Effects of heterologous expression of human cyclic nucleotide Phosphodiesterase 3A (hPDE3A) on redox regulation in yeast.

Oxidative stress plays a pivotal role in pathogenesis of cardiovascular diseases and diabetes, however, the roles of Protein Kinase A (PKA) and human Phosphodiesterase 3A ( hPDE3A ) remain unknown. Here we show that yeast expressing wild type (WT) hPDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous H2O2, respectively. H2O2-stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A (OxiS1), compared to yeast expressing WT hPDE3A (OxiR1). In OxiR1, YAP1 and YAP1 -dependent anti-oxidant genes were upregulated, accompanied by reduction of thioredoxin peroxidase. In OxiS1, expression of YAP1 and YAP1 -dependent genes was impaired, and the thioredoxin system malfunctioned. H2O2 increased cAMP-hydrolyzing activity of WT hPDE3A, but not K13R hPDE3A, through PKA-dependent phosphorylation of hPDE3A, which was correlated with its ubiquitinylation. The changes in anti-oxidant gene expression did not directly correlate with differences in cAMP/PKA signaling. Despite differences in their capacities to hydrolyze cAMP, total cAMP levels among OxiR1, OxiS1, and mock were similar, PKA activity, however, was lower in OxiS1 than in OxiR1 or mock. During exposure to H2O2, however, Sch9p activity, a TORC1-regulated Rps6 kinase and negative-regulator of PKA, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol, with PKA activation. In OxiS1, Sch9p activity was unchanged during exposure to H2O2, consistent with reduced activation of PKA. These results suggest that, during oxidative stress, TOR-Sch9 signaling might regulate PKA activity, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress.