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

MORENO, SILVIA; GALELLO, FIORELLA, RINALDI, JIMENA, GONZÁLEZ-BARDECI, NICOLÁS, PEREYRA, ELBA, ROSSI, SILVIA

Puerto Madryn, Argentina

Congreso; XLVI SAIB Reunión Anual; 2010

SAIB Sociedad Argentina de Bioquímica y Biología Molecular

@font-face { font-family: "Times"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0cm 0cm 0.0001pt; font-size: 12pt; font-family: "Times New Roman"; }div.Section1 { page: Section1; } 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 subfillum 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 has shown to interact with several proteins; from these some were selected making theoretical prediction of putative AKAPs domains. Determinants and strength of the interaction with Bcy1 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 affiity interaction, making a difference with mammalian AKAPs.