Distinct phosphorylation events of the catalytic subunit of protein kinase A in

V.RECOUVREUX, V.TUDISCA, S.MORENO Y P.PORTELA

Rosario, Santa Fe, Argentina

Congreso; 42 Reunion Anual SAIB; 2006

SAIB

catalytic subunits are regulated by multiple phosphorylations both in the activation loop and at allosteric sites. In S. cerevisiae, the PKA has three TPK1, TPK2 and TPK3 genes encoding the catalytic subunit. Genetic and kinetic experiments indicate an intramolecular phosphorylation mechanism for the Tpk1 isoform during glucose stimulus. We used MALDI-TOF/TOF to identify which aminoacids are phosphorylated in Tpk1 post glucose stimulus. We have identified Ser 32, 43, 179 and 322 as in vivo phosphorylation sites. We constructed strains carrying an inactive version of Tpk1 (tpk1K116R) or a mutant tpk1S179A. In situ and in vitro kinase assays indicate that the phosphorylation in Ser179 contributes to catalytic activity. Immunological analysis showed that inactive Tpk1 is phosphorylated on Ser and Thr241, suggesting an additional intermolecular mechanism of phosphorylation by a heterologous kinase. Additionally, using pull-down assays and mass spectrometry we found that the holoenzyme can consist of different isoforms of catalytic subunit bound to a regulatory subunit dimer in vivo. These results collectively suggest that two phosphorylation pathways can operate on TPK1: i)Tpk1 itself through autophosphorylation in response to glucose stimulus and ii)Tpk1 through Ser/Thr241 phosphorylation by heterologous protein kinase.