IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
STRUCTURALLY EXPOSED SITES AND INTERACTORS OF BCY1, THE REGULATORY SUBUNIT OF PROTEIN KINASE A FROM Saccharomyces cerevisiae, ANALYZED BY MASS SPECTROMETRY
Autor/es:
FERNANDEZ JORGE GERMAN; ENZO TOFOLON; MORENO SILVIA; ROSSI SILVIA; NEME TAUIL, RICARDO; MARIA PIA VALACCO
Lugar:
Buenos Aires
Reunión:
Congreso; Reunion conjunta de Biociencias y 53 reunion anual SAIB; 2018
Institución organizadora:
SAIB y otras 9 sociedades de Biociencia
Resumen:
Protein kinase A is an inactive tetramer formed by a dimer of regulatorysubunit (R2) bound to two monomers of catalytic subunit (C).When two molecules of cAMP bind to each R, the affinity betweenR 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 thesignal transduction pathway. In yeast, PKA is also a tetramer; theN-terminus of Bcy1 (R subunit) is responsible for dimerization andfor its own localization. No AKAPs have been described yet exceptfor 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 bandsderived from aged Bcy1 by MALDI TOF-TOF and nano-LC-ESI-Orbitrap (QExactive). On the other hand, to investigate the oligomericof structure Bcy1 in vivo and search for in vivo interactors by overexpressionin yeast of its dimerization/docking domain, 1-50 Bcy1(DD), tagged with thioredoxin-His in its N-terminus and analysis ofthe pulled down proteins after Ni-agarose.The first approach indicates that Bcy1 has clear points of endogenousproteolysis that speak about its folding.The results of the other approach suggest tetramer formation invivo because of the intact endogenous Bcy1 presence in the pulleddown using overexpressed His-Tagged DD domain. Together withBcy1 a good number of proteins were also pulled down. Some ofthese proteins were selected for future studies as putative Bcy1 interactorsaccording to rigurous criteria.In conclusion, we have obtained information regarding the apparentlyexposed/labile sites of Bcy1 and the potential Bcy1 interactors,and demonstrate the tetramer formation in vivo.Keywords: PKA, Bcy1, mass spectrometry, interactors, proteomics