IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
DIMERIZATION AND DOCKING DOMAIN OF BCY1, THE REGULATORY SUBUNIT OF PKA FROM Saccharomyces cerevisiae.
Autor/es:
TOFOLON, E; GONZALEZ BARDECI, N; ROSSI, S; MORENO, S
Lugar:
Rosario, Santa Fe
Reunión:
Congreso; L Reunion SAIB; 2014
Institución organizadora:
SAIB
Resumen:
Protein kinases A (PKA) are formed by a dimer of regulatory subunit (R2), which binds cAMP, bound to two catalytic subunits (C). In mammals, the N-terminus of R2 is responsible for dimerization and for binding of PKA to AKAPs. Our model is the R (Bcy1) subunit of PKA from S. cerevisiae. Previously, we have reported that: a) a mutant of Bcy1 lacking the first 85 amino acids, no longer binds to specific interactors; b) a bacterial recombinant construct of Bcy1 (1-50) contains the dimerization domain (DD) which has been structurally characterized. Using the following techniques: in vitro crosslinking with EGS, size exclusion chromatography, sucrose gradient sedimentation and static light scattering, we demonstrated that the predominant form of the recombinant DD in solution is tetrameric. This result is in accordance with the packing of the DD in the crystal structure. We have expressed the DD construct in a yeast vector fused to a tag of thioredoxin, His tag, and enterokinase cleavage site; studied the overexpression conditions, as well as its purification. We compared the protein profiles of Ni-agarose bound WT versus WT+DD overexpressing extracts and demonstrated that DD binds a differential set of proteins, which are competed by incubation with a purified preparation of whole Bcy1. These results indicate that in Bcy1 (1-50) resides not only the dimerization domain but an interacting domain.