N-terminal domain of the regulatory subunit of PKA from fungi in dimerization and tethering

SILVIA MORENO; FIORELLA GALELLO; NICOLÁS GONZÁLEZ BARDECI; JIMENA RINALDI; SILVIA ROSSI

Puerto Madryn

Congreso; XLVI Reunion Nacional de SAIB; 2010

SAIB

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 characteristics of dimerization domains (D/D) both in primary and secondary structure. A subphyllum of ascomycetes, exemplified by Neurospora crassa, 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 and mass spectrometry, Bcy1 was shown to interact with several proteins; from these some were selected making theoretical predictions of putative AKAPs domains.  Determinants and strength of the interactions with Bcy-1 and the role of the N-terminus in these interactions were assayed by peptide array. The best interactors were Ira2 and Hsp60, being positive charges necessary for high affinity interaction, marking a difference with mammalian AKAPs.