Role of the N-terminal regulatory subunit of protein kinase A from fungi in dimerization and tethering to proteins

MORENO, SILVIA; GALELLO, FIORELLA; RINALDI, J; BARDECI, N; ROSSI, S

Oslo

Congreso; 3rd International Meeting on Anchored cAMP Signalling Pathways; 2010

Among fungi both ascomycetes and basidiomycetes have only one isoform of regulatory subunit (R) of PKA, while zygomycetes have four isoforms. A bioinformatic comparison of the divergent amino termini of the R subunits from fungi with those of mammals showed a conserved region with conserved characteristics of dimerization domains (D/D) both in primary and secondary structure. Although Neurospora crassa as other ascomycetes lacked this region and showed to have a monomeric R subunit. We studied in detail the N-terminus from Saccharomyces cerevisiae (ascomycete) and Mucor circinelloides (zygomycete).In the S.cerevisiae dimeric R (Bcy1) subunit the deletion of the D/D yielded a monomeric isoform.  A comparison of the D/D region of  Bcy1 and the four R subunits from M. circinelloides with the D/D domains of RI and RII predicts possible interacting surfaces for these subunits with other proteins. By pull-down assay Bcy1 was shown to interact with D-AKAP2. The addition of the permeable version of Ht-31 to M.circinelloides growth medium, had significant phenotypic consequences. Possible interactors of Bcy1 were selected making theoretical predictions of putative AKAPs from the yeast genome (in collaboration with W. from Taylor’s lab).  Determinants and strength of the interactions with Bcy-1  and the role of N-terminal in these interactions were assayed by peptide array