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

Mar del Plata

Congreso; Sociedad Argentina de Investigación Bioquimica y Biología Molecular; 2005

SAIB

Protein kinase A is a tetramer composed of a dimer of regulatory subunits (R) and two monomeric catalyticsubunits (C). In the holoenzyme C interacts with the pseudosubstrate site in R. This pseudosubstrate site andadditional contact sites in R render an affinity in the nanomolar range. The affinities between R and C subunits ofPKA 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 thatare responsible for the different affinity. The wild type R subunit and two mutant R, R1 laking the N-terminaland R2 lacking a region of acidic residues were cloned and expressed in a S.cerevisiae strain tpk2- tpk3- bcy1-(133). Interaction between wt R or mutants R, and homologous or heterologous C subunit was evaluated by invivo and in vitro assays. In vivo assays were made in yeast analyzing differences in phenotypes and growthbetween the different transformants. In vitro assays were made assaying the inhibition of C homologous orheterologous by each R subunit and dissociation of holoenzymes reconstituted. The IC50 for wtR was lower thanthose for mutant Rs. Dissociation of reconstituted holoenzyme showed that the concentration of cAMP requiredto dissociate the wtR-C holoenzyme was higher than the concentration requeried to dissociate the mutant R-Choloenzyme These results indicates that the acidic residues region may be important in the high affinityinteraction R-C found in M.rouxii and M.circinelloides PKA