“Proteins interacting with the regulatory subunit of PKA from Saccharomyces cerevisiae”

FIORELLA GALELLO; SILVIA MORENO; SILVIA ROSSI

Mar del Plata, Bs As, Argentina

Congreso; XLIII Reunión Annual SAIB; 2007

SAIB

The cAMP-dependent protein kinase (PKA) is activated by cellular events that induce the cAMP synthesis and is involved in many specific signalling transduction pathways. The specificity of PKA is determined not only by the molecular recognition of a specific peptide sequence surrounding the phosphorylated residue of a substrate, the substrate recruitment plays a critical role in determining substrate preference. PKA localization in subcellular compartments by anchoring proteins (AKAPs) contributes to this specificity. The amino terminus of regulatory subunits (BCY1) contains a dimerization/docking (D/D) domain that mediates the dimerization between two R subunits and the binding to AKAPs. Our aim is to identify and characterize yeast AKAPs to study PKA localization and to evaluate their participation in PKA activation. In silico analysis of BCY1 amino terminus predicts a classical D/D domain for this protein. We have isolated and identified putative anchoring proteins through MALDI-TOF-TOF using three strategies: Purification of BCY-TAP and analysis of associated proteins;purification of TPK1-TAP and dissociation of the bound complex containing BCY1 and associated proteins with cAMP and NaCl and further identification of proteins in the flow-through; purification of overexpressed BCY through cAMPagarose and analysis of  bound proteins