A proteomic approach to protein kinase A signalling in yeast

V.RECOUVREUX, V.TUDISCA, P.PORTELA Y S.MORENO

Pilar, Buenos Aires

Congreso; Primer Congreso Anual Iberoamericano de Proteómica,; 2007

LAHUPO

Our main interest is to understand the activation of protein kinase A (PKA) by cAMP within the cell, using Saccharomyces cerevisiae as a model system. PKA is a heterotetramer formed by two regulatory R subunits (BCY1) and two catalytic C subunits (TPK1, TPK2, TPK3). cAMP binding to the R subunit in the holoenzyme decreases the affinity between R and C, with the consequent activation of the C subunit. At present, several experimental data provide evidences for a specificity of signaling through the three PKA catalytic subunits, such as unique substrate profile recognition and subcellular localization. In order to analyze if the hybrid holoenzyme (a holoenzyme molecule constituted by a dimer of Bcy1 associated to Tpk1 and Tpk2 or Tpk3 catalytic subunit isoforms) could be formed in vivo we analyzed the kinase activity associated to a catalytic inactive version of Tpk1 fused to TAP epitope (tpk1dead –TAP). Tpk1-TAP or tpk1dead –TAP were purified by affinity chromatography with or without incubation with cAMP-NaCl. The complexes inmunoprecipitated were subjected to in vitro kinase activity measurements and MALDI-TOF-TOF analysis.   We observed kinase activity associated to tpk1dead –TAP only when Bcy1 was present into the complex. The analysis by MALDI-TOF-TOF of this complex showed that the proteins associated to tpk1dead –TAP where Bcy1 and Tpk2. These results suggest the presence of different catalytic subunits associated by BCY1 in the cell.