PROTEIN SUBSTRATES OF PKA IN SACCHAROMYCES CEREVISAE

GALELLO, F; ROSSI, S

Galello, F.; Rossi, Sosario, Santa Fe , Argentina

Congreso; SAIB 41st Annual Meeting XLII Reunión Anual -Argentine Society for Biochemistry and Molecular Biology- -Sociedad Argentina de Investigación Bioquímica y Biología Molecular-; 2006

SAIB

The PKA is the most well characterized member of the Ser/Thr protein kinase family. It was widely accepted that cAMP activates PKA however, recent experiments suggest that the substrate would play an important rol in the activation of the holenzyme.The effectiveness of protein phosphorylation by PKA is belived to depend on the primary structure of the protein around the phosphorylation site.Several PKA substrates have been described but is needed to demonstrate that the candidates proteins identified are indeed PKA substrates. Among all the yeast ORF which have a consensus RRXS sequence of 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 and compared with kemptide. Although all of them present the canonical RRXS,only three of them were substrates.Small differences in peptide sequences resulted in significative Kesp difference.The precense of basic residues in -5 position seems to be important for the phosphorylation.The specificity determinants that contribute to the recognition of substrates by PKA described until now is R at the p-6, p-3 and p-2 position. The phosphorylation behaviour of the proteins were compared with the peptides