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

Abstract

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. 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 increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 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 pde1Delta. The difference between the Pde1(ala252) allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2(val19) pde2Delta background, the Pde1(ala252) allele caused nearly the same hyperaccumulation of cAMP as pde1Delta, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation 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 phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1(ala252) enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation 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. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

Figure 1

Intracellular cAMP level as a function of time after addition of 100 mM glucose (A and C) or 2 mM 2,4-dinitrophenol (B and D). (A and B) Phosphodiesterase-deficient strains: wild-type strain (W303–1A) (•); pde1Δ strain (PM941) (○); pde2Δ strain (PM942) (▴); and pde1Δ pde2Δ strain (PM943) (▵). (C and D) Strains with overexpression of PDE1 or PDE2: wild-type strain + YEplac195 (PM850) (•); wild-type strain + YEpPDE1 (PM851) (○); wild-type strain + YEpPDE2 (PM852) (▴).

Figure 2

Cells of a wild-type strain (W303–1A) transformed with either YEpPDE1 (PM851), YEpPDE2 (PM852), or the empty vector YEplac195 (PM850) were grown in SD glucose-uracil medium until OD₆₀₀ = 2.0–2.5, after which the OD was adjusted to 2 with the same medium. The cell suspension was heat shocked for 30 min at 50°C in a water bath. Treated and untreated cell suspensions were spotted on YPD plates and incubated at 30°C for 2 d. Serial dilutions were made with a factor of 10.

Figure 3

Intracellular cAMP level as a function of time after addition of 100 mM glucose (A) or 2 mM 2,4-dinitrophenol (B) in strains in which PDE1 has been replaced by pde1^ala252: wild-type strain (W303–1A) (•); pde1Δ strain (PM941) (○); pde1^ala252 strain (PM581) (▴); and pde1^ala252 pde2Δ strain (PM582) (▵). (C) Heat shock resistance of strains carrying different alleles of the PDE genes. Cells were grown in YPD medium until OD₆₀₀ = 2.0–2.5, after which the OD was adjusted to 2 with the same medium. The cell suspension was heat shocked for 20 min at 50°C in a water bath. Treated and untreated cell suspensions were spotted on YPD plates and incubated at 30°C for 2 d. Serial dilutions were made with a factor of 10. (D) Basal cAMP level during exponential growth on YPD medium in RAS2^val19 strains with deletion and/or modification of the phosphodiesterase genes.

Figure 4

Intracellular cAMP level as a function of time after addition of 100 mM glucose to cells of a PKA-attenuated strain (tpk1^w1 tpk2Δ tpk3Δ bcy1Δ) expressing an additional copy of distinct Pde1 alleles or Pde2 from the centromeric plasmid YCplac33. The cells were pregrown into exponential phase on SDglucose medium and resuspended after washing with water in the same medium without glucose. Control strain: empty YCplac33 (PM975) (•); YCplac33 + PDE1 (PM976) (○); YCplac33 + pde1^ala252 (PM977) (▴); YCplac33 + pde1^asp252 (PM 979) (▵); YCplac33 + PDE2 (PM978) (▪).

Figure 5

Purification and phosphorylation of Pde1. (A) SDS-PAGE and protein silver staining of highly purified Pde1 preparation. Pde1 has a molecular mass of 42.6 kDa. (B) Phosphorylation of wild-type Pde1 (250 ng) with PKA and [γ³²P]-labeled ATP results in incorporation of label into a band with the same molecular mass as Pde1. In control incubations, either the kinase or Pde1 was omitted. Samples were analyzed by SDS-PAGE and autoradiography. The arrow denotes the migration position of Pde1 (42.6 kDa). The right panel shows phosphorylation of Pde1^ala252, which results in much weaker incorporation of label at the same position (see RESULTS). This panel was exposed about 2.5 times longer than the panel of the wild-type strain. (C) A purified preparation of Pde1 was incubated with ATPMg and PKA as described in MATERIALS AND METHODS. Both samples and a negative control (buffer) were subsequently incubated in 100 μl buffer A containing 50 nmol (500 μM) cAMP. At the indicated time points aliquots were taken and assayed for cAMP. Purified Pde1 not treated with PKA (•) or treated with PKA (○); negative control, absence of purified Pde1 (▴). A typical result is shown. (D) HA-tagged Pde1 was immunoprecipitated from glycerol-grown ³²P-labeled cells of a wild-type strain (W303–1A) and a Pde1^ala252 strain, before or after addition of 2% glucose, and analyzed by electrophoresis and autoradiography. The arrow denotes the migration position of Pde1 or Pde1^ala252; background signals correspond to unspecifically labeled protein A.

Figure 6

Effect of PKA treatment on Pde1 activity in total crude cell extracts from a wild-type strain (W303–1A) and a strain in which PDE1 has been replaced by pde1^ala252 (PM581).