Noncatalytic domains outside the active cleft and regulation of the phosphorylating activity of protein kinase A

RINALDI, J., ROSSI, S. AND MORENO, S.

Iguazu, Misiones

Congreso; XL Reunion Anual de SAIB; 2004

SAIB

There are great differences among species in the affinity between regulatory (R) and catalytic (C) subunits of cAMP dependent protein kinase. These differences can not be explained by the present structural data. In order to analyse how the R subunit interacts with C beyond its autophosphorylation site we studied the effects of several peptides on the phosphorylating activity of Mucor rouxii and bovine heart C subunits. The peptide sequences (18 aa) correspond to the autophosphorylation site and the hydrophobic residue-rich region toward the amino-termini of M rouxii and S. cerevisiae R subunits. The non-phosphorylable peptides (Ala instead of Ser) have a dual effect on kemptide phosphorylation: not only do they inhibit C activity, probably by competition for the active site, but also activate. The phosphorylable peptides (P) have higher kt.\ and higher v MAX on bovine heart C, while VMA¡( is higher and kM IS lower on M rouxii when compared to kemptlde. AII parameters change in the same degree for both series, with exception of v k!AX on bovine heart C: it is twice higher for PMrouxii than for J'Scerevisiae. Peptides containing only the autophosphorylaton site (8aa) have a VMAX similar to kemptide for both series of peptide and both enzymes. On the other hand, preliminary results show that a peptide (10 aa) that corresponds only to the hydrophobic residue-rich region of M. rouxii produces activation on M. rouxii, when added simultaneously with kemptide, while .no effect is observed on bovine heart C. These results can be interpreted as noncatalytic domains outside the active cleft regulate the phosphorylating activity of C by the interaction with peptides derived from R sequence.