In situ assay of protein kinase A activity as a mesure of the endogenous activation state

PAULA PORTELA; PATRICIA JACOBO; SILVIA MORENO

Villa Carlos Paz

Congreso; XXXVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2002

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Protein kinase A (PKA) activity was measured in situ in permeabilised Saccharomyces cerevisiae cells -/+ exogenously added cAMP. Four strains predicted to have differential PKA-dependent phenotypes were used. The measured phenotypes were: trehalose, glycogen, PKA-promoter dependent activity, and stationary phase survival. The endogenous PKA activity (measured in situ in the absence of exogenous cAMP) showed the best correlation with the phenotypes and we proposed it is good estimate of in vivo PKA activity. To verify this proposal endogenous PKA activity was followed after the addition of glucose to cells growing on a non-fermentable carbon source, a physiological condition known to produce a transient peak of cAMP during the first 5 min. Endogenous PKA activity showed a transient peak, followed in time the peak of cAMP. Surprisingly, the PKA activity measured in the presence of 10 mM cAMP, also showed a peak of activity. Western blots for R and C subunits and their corresponding activities, assayed in crude extracts, showed constant levels of both subunits during the 15 min after glucose addition. These results suggest a different state of activability ok PKA within the cell along the transient rise of cAMP. An additional results is that the relative proportion of phosphorylated species of the PKA catalytic subunit (TPK1) assessed by differential mobility on native PAGE, changed during the peak of cAMP in a PKA activity-dependent manner.