PROTEINS INTERACTING WITH REGULATORY SUBUNIT OF PKA FROM SACCHAROMYCES CEREVISIAE

F.GALELLO, S.MORENO Y S.ROSSI

Pilar Buenos Aires

Congreso; Primer Congreso Anual Iberoamericano de Proteómica; 2007

LAHUPO

PKA is a heterotetramer formed by two regulatory R subunits (BCY1) and two catalytic C subunits (TPK1, TPK2, TPK3). 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. To ensure signalling fidelity, PKA must be specific and act only on a defined subset of cellular targets. In yeast few PKA substrates have been defined. 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 R subunits contains a dimerization/docking domain that mediates the homodimerization between two R subunits and the binding to AKAPs. No anchoring proteins have been characterized yet in yeast. 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 dimerization/docking domain for this protein.We have isolated and identified putative anchoring proteins through MALDI-TOF-TOF using three strategies: 1) Purification of BCY-TAP and analysis of associated proteins, 2) 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; 3) Purification of overexpressed BCY through cAMP-agarose and analysis of  bound proteins. To evaluate if the identified proteins were bound to the holoenzyme through TPK1 or BCY1, the approach 3 was performed using strains which have deleted two of the three isoforms of catalytic subunits and the associated proteins were analyzed.