Activation of PKA by substrates from Saccharomyces cerevisiae

GALELLO, FIORELLA; PAUTASSO, CONSTANZA; MORENO, SILVIA; ROSSI, SILVIA

San Diego, CA, Estados Unidos

Congreso; ASBMB Annual Meeting; 2008

The American Society for Biochemistry and Molecular Biology (ASBMB)

The effectiveness of protein phosphorylation by kinases is believed to depend on the primary structure of the protein around the phosphorylation site. Our aim is to assess the best consensus sequence for yeast Protein kinase A and the participation of the substrate in yeast holoenzyme activation. We used the proteins Pyk1, Pyk2 and Nth1, which have been described as PKA substrates in yeast and have consensus RRXS sequence for PKA phosphorylation. Five synthetic peptides including the consensus phosphorylation sequences from these proteins were phosphorylated in vitro. Three of them behaved as good substrates. Small differences in peptide sequences resulted in important Km and Vmax differences. Peptide array was designed to verify the residues responsible for these differences. The substrate role in the activation of holoenzyme was also investigated. Activity of purified PKA in presence of different concentrations of cAMP and different peptides was assayed. The cAMP A0.5 was different with each substrate and substrate concentration, indicating that the primary structure of the substrate plays an important role in the activation mechanism. The better the substrate the higher the activation. The activation of PKA was also different when the activation assays were made using the whole protein as substrate and compared with the peptide as substrate.