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Vol. 10, Issue 1, 91-104, January 1999
Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit
Leuven, B-3001 Leuven-Heverlee, Flanders, Belgium
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 Pde1ala252
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
. The difference between the
Pde1ala252 allele and wild-type Pde1 was strongly dependent
on PKA activity. In a RAS2val19
pde2 background, the Pde1ala252 allele caused
nearly the same hyperaccumulation of cAMP as pde1, 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
Pde1ala252 mutant. Pde1 belongs to a separate class of
phosphodiesterases and is the first member shown to be phosphorylated.
However, in vitro the Pde1ala252 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
Pde1ala252, 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.
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