Protein kinase A phosphorylation of human phosphodiesterase 3B promotes 14-3-3 protein binding and inhibits phosphatase-catalyzed inactivation.

Recent studies confirm that intracellular cAMP concentrations are nonuniform and that localized subcellular cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is important in maintaining these cAMP compartments. Human phosphodiesterase 3B (HSPDE3B), a member of the PDE3 family of PDEs, represents the dominant particulate cAMP-PDE activity in many cell types, including adipocytes and cells of hematopoietic lineage. Although several previous reports have shown that phosphorylation of HSPDE3B by either protein kinase A (PKA) or protein kinase B (PKB) activates this enzyme, the mechanisms that allow cells to distinguish these two activated forms of HSPDE3B are unknown. Here we report that PKA phosphorylates HSPDE3B at several distinct sites (Ser-73, Ser-296, and Ser-318), and we show that phosphorylation of HSPDE3B at Ser-318 activates this PDE and stimulates its interaction with 14-3-3 proteins. In contrast, although PKB-catalyzed phosphorylation of HSPDE3B activates this enzyme, it does not promote 14-3-3 protein binding. Interestingly, we report that the PKA-phosphorylated, 14-3-3 protein-bound, form of HSPDE3B is protected from phosphatase-dependent dephosphorylation and inactivation. In contrast, PKA-phosphorylated HSPDE3B that is not bound to 14-3-3 proteins is readily dephosphorylated and inactivated. Our data are presented in the context that a selective interaction between PKA-activated HSPDE3B and 14-3-3 proteins represents a mechanism by which cells can protect this enzyme from deactivation. Moreover, we propose that this mechanism may allow cells to distinguish between PKA- and PKB-activated HSPDE3B.

Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system.

Cyclic AMP (cAMP) and cGMP regulate a myriad of cellular functions, such as metabolism, contractility, motility, and transcription in virtually all cell types, including those of the cardiovascular system. Considerable effort over the last 20 years has allowed identification of the cellular components involved in the synthesis of cyclic nucleotides, as well as effectors of cyclic nucleotide-mediated signaling. More recently, a central role for cyclic nucleotide phosphodiesterase (PDE) has also been elaborated in many cell types, including those involved in regulating the activities of the cardiovascular system. In this review, we introduce the PDE families whose members are expressed in cells of the cardiovascular system including cardiomyocytes, vascular smooth muscle cells, and vascular endothelial cells. Because cell behavior is a dynamic process influenced by numerous factors, we will attempt to emphasize how changes in the activity, expression, and targeting of PDE influence cyclic nucleotide-mediated regulation of the behavior of these cells.

Reduced phosphodiesterase 3 activity and phosphodiesterase 3A level in synthetic vascular smooth muscle cells: implications for use of phosphodiesterase 3 inhibitors in cardiovascular tissues.

Vascular smooth muscle cells (VSMC) in situ function to control contraction and are said to express a contractile phenotype. However, during development or in response to vascular damage, VSMC proliferate and express a more synthetic phenotype. A survey of literature values for contractile and synthetic VSMC phosphodiesterase (PDE) 3 and PDE4 activities identified a marked difference in the PDE3 and PDE4 activities of these cells. In this study, a comparison of PDE3 and PDE4 activities in contractile and synthetic VSMC demonstrates that a reduced PDE3/PDE4 activity ratio in synthetic VSMC correlates with a reduced PDE3 activity and is associated with marked reductions in PDE3A mRNA and protein levels. Because we show that similar reductions in PDE3 activity and PDE3A levels occur upon culture of human aortic VSMC and that this phenomenon associates with the phenotypic switch that occurs to VSMC in response to vascular damage, our findings are presented in the context that PDE3 inhibition might be expected to selectively alter functions of contractile VSMC.

Altered phosphodiesterase 3-mediated cAMP hydrolysis contributes to a hypermotile phenotype in obese JCR:LA-cp rat aortic vascular smooth muscle cells: implications for diabetes-associated cardiovascular disease.

Cardiovascular diseases represent a significant cause of morbidity and mortality in diabetes. Of the many animal models used in the study of non-insulin-dependent (type 2) diabetes, the JCR:LA-cp rat is unique in that it develops insulin resistance in the presence of obesity and manifests both peripheral and coronary vasculopathies. In this animal model, arterial vascular smooth muscle cells (VSMCs) from homozygous obese (cp/cp) rats, but not from age-matched healthy (+/+ or + /cp, collectively defined +/?) littermates, display an " activated" phenotype in vitro and in vivo and have an elevated level of cAMP phosphodiesterase (PDE) activity. In this report, we confirm that cp/cp rat aortic VSMCs have an elevated level of PDE3 activity and show that only particulate PDE3 (PDE3B) activity is elevated. In marked contrast to results obtained in + /? VSMCs, simultaneous activation of adenylyl cyclase and inhibition of PDE3 activity in cp/cp VSMCs synergistically increased cAMP. Although PDE3 inhibition did not potentiate the antimigratory effects of forskolin on +/? VSMCs, PDE3 inhibition did markedly potentiate the forskolin-induced inhibition of migration of cp/cp-derived VSMCs. Although PDE3 activity was elevated in cp/cp rat aortic VSMCs, levels of expression of cytosolic PDE3 (PDE3A) and PDE3B in +/? and cp/cp VSMCs, as well as activation of these enzymes following activation of the cAMP-protein kinase A signaling cascade, were not different. Our data are consistent with an increased role for PDE3 in regulating cAMP-dependent signaling in cp/cp VSMCs and identify PDE3 as a cellular activity potentially responsible for the phenotype of cp/cp VSMCs.