A novel activating effect of the regulatory subunit of protein kinase A on catalytic subunit activity

The strength of the interaction between the catalytic and regulatory subunits in protein kinase A differs among species. The linker region from regulatory subunits is non-conserved. To evaluate the participation of this region in the interaction with the catalytic subunit, we have assayed its effect on the enzymatic properties of the catalytic subunit. Protein kinase A from three fungi, Mucor rouxii, Mucor circinelloides and Saccharomyces cerevisiae have been chosen as models. The R-C interaction is explored by using synthetic peptides of 8, 18 and 47 amino acids, corresponding to the R subunit autophosphorylation site plus a variable region toward the N terminus (0, 10, or 39 residues). The K_m of the catalytic subunits decreased with the length of the peptide, while the V_max increased. Viscosity studies identified product release as the rate limiting step for phosphorylation of the longer peptides. Pseudosubstrate derivatives of the 18 residue peptides did not display a competitive inhibition behavior toward either kemptide or a bona fide protein substrate since, at low relative pseudosubstrate/substrate concentration, stimulation of kemptide or protein substrate phosphorylation was observed. The behavior was mimicked by intact R. We conclude that in addition to its negative regulatory role, the R subunit stimulates C activity via distal interactions.