on Yeasts; 2004

 In the unicellular eukaryote Saccharomyces cerevisiae, the cAMP-dependent protein kinase (PKA) is responsive to fermentable carbon sources. The addition of glucose to cells growing on a non-fermentable carbon source results in a transient increase in the cAMP levels. The objective of our group is to study the mechanism of activation of PKA. Kinase activity was measured in vivo using permeabilized yeast cells, after addition of glucose to cells growing on glycerol.    In wild type strains, in vivo PKA activity followed in time the peak of cAMP showing low or non-detectable values, depending on the genetic background. The in vivo PKA activity measured in the presence of 10 mM cAMP, always showed a peak of activity correlating with the cAMP peak. The level of cAMP intracellularly bound to regulatory subunit followed the course of PKA activation. Western blots for regulatory and catalytic subunits and their corresponding activities, assayed in partially purified samples, showed constant levels of both subunits. These results suggest a different state of activability of PKA within the cells, as a consequence of binding of cAMP along the transient rise of cAMP triggered by glucose addition. The relative proportion of phosphorylated species of the catalytic subunit (TPK1), changed during the transition from non-fermentable to fermentable carbon source, in a peak of cAMP and PKA activity-dependent manner. The phosphorylation state of TPK1 was found carbon source dependent: glycerol-grown cells have less phosphorylated isoforms than glucose-grown cells. Preliminary kinetic analysis of crude extracts, carrying phosphorylated isoforms before and after phosphatase treatment showed that the phosphorylation of TPK1 decreases the Km for kemptide. A preliminar speculation from these results is that the phosphorylation state of the catalytic subunit of PKA after the transient peak of cAMP, might increase its affinity toward target substrates, thus shifting the post translational modification of those substrates toward their phosphorylated forms, necessary to sustain the fermentative metabolism triggered upon glucose addition.