The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomycescerevisiae has a specific function in controlling agonist-induced cAMP signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiester ase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP i ncreases. These results indicate a specific role for Pde1 in controlling gl ucose and intracellular acidification-induced cAMP signaling. Elimination o f a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine(252) into alanine resulted in a Pde1(ala252) allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1 Delta. The d ifference between the Pde1(ala252) allele and wild-type Pde1 was strongly d ependent on PKA activity. In a RAS2(val19) pde2 Delta background, the pde1( ala252) allele caused nearly the same hyperaccumulation of cAMP as pde1 Del ta while its expression in a PKA-attenuated strain caused the same reductio n in cAMP hyperaccumulation as wild-type Pde1. These results suggest that s erine252 might be the first target site for feedback inhibition of cAMP acc umulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the pde1(ala252) mutant. Pde1 belongs to a separate class of phosphodies terases and is the first member shown to be phosphorylated. However, in vit ro the Pde1(ala252) enzyme had the same catalytic activity as wild-type Pde 1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivatio n of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1(ala252), and phosphate incorporation was strongly reduced. Apparent ly, phosphorylation of Pde1 does not change its intrinsic activity or affin ity for cAMP but appears to be important in vivo for protein-protein intera ction or for targeting Pde1 to a specific subcellular location. The PKA rec ognition site is conserved in the corresponding region of the Schizosacchar omyces pombe and Candida albicans Pde1 homologues, possibly indicating a si milar control by phosphorylation.