Fast detection of PKA activity in Saccharomyces cerevisiae cell population using AKAR fluorescence resonance energy transfer probes

Abstract

In Saccharomyces cerevisiae, the protein kinase A (PKA) plays a central role in the control of metabolism, stress resistance and cell cycle progression. In a previous work, we used a FRET-based A-kinase activity reporter (AKAR3 probe) to monitor changes in PKA activity in vivo in single S. cerevisiae cells. Since this procedure is quite complex and time-consuming, in this work we used the AKAR3 probe (evenly distributed within the cells) and the plate reader Victor-X3™ (Perkin Elmer®) to measure PKA activity in vivo in a whole cell population. We show that in wild type strains, the FRET increases after addition of glucose to glucose-starved cells, while no changes are observed when this sugar is added to strains with either absent or attenuated PKA activity. Moreover, using the pm-AKAR3 probe, mainly expressed at the plasma membrane and partially at the vacuolar membrane, we could monitor PKA activity from the starting site of the signal to internal regions, where the signal is propagated. Finally, we also show evidence for direct activation of PKA by glucose, independent of cAMP. In conclusion, our data show that AKAR3 and pm-AKAR3 probes are useful biosensors to monitor PKA activity in a S. cerevisiae cell population using a plate reader.

Keywords: Fluorescence resonance energy transfer; Protein kinase activity; Saccharomyces cerevisiae; Yeast; cAMP/PKA pathway.