Structural aspects of Bcy1, the regulatory subunit of protein kinase A from Saccharomyces cerevisiae approached by proteomic methods

JORGE GERMÁN FERNÁNDEZ; ENZO TOFOLON; SILVIA ROSSI; MARIA PIA VALACCO; RICARDO MARTIN NEME TAUIL; SILVIA MORENO DE COLONNA

Campinas

Workshop; Sao Paulo School of Advanced Science on Mass Spectrometry-based Proteomics; 2017

Brazilian Bioscience National Laboratory

Protein kinase A is an inactive tetramer formed by a dimer of regulatory subunit ( R) 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 dimerization and for anchoring to AKAPs and thus localize 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 (a). We have crystallized two domains of Bcy1: its carboxi-terminus (b) and its first 50 aa., which proved to be a dimer of dimers both in crystal (in prepration) and in solution (SAXS) (c). Three of our recent objectives have been approached by proteomic methods: 1) obtain information on exposed sites in Bcy1 to reconstruct/model the whole structure; 2) inquire whether Bcy1 can also form a tetramer in vivo; 3) search for in vivo interactors of Bcy1.The approaches used are: 1) study of three proteolytic bands derived from aged Bcy1 by MALDI TOF TOF and by nano-LC-ESI-Orbitrap (QExactive); 2 and 3) overexpression in yeast of 1-50 Bcy1 (DD), tagged with thioredoxin-His in its N-terminus and analysis of the pulled down proteins after Ni-agarose* .The results of (1) indicate that Bcy1 has clear points of endogenous proteolysis that speak about its folding, schematized in Fig 1. The results of (2) show that the overexpressed His-tagged DD domain pulled down intact endogenous Bcy1, suggesting tetramer formation in vivo. Together with Bcy1 a good number of proteins were also pulled-down (3). A subset were selected for future studies as putative Bcy1 interactors according to the following criteria: 1) identification with at least one high quality peptide in at least 3/4 independent replicates; 2) absence in control experiments; 3) absence from CRAPOME, 4) absence of Zn fingers. The list of hits was contrasted to those present in APID and SGD databases; two interactors were present in the databases as physical or genetic interactors.