Study of the interaction between the R and C subunits of the protein kinase A in the fungus Mucor circinelloides

OCAMPO, J., MORENO, S. AND ROSSI, S.

Pinamar, Bs.As

Congreso; 10ma Reunion Internacional de PABMB y XLI Reunion Anual de SAIB; 2005

SAIB

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 ofPKA are quite different depending on the specie, finding the highest values in Mrouxií and Mcircinelloides. 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, Rl laking the N-terminal and R2 lacking a region of acidic residues were cloned and expressed in aS.cerevisiae strain tpk2´ tpk3´ bcyl´ (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 ofC homologous or heterologous by each R subunitand dissociation ofholoenzymes reconstituted. The IC50 for wtR was lower than those for mutant Rs. Dissociation of reconstituted holoenzyme showed that the concentration of cAMP required to di_sociate the wtR-C holoenzyme was higher than the concentration requeried to dissociate the mutant R-C holoenzyme These results indicatesthat the acidic residues region may be important in the high affinity interaction R­C found in Mrouxií and Mcircinelloides PKA.     .