Regulation of PKA activity by an autophosphorylation mechanism in Saccharomyces cerevisiae.

PKA (cAMP-dependent protein kinase) activity, as well as that of other AGC members, is regulated by multiple phosphorylations of its catalytic subunits. In Saccharomyces cerevisiae, the PKA regulatory subunit is encoded by the gene BCY1, 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 identified Ser179 as a target residue. Tpk1 is phosphorylated on Ser179 in vivo during glucose stimulus. Reduction of the activation loop Thr241 phosphorylation increases Ser179 autophosphorylation. To evaluate the role of phosphorylation on Ser179, we made strains expressing tpk1S179A or tpk1S179D as the sole PKA kinase source. Our results suggest that Ser179 phosphorylation increases the reactivity towards the substrate without affecting the formation of the holoenzyme. Phenotypic readout analysis showed that Ser179 phosphorylation increases in vivo PKA activity, reducing cell survival, stress and lifespan. Ser179 phosphorylation increases Tpk1 cytoplasmic accumulation in glucose-grown cells. These results describe for the first time that an autophosphorylation mechanism on Tpk1 controls PKA activity in response to glucose availability.

PKA isoforms coordinate mRNA fate during nutrient starvation.

A variety of stress conditions induce mRNA and protein aggregation into mRNA silencing foci, but the signalling pathways mediating these responses are still elusive. Previously we demonstrated that PKA catalytic isoforms Tpk2 and Tpk3 localise with processing and stress bodies in Saccharomyces cerevisiae. Here, we show that Tpk2 and Tpk3 are associated with translation initiation factors Pab1 and Rps3 in exponentially growing cells. Glucose starvation promotes the loss of interaction between Tpk and initiation factors followed by their accumulation into processing bodies. Analysis of mutants of the individual PKA isoform genes has revealed that the TPK3 or TPK2 deletion affects the capacity of the cells to form granules and arrest translation properly in response to glucose starvation or stationary phase. Moreover, we demonstrate that PKA controls Rpg1 and eIF4G(1) protein abundance, possibly controlling cap-dependent translation. Taken together, our data suggest that the PKA pathway coordinates multiple stages in the fate of mRNAs in association with nutritional environment and growth status of the cell.

The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae.

PDK1 (phosphoinositide-dependent protein kinase 1) phosphorylates and activates PKA (cAMP-dependent protein kinase) in vitro. Docking of the HM (hydrophobic motif) in the C-terminal tail of the PKA catalytic subunits on to the PIF (PDK1-interacting fragment) pocket of PDK1 is a critical step in this activation process. However, PDK1 regulation of PKA in vivo remains controversial. Saccharomyces cerevisiae contains three PKA catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh [PKB (protein kinase B)-activating kinase homologue] protein kinases phosphorylate the activation loop of each Tpk in vivo with various efficiencies. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of PKA. Comparative analysis of the in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through an HM-PIF pocket interaction. Unlike Tpk1, mutagenesis of the activation loop site in Tpk2 does not abolish in vitro phosphorylation, suggesting that Tpk2 contains other, as yet uncharacterized, Pkh1 target sites. Tpk3 is poorly phosphorylated on its activation loop site, and this is due to the weak interaction of Tpk3 with Pkh1 because of the atypical HM found in Tpk3. In conclusion, the results of the present study show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.