Functional Characterization Of The High-affnity CAMP Phosphodiesterase, Pde2p, In Saccharomyces Cerevisiae

Throughout the eukaryotic kingdom cyclic AMP signaling is known to modulate a variety of cellular functions. The only way to inactivate cAMP is to degrade it through the action of cAMP phosphodiesterases (PDEs), which are thus poised to play a key regulatory role. This work deals with the localization and the protein level regulation of Pde2, the high-affinity phosphordiesterase in yeast Saccharomyces cerevisiae. The results indicate an important role of Pde2 in feedback inhibition of the cAMP-PKA pathway through the regulation of PKA. They may also indicate a role of Pde2 in cross-talks between cAMP-PKA pathway and other pathways involved in nutrients availability, stress responses, growth and longevity of the cell.In PKA-attenuated strain, Pde2 was evenly distributed over the nucleus and the cytoplasm. In contrast, in strains with elevated PKA activity it was more concentrated in the nucleus than in wild-type strain independent on the carbon source. To explore the physiological role of PKA-regulated Pde2 localization, the localization pattern of Pde2 was studied with that of PKA. Pde2, localized in the same compartment with Bcy1 (the regulatory subunit of PKA), may down-regulate cAMP signaling more efficiently by differential degradation of cAMP, thereby in infavor of the reassociation of Bcy1 with Tpk which inhibits activation of the latter. The PKA-regulated Pde2 localization indicates a new feedback inhibition mechanism to down-regulate the cAMP signaling pathway.Experimental data of this work also demonstrated a role of PKA in regulating the protein level of Pde2. Activated PKA could exert its positive effect on Pde2 protein level through one or more post-transcriptional mechanisms, including mRNA stability, translational efficacy, or protein degradation. The positive regulation could also be transcriptional. In the situation of lower PKA activity, regulation is mainly exerted on mRNA level. Howerver, in the situation of higher PKA activity, it is through promotion of translational or/and inhibition of protein degradation. The positive correlation of PKA activity with Pde2 protein level is in consistent with the fact that high PKA activity in some way is required for efficient breakdown of cAMP by the phosphodiesterases, and thus indicates another mechanism of PKA-regulated feedback inhibition on cAMP accumulation through cAMP degradation.In this work, we also investigated the effect of stop codon readthrough on the localization, concentration and function of Pde2 protein. Tranlational readthrough of the stop codon extends the protein by 22 amino acids. The resulting Pde2Lp could also be concentrated in the nucleus. Howerver, protein level of Pde2Lp was much lower than that of the wild-type protein, and thus was insufficient in cAMP degradation.In the last part of this work, we show that two protein kinases, Sch9 and Yak1, are invoved in regulation of Pde2 localization, with unknown mechanisms. In sch9Δstrain, Pde2 was localized exclusively in the cytoplasm, while Bcy1 was nuclear. Accoding to previous results, the localization of Pde2 and Bcy1 in different compartments may results in elevated PKA activity. Deletion of YAK1 could repress the aberrant localization by deletion of SCH9. In addition, Pde2 was concentrated in the nucleus in yak1Δcells grown on non-fermentable carbon sources. Regulation of Pde2 localization by these two kinases may involve in down-regulation of PKA.