?Activation of PKA by differents substrates from Saccharomyces cerevisiae?

FIORELLA GALELLO; CONSTANZA PAUTASSO; SILVIA MORENO; SILVIA ROSSI

Mar del Plata, Bs As, Argentina

Congreso; XLIII Reunión Annual SAIB; 2007

SAIB

The effectiveness of protein phosphorylation by kinases is believed to depend on the primary structure of the protein around the phosphorylation site. Protein kinase A is a cAMP dependent kinase and it is widely accepted that the cyclic nucleotide activates the kinase. However recent experiments suggest that the substrate would play an important role in the activation of the holoenzyme. Among all the yeast ORF which have a consensus RRXS sequence for PKA phosphorylation, we chose 10 and probed their phosphorylation in vitro by yeast PKA. Only three of them were effectively phosphorylated: Pyk1, Pyk2 and Nth1. Synthetic peptides including the consensus phosphorylation sequences from these proteins were phosphorylated in vitro. Although all of them present the canonical RRXS, only three of them were substrates. Small differences in peptide sequences resulted in important Km and Vmax differences. The substrate role in the activation of the holoenzyme was investigated. Holoenzyme PKA was purified and its activation in presence of different cAMP amounts and different peptides was assayed. The cAMP A0.5 was different for each substrate, and different with the substrate concentration, indicating that the substrate primary structure plays an important role in the activation mechanism. The activation of PKA was also different when the activation assays were made with Pyk1 whole protein.