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

CLARA ANDREA SOLARI; VANESA TUDISCA; MARCELO PUGLIESSI; ALEJANDRO NADRA; SILVIA MORENO; PAULA PORTELA

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 2014 vol. 462 p. 567 - 579

0264-6021

PKA (cAMP-dependent protein kinase) activity, as well as that ofother AGC members, is regulated by multiple phosphorylationsof its catalytic subunits. In Saccharomyces cerevisiae, the PKAregulatory subunit is encoded by the gene BCY1, and the catalyticsubunits are encoded by three genes: TPK1, TPK2 and TPK3.Previously,we have reported that, following cAMP/PKA pathwayactivation, Tpk1 increases its phosphorylation status. Now, in vivogenetic and in vitro experiments indicate an autophosphorylationmechanism for Tpk1. Using array peptides derived from Tpk1,we identified Ser179 as a target residue. Tpk1 is phosphorylated onSer179 in vivo during glucose stimulus. Reduction of the activationloop Thr241 phosphorylation increases Ser179 autophosphorylation.To evaluate the role of phosphorylation on Ser179, wemade strainsexpressing tpk1S179A or tpk1S179D as the sole PKA kinase source.Our results suggest that Ser179 phosphorylation increases thereactivity towards the substrate without affecting the formationof the holoenzyme. Phenotypic readout analysis showed thatSer179 phosphorylation increases in vivo PKA activity, reducingcell survival, stress and lifespan. Ser179 phosphorylation increasesTpk1 cytoplasmic accumulation in glucose-grown cells. Theseresults describe for the first time that an autophosphorylationmechanism on Tpk1 controls PKA activity in response to glucoseavailability.