The cAMP-specific phosphodiesterase PDE4D3 is regulated by phosphatidic acid binding - Consequences for cAMP signaling pathway and characterization of a phosphatidic acid binding site

Hormones and growth factors induce in many cell types the production of pho sphatidic acid (PA), which has been proposed to play a role as a second mes senger. We have previously shown in an acellular system that PA selectively stimulates certain isoforms of type 4 cAMP-phosphodiesterases (PDE4), Here we studied the effect of endogenous PA on PDE activity of transiently tran sfected MA10 cells overexpressing the PA-sensitive isoform PDE4D3, Cell tre atment with inhibitors of PA degradation, including propranolol, induced an accumulation of endogenous PA accompanied by a stimulation of PDE activity and a significant decrease in both cAMP levels and protein kinase A activi ty. Furthermore, in FRTL5 cells, which natively express PDE4D3, pretreatmen t with compounds inducing PA accumulation prevented both cAMP increase and cAMP-responsive element-binding protein phosphorylation triggered by thyroi d-stimulating hormone. To determine the mechanism of PDE stimulation by PA, endogenous phospholipids were labeled by preincubating MA10 cells overexpr essing PDE4D3 with [P-32]orthophosphate. Immunoprecipitation experiments sh owed that PA was specifically bound to PDE4D3, supporting the hypothesis th at PDE4D3 activation occurs through direct binding of PA to the protein. PA binding site on PDE4D3 was characterized by engineering deletions of selec ted regions in the N-terminal regulatory domain of the enzyme, Deletion of amino acid residues 31-59 suppressed both PA-activating effect and PA bindi ng, suggesting that this region rich in basic and hydrophobic residues cont ains the PA binding site. These observations strongly suggest that endogeno us PA can modulate cAMP levels in intact cells, through a direct activation of PDE4D3.