PERSONAL DE APOYO
PAUTASSO Maria Constanza
congresos y reuniones científicas
Título:
ACTIVATION OF PKA BY DIFFERENT SUBSTRATES FROM SACCHAROMYCES CEREVISIAE
Autor/es:
GALELLO, FIORELLA; PAUTASSO, CONSTANZA; ROSSI, SILVIA
Lugar:
Mar del Plata, Buenos Aires
Reunión:
Congreso; XLIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007
Institución organizadora:
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Resumen:
It was widely accepted that cAMP activates PKA, however recent experiments suggest that the substrate would play an important role in the activation of the holenzyme. The effectiveness of protein phosphorylation by PKA is believed to depend on the primary structure of the protein around the phosphorylation site. Several PKA substrates have been described but it is necessary 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. Although all of them present the canonical RRXS, only three of them were substrates. Small differences in peptide sequences resulted in significative Km and Vmax differences. The role the substrate plays in the activation of the holoenzyme was investigated. Holoenzyme PKA was purified and its activation in presence of cAMP and different peptide substrate was assayed. The cAMP A0.5 was different for each substrate, indicating that the substrate primary structure plays an important role in the activation mechanism. The activation of PKA was also studied in presence of cAMP and Pyk1 whole protein.