ACTIVATION OF PKA BY DIFFERENT SUBSTRATES FROM SACCHAROMYCES CEREVISIAE

GALLELO FIORELLA; MORENO SILVIA; ROSSI SILVIA

Mar del Plata, Buenos Aires

Congreso; 43 Reunion Anual 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 the kinase cAMP dependent and it is widely accepted that the cyclic nucleotide activates the kinase. However recent experiments suggest that the substrate plays an important role in the activation of the holenzyme. 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 were different for each substrate, and different with the substrate concentration indicating that the substrate primary sequence 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.