MOLECULAR MECHANISM OF PROTEIN KINASE A PHOSPHORYLATION DURING TRANSITlON FROM RESPIRATORY TO FERMENTATlVE METABOLISM IN SACCHAROMYCES CEREVISIAE

PUGLIESSI, M., MORENO, S. AND PORTELA, P

Pinamar, Buenos Aires

Congreso; 10 Reunion Internacional PABMB y XLI Reunion Nacional SAIB; 2005

PABMB y SAIB

In S. cerevisiae, the cAMP dependent protein kinase (PKA) has three partially redundant TPKI, TPK2 and TPK3 genes encoding the catalytic subunits. Glucose-dependent activation of PKA activity changes the phosphorylation State of its Tpkl. Strains carrying inactive Tpkl isoform (tpkIK116R or tpkIwJ) were transformed with plasmids expressing Tpk2, Tpk3 or TpkI-HA. A cAMP peak was triggered by glucose addition to glycerol-growing cells; during this peak we measured PKA activation in situ in permeabilized cells,PKA-dependent phenotypes and the phosphorylation state of tpkl, followed by native westem blot. The phosphorylation state ofthe inactive Tpkl molecule did not change upon PKA activation, remaining in a low phosphorylation state. The results suggest an intramolecular phosphorylation mechanism of Tpkl. Peptides were designed based on TpkI sequence in order to identify which aminoacid is phosphorylated in Tpkl. Eight peptides containing these sites were synthesized. In situ and in vitro assays using permeabilizedcells or purified TpklTAP respectively, indicate that only one peptide (LLRKSQRFP) was substrate for the kinase, suggesting that S 179 is a potential phosphorylation site in the whole Tpkl protein.