STRUCTURALLY EXPOSED SITES AND INTERACTORS OF BCY1, THE REGULATORY SUBUNIT OF PROTEIN KINASE A FROM Saccharomyces cerevisiae, ANALYZED BY MASS SPECTROMETRY.

TOFOLÓN, ENZO A; MORENO S; VALACCO P; NEME TAUIL R; FERNÁNDEZ G; ROSSI S

Capital Federal

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Protein kinase A is an inactive tetramer formed by a dimer of regulatory subunit (R2) bound to two monomers of catalytic subunit (C). When two molecules of cAMP bind to each R, the affinity between R and C is decreased and active C subunit is released. In mammals, the N-terminus of R subunits (DD) is responsible for the homo-dimerization and for anchoring the AKAPs and thus localizes the signal transduction pathway. In yeast, PKA is also a tetramer; the N-terminus of Bcy1 (R subunit) is responsible for dimerization and for its own localization. No AKAPs have been described yet except for our own preliminary report.Our study was carried out by proteomic approaches. On one hand, the goal was to obtain information on exposed sites in Bcy1 to reconstruct/model the whole structure, studying three proteolytic bands derived from aged Bcy1 by MALDI TOF-TOF and nano-LC-ESI-Orbitrap (QExactive). On the other hand, to investigate the oligomeric structure of Bcy1 in vivo and search for in vivo interactors by overexpression in yeast of its dimerization/docking domain, 1-50 Bcy1 (DD), tagged with thioredoxin-His in its N-terminus and analysis of the pulled down proteins after Ni-agarose.The first approach indicates that Bcy1 has clear points of endogenous proteolysis that speak about its folding. The results of the other approach suggest tetramer formation in vivo because of the intact endogenous Bcy1 presence in the pulled down using overexpressed His-Tagged DD domain. Together with Bcy1 a good number of proteins were also pulled down. Some of these proteins were selected for future studies as putative Bcy1 interactors according to rigurous criteria.In conclusion, we have obtained information regarding the apparently exposed/labile sites of Bcy1 and the potential Bcy1 interactors, and demonstrate the tetramer formation in vivo.