Implications of phosphorylation on S179 of the C subunit isoform Tpk1 from yeast PKA

CLARA SOLARI; VANESA TUDISCA; SILVIA MORENO; PAULA PORTELA

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

SAIB

Many protein kinases are themselves phosphoproteins, and their biological function and activity are frequently regulated by phosphorylation. PKA regulatory subunit is encoded by the BCY1 gene, and the catalytic subunits are encoded by three genes: TPK1, TPK2 and TPK3. Previously we have reported that following cAMP‐PKA pathway activation, Tpk1 changes its phosphorylation status toward more phosphorylated isoforms by an intramolecular phosphorylation mechanism. Tpk1 increases its specific activity toward kemptide. Using mass spectrometry and array peptides derived from Tpk1 we identified mainly Ser179 as a putative target residue. Here we report the in vivo role of Ser179 phosphorylation of Tpk1 by analysis of several readouts of PKA. We have constructed strains containing Tpk1 wt or Tpk1S179A or Tpk1S179D as sole source of PKA activity. Our results show that Tpk1S179D strain is deficient on non‐fermentable carbon source growth, glycogen content, tolerance to heat stress, growth on rapamycin containing medium and shows a reduced life span on stationary phase. Tpk1‐GFP and Tpk1S179D‐GFP show a nucleo‐cytoplasmic distribution in glucose growing cells whereas Tpk1S179A ?GFP was localized in the nucleus. Our results suggest that the unphosphorylated state of Ser179 reduces the Tpk1 kinase activity improving the respiratory metabolism, survival to stress and life span of the yeast cells.