Eva Degerman

Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3)

cAMP and cGMP mediate biological responses initiated by diverse extracellular signals. By catalyzing hydrolysis of the 39–59phosphodiester bond of cyclic nucleotides, cyclic nucleotide phosphodiesterases (PDEs) regulate intracellular concentrations and effects of these second messengers. PDEs include a large group of structurally related enzymes (reviewed in Refs. 1–3). These enzymes belong to at least seven related gene families (PDEs 1–7) (Fig. 1), which differ in their primary structures, affinities for cAMP and cGMP, responses to specific effectors, sensitivities to specific inhibitors, and mechanisms of regulation (1–3). Most families are comprised of more than one gene; 14 different PDE genes have been identified. Within different families, tissue-specific mRNAs are generated from the same gene by the use of different transcription initiation sites or by alternative mRNA splicing. Although some aspects of different PDE families will be discussed, this review emphasizes the PDE3 family, including structure-function information and regulation of the adipocyte PDE3, which plays a key role in the antilipolytic action of insulin. Mammalian PDEs share a common structural organization, with a conserved catalytic core (;270 amino acids) usually located in the C-terminal half (Fig. 1) (4). This region is much more similar within an individual PDE family (.80% amino acid identity) than between different PDE families (;25–40% identity) (1–4). The catalytic core is thought to contain common structural elements important for hydrolysis of the cyclic nucleotide phosphodiester bond, as well as family-specific determinants responsible for differences in substrate affinities and inhibitor sensitivities among the different gene families. It contains a PDE-specific sequence motif, HD(X)2H(X4)N, and two consensus Zn -binding domains, the second of which overlaps the PDE motif (3, 5). PDE5 contains tightly bound Zn, which supports catalytic activity (5). The precise role of Zn or other divalent cations in catalytic function of other PDEs has not been defined. Mutagenesis of the first histidine of the PDE sequence motif abolished activity of a recombinant PDE4 expressed in Escherichia coli (6). Histidineand sulfhydryl-modifying reagents inhibited PDE3 activity (7). The widely divergent N-terminal portions of PDEs (Fig. 1) contain determinants that confer regulatory properties specific to the different gene families, e.g. calmodulin-binding domains (PDE1); two non-catalytic cyclic nucleotide-binding domains (PDEs 2, 5, and 6); N-terminal membrane-targeting (PDE4) or hydrophobic membrane-association (PDE3) domains; and calmodulin (PDE1)-, cyclic AMP (PDEs 1, 3, and 4)-, and cGMP (PDE5)-dependent protein kinase phosphorylation sites, etc. (Fig. 1) (1–3). Most cells contain representatives of several PDE families in different amounts, proportions, and subcellular locations (1–3). In some instances a specific PDE regulates a unique cellular function, e.g. photoreceptor PDE6 in cGMP-dependent initiation of visual transduction. In individual cells, different PDEs, with their different responses to regulatory signals, participate in integrating multiple inputs in the complex modulation and termination of cyclic nucleotide signals and responses, e.g. their magnitude and duration, their functional and spatial compartmentation, and their attenuation by short-term feedback or long-term desensitization.

Identification of Novel Phosphorylation Sites in Hormone-sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitrophosphorylation of HSL containing Ser → Ala mutations in residues 563 and 565 (S563A,S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Regulation and function of the cyclic nucleotide phosphodiesterase (PDE3) gene family

Publisher Summary This chapter discusses some general information about cyclic nucleotide phosphodiesterases (PDEs). It also discusses the PDE3 gene family, emphasizing the molecular biology, structure/function relationships, and cellular regulation and functional roles of PDE3s, as well as physiological/pharmacological actions, therapeutic applications, and potential benefits of PDE3 inhibitors. The major cause of concern in the use of PDE3 inhibitors as therapeutic agents is the potential for increased mortality in patients with known heart disease. Although caution is certainly warranted in this context, conclusions should not be indiscriminately applied to all PDE3 inhibitors. The pharmacological profiles of newer PDE3 inhibitors differ from those of the PDE3 inhibitors used in earlier heart failure clinical trials. Although milrinone and cilostazol are similar in potency as inhibitors of PDE3, milrinone had greater effects than cilostazol on increasing both cyclic adenosine monophosphate (cAMP) and contractility in isolated rabbit cardiomyocytes. The ability to target PDE3 inhibitors to specific isoforms in specific intracellular compartments and/or specific cells may be critical for improvement in efficacy and safety. The acute benefits and chronic adverse actions of PDE3 inhibitors in patients, with heart failure, may result from the phosphorylation of different substrates of Protein kinase A (PKA) in different intracellular compartments. Newer PDE3 inhibitors that target a specific isoform in the appropriate compartment could potentially confer beneficial hemodynamic effects without adverse effects on mortality.

