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

MORENO, SILVIA; TOFOLON ENZO; VALACCO, PIA; NEME TAUIL, RICARDO; FERNANDEZ JORGE GERMAN; ROSSI, SILVIA

Potsdam

Congreso; Proteomic Forum 2017; 2017

Deutsche Gesellschaft für Proteomforschung

Proteinkinase A is an inactive tetramer formed by a dimer of regulatory subunit ( R) boundto two monomers of catalytic subunit (C). When two molecules of cAMP bind toeach 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 thedimerization and for anchoring to AKAPs and thus localize the signaltransduction pathway. In yeast PKA is also a tetramer; the N-terminus of Bcy1(R subunit) is responsible for dimerization and for its own localization. NoAKAPs have been described yet except for our own preliminary report (a). Wehave crystallized two domains of Bcy1: its carboxi-terminus (b) and its first50 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 beenapproached by proteomic methods: 1) obtain information on exposed sites in Bcy1to reconstruct/model the whole structure; 2) inquire whether Bcy1 can also forma tetramer in vivo; 3) search for in vivo interactors of Bcy1.Theapproaches used are: 1) study of three proteolytic bands derived from aged Bcy1by 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 itsN-terminus and analysis of the pulled down proteins after Ni-agarose* .The resultsof (1) indicate that Bcy1 has clear points of endogenous proteolysis that speakabout its folding, schematized in Fig 1. The results of (2) show that theoverexpressed His-tagged DD domain pulled down intact endogenous Bcy1, suggestingtetramer formation in vivo. Togetherwith Bcy1 a good number of proteins were also pulled-down (3). A subset wereselected for future studies as putative Bcy1 interactors according to thefollowing criteria: 1) identification with at least one high quality peptide inat least 3/4 independent replicates; 2) absence in control experiments; 3)absence from CRAPOME, 4) absence of Zn fingers. The list of hits was contrastedto those present in APID and SGD databases; two interactors were present in thedatabases as physical or genetic interactors. a- J Proteomics (2014) 23:109:261-75b- J Struct Biol(2016) 193:141-54c- Structure (2010)18:1471-82 Fig 1- Bcy1partial proteolysis visualized by SDS-PAGE and scheme of results with arrowspointing sites of proteolysis