Analysis of N – termini of fungal protein kinase A regulatory subunits predicts a D/D domain for dimerization and docking to anchoring proteins

NICOLAS GONZALEZ BARDECI; JIMENA RINALDI; SILVIA ROSSI; SILVIA MORENO

Salta

Congreso; 3rd LAPSM (Latinamerican Protein Society Meeting),; 2010

LAPSM

       Protein kinase A (PKA) regulatory (R) subunit from mammals has been widely studied from a structural point of view. The four known isoforms possess a well defined domain architecture consisting of two conserved tandem cAMP binding sites at C-terminus,  and a docking/dimerization domain (D/D) at N-terminus. This domain is responsible for the dimeric nature of R for its docking to A Kinase Anchoring Proteins (AKAPs). Both domains are connected by a variable linker region, which contains an inhibitory sequence for PKA catalytic subunit. Solution and crystal structures for the D/D of mammalian Rs have been obtained.        Structure of R subunits from organisms other than mammals is still poorly understood. In this work we analyze the N- terminal domain of fungal Rs. Multiple sequence alignment shows that most of the main determinants known for R dimerization in mammals are present at the N-terminus from many fungi, although with some unique characteristics. The existence of a D/D is therefore predicted for fungi. We concentrate on Saccharomycetales and Mucorales. Chemical crosslinking experiments show that BCY1, the R subunit from Saccharomyces cerevisiae, exists as a dimer, via its N - terminus. Surfaces for docking to putative AKAP proteins seem to be quite different from that of mammals, according to its secondary structure prediction and modelling of the N-terminus of BCY1 and of the four isoforms from the zygomycete Mucor circinelloides. A pull down experimento shows that the N–terminus of yeast R subunit interacts with the R binding domain of a mammalian AKAP