Mechanisms Involved in the Regulation of Free Fatty Acid Release from Isolated Human Fat Cells by Acylation-stimulating Protein and Insulin

The effects of acylation-stimulating protein (ASP) and insulin on free fatty acid (FFA) release from isolated human fat cells and the signal transduction pathways to induce these effects were studied. ASP and insulin inhibited basal and norepinephrine-induced FFA release by stimulating fractional FFA re-esterification (both to the same extent) and by inhibiting FFA produced during lipolysis (ASP to a lesser extent than insulin). Protein kinase C inhibition influenced none of the effects of ASP or insulin. Phosphatidylinositol 3-kinase inhibition counteracted the effects of insulin but not of ASP. Phosphodiesterase 3 (PDE3) activity was stimulated by ASP and insulin, whereas PDE4 activity was slightly increased by ASP only. Selective PDE3 inhibition reversed the effects of both ASP and insulin on fractional FFA re-esterification and lipolysis. Selective PDE4 inhibition slightly counteracted the ASP but not the effect of insulin on fractional FFA re-esterification and did not prevent the action of ASP or insulin on lipolysis. Thus, ASP and insulin play a major role in regulating FFA release from fat cells as follows: insulin by stimulating fractional FFA re-esterification and inhibiting lipolysis and ASP mainly by stimulating fractional FFA re-esterification. For both ASP and insulin these effects on FFA release are mediated by PDE3, and for ASP PDE4 might also be involved. The signaling pathway preceding PDE is not known for ASP but involves phosphatidylinositol 3-kinase for insulin.

Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes.

In adipocytes, protein kinase B (PKB) has been suggested to be the enzyme that phosphorylates phosphodiesterase 3B (PDE3B), a key enzyme in insulins antilipolytic signalling pathway. In order to screen for PKB phosphatases, adipocyte homogenates were fractionated using ion-exchange chromatography and analysed for PKB phosphatase activities. PKB phosphatase activity eluted as one main peak, which coeluted with serine/threonine phosphatases (PP)2A. In addition, adipocytes were incubated with inhibitors of PP. Incubation of adipocytes with 1 microM okadaic acid inhibited PP2A by 75% and PP1 activity by only 17%, while 1 microM tautomycin inhibited PP1 activity by 54% and PP2A by only 7%. Okadaic acid, but not tautomycin, induced the activation of both PKBalpha and PKBbeta. Finally, PP2A subunits were found in several subcellular compartments, including plasma membranes (PM) where the phosphorylation of PKB is thought to occur. In summary, our results suggest that PP2A is the principal phosphatase that dephosphorylates PKB in adipocytes.

Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B–null mice

Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.

Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase.

Insulin stimulation of adipocytes results in serine phosphorylation/activation of phosphodiesterase 3B (PDE 3B) and activation of a kinase that phosphorylates PDE 3B in vitro, key events in the antilipolytic action of this hormone. We have investigated the role for p70 S6 kinase, mitogen-activated protein kinases (MAP kinases), and protein kinase B (PKB) in the insulin signaling pathway leading to phosphorylation/activation of PDE 3B in adipocytes. Insulin stimulation of adipocytes resulted in increased activity of p70 S6 kinase, which was completely blocked by pretreatment with rapamycin. However, rapamycin had no effect on the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B. Stimulation of adipocytes with insulin or phorbol myristate acetate induced activation of MAP kinases. Pretreatment of adipocytes with the MAP kinase kinase inhibitor PD 98059 was without effect on the insulin-induced activation of PDE 3B. Furthermore, phorbol myristate acetate stimulation did not result in phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B. Using Mono Q and Superdex chromatography, the kinase that phosphorylates PDE 3B was found to co-elute with PKB, but not with p70 S6 kinase or MAP kinases. Furthermore, both PKB and the kinase that phosphorylates PDE 3B were found to translocate to membranes in response to peroxovanadate stimulation of adipocytes in a wortmannin-sensitive way. Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B.

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes. Studies using the selective inhibitor wortmannin.

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC50≈ 100 nM), the insulin‐induced activation and phosphorylation of cGMP‐inhibited cAMP phosphodiesterase (cGI‐PDE) as well as the activation of the insulin‐stimulated cGI‐PDE kinase (IC50≈ 10–30 nM). No direct effects of the inhibitor on the insulin‐stimulated cGI‐PDE kinase