A proteomic approach to protein kinase A signalling in yeast

MORENO, S.

Valencia, España

Congreso; 2nd Congress of the Spanish Proteomic Society and 1st Congress of the European Proteomics Association; 2007

SEPROT

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 an 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. Under non equilibrium conditions, in vitro, this leads to the dissociation of the inactive holoenzyme complex into R2 and active C. However, the dissociation in vivo is not so evident since the holoenzyme is highly concentrated and localized  Upon addition of glucose to yeast cells growing in glycerol a transient peak of cAMP is produced. We have followed activation of PKA using an in situ assay in permeabilized cells. In response to its activation the phosphorylation state of TPK1 is shifted to more phosphorylated isoforms. The phosphorylation sites were identified by mass spectrometry, using a strain contaning the TPK1 isoform TAP-tagged in its chromosomal copy. The phosphorylated TPK1 has a higher specific activity towards peptide substrates. The change in phosphorylation pattern is triggered by an intramolecular phosphorylation process. The phosphorylation pattern remains as long as there is glucose to ferment.  The purification of TPK1-TAP and further analysis by MALDI-TOF-TOF of the accompanying principal bands, uncovered the existence of hybrid holoenzymes composed of a dimer of R subunit bound to TPK1 and TPK2. The role of this hybrid enzyme is under study. The amino terminus of R subunits contains a dimerization/docking domain that mediates the homodimerization between two R subunits and the binding to AKAPs (A Kinase Anchoring Proteins). No anchoring proteins have been characterized yet in yeast. In silico analysis of the BCY1 amino terminus predicts a classical dimerization/docking domain for this protein. We have isolated and identified putative anchoring proteins through MALDI-TOF-TOF using three stategies: 1) Purification of BCY1-TAP and analysis of associated proteins; 2) Purification of TPK1-TAP and dissociation of the bound holoenzyme complex containing BCY1, TPK2, and associated proteins, with cAMP and NaCl and further identification of proteins in the flow-through; 3) Purification of overexpressed BCY1 (wild type and with an amino terminal deletion as control) through cAMP agarose, and analysis of the bound proteins.  The new proteomic approach has been possible due to the recent acquisition of an Ultraflex II MALDI-TOF-TOF spectrometer, and the creation of the first macromolecular mass spectrometry center in Argentina.