Regulation of PKA activity by an auto-phosphorylation mechanism in Saccharomyces cerevisiae.

SOLARI, CLARITA; TUDISCA VANESA; PUGLIESSI; MARCELO; NADRA ALEJANDRO; MORENO SILVIA; PAULA PORTELA

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Año: 2014

0264-6021

The cAMP dependent Protein Kinase activity, as well as other AGC members, is regulated by multiple phosphorylations both in the activation loop and at allosteric sites of its catalytic subunits. In Saccharomyces cerevisiae 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 increases its phosphorylation status. Now, in vivo genetic and in vitro experiments indicate an autophosphorylation mechanism for Tpk1. Using array peptides derived from Tpk1 we identify Ser179 as a target residue. Tpk1 is phosphorylated on Ser179 in vivo during glucose stimulus and after reduction of Thr241 residue activation loop phosphorylation. To evaluate the phosphorylation role on Ser179 we made strains expressing tpk1S179A or tpk1S179D as sole PKA kinase source. Our results suggest that Ser179 phosphorylation increases the reactivity toward substrate without affecting the formation of holoenzyme complex. Phenotypic readouts analysis shows that Ser179 phosphorylation increases in vivo PKA activity reducing cell survival to stress and life span. Ser179 phosphorylation increases Tpk1 cytoplasmic accumulation in glucose-growing cells. These results describe for the first time that the autophosphorylation mechanism on Tpk1 controls PKA activity in response to glucose availability.