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

NICOLÁS GONZÁLEZ BARDECI, JIMENA RINALDI, SILVIA ROSSI, SILVIA MORENO

Salta, Argentina

Congreso; Latin American Protein Society Meeting; 2010

Latin American Protein Society

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 domain (D/D) at the N-terminus. This domain is responsible for the dimeric nature of R and 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 structure for the D/D of mammalian Rs have been obtained. Structure of Rs from organism other than mammals is still poorly understood. In this work we analyze the N-termini 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 of 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 cross-linking experiments show that BCY1, the R subunit of 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 modeling of the N-terminus of BCY1 and of the four isoforms from zygomycete Mucor circinelloides. Pull down experiments show that the N-terminus of yeast R subunit interacts with the R binding domain of mammalian AKAP.