Premium
Regulation of PKA catalytic activity by phosphorylation at multiple sites during the fermentative‐respiratory transition in Saccharomyces cerevisiae
Author(s) -
Moreno Silvia,
Tudisca Vanesa,
Portela Paula
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.317
Subject(s) - phosphorylation , autophosphorylation , serine , kinase , saccharomyces cerevisiae , protein subunit , biochemistry , microbiology and biotechnology , chemistry , biology , yeast , protein kinase a , gene
In the AGC family of serine/threonine kinases, the activities of catalytic subunits are regulated by multiple phosphorylation both in the activation loop and at allosteric sites. In yeast, PKA has three catalytic subunits, TPK1, TPK2 and TPK3. We describe a transient change in the phosphorylation state of Tpk1p during in vivo cAMP pathway activation evoked by glucose addition to glycerol‐growing cells. Phosphorylation of Tpk1 increases its Ksp toward kemptide. Genetic and in vitro kinetic experiments indicate an intramolecular phosphorylation mechanism for Tpk1 during glucose stimulus. PDK1 controls several protein kinases through phosphorylation on a conserved Thr in the activation loop. S.cerevisiae contains three PDK1 homologues: Pkh1, Pkh2 and Pkh3. We show that Pkh1 is capable of phosphorylating Thr241 of Tpk1 in vivo permitting the interaction with the regulatory subunit Bcy1. Using pkhD398G, pkh2 delta mutant we studied the effect of inactivation of Pkh activity over several phenotypes controlled by Tpk1. These experiments point to the Pkh kinase as an upstream negative regulator of the cAMP‐PKA pathway in yeast. The results suggest at least two phosphorylation pathways operating on TPK1: i) Tpk1 itself through autophosphorylation in response to glucose stimulus and ii) Tpk1 through Thr241 phosphorylation by Pkh. Research supported by University of Buenos Aires, CONICET and ANPCYT grants.