STUDY OF THE INTERACTION BETWEEN THE R AND C SUBUNITS OF THE PROTEIN KINASE A IN THE FUNGUS MUCOR CIRCINELLOIDES

OCAMPO J; MORENO S; ROSSI S.

Pinamar, Buenos Aires

Congreso; SAIB 41st Annual Meeting XLI Reunión Anual -Argentine Society for Biochemistry and Molecular Biology- -Sociedad Argentina de Investigación Bioquímica y Biología Molecular-; 2005

SAIB-SAN

Protein kinase A is a tetramer composed of a dimer of regulatory subunits (R) and two monomeric catalytic subunits (C). In the holoenzyme C interacts with the pseudosubstrate site in R. This pseudosubstrate site and additional contact sites in R render an affinity in the nanomolar range. The affinities between R and C subunits of PKA are quite different depending on the specie, finding the highest values in M.rouxii and M.circinelloides. Our hypothesis is that in the amino-termini region of R subunits exist elements that interact with C subunits that are responsible for the different affinity. The wild type R subunit and two mutant, R1 laking the N-terminal and R2 lacking a region of acidic residues were cloned and expressed in a S.cerevisiae strain tpk2- tpk3- bcy1- (133). Interaction between wtR or mutants R, and homologous or heterologous C subunit was evaluated by in vivo and in vitro assays. In vivo assays were made in yeast analyzing differences in phenotypes and growth between the different transformants. In vitro assays were made assaying the inhibition of C homologous or heterologous by each R subunit and dissociation of holoenzymes reconstituted. The IC50 for wtR was lower than those for mutant Rs. Dissociation of reconstituted holoenzyme showed that the concentration of cAMP required to dissociate the wtR-C holoenzyme was higher than the concentration requeried to dissociate the mutant R-C holoenzyme These results indicates that the acidic residues region may be important in the high affinity interaction RC found in M.rouxii and M.circinelloides PKA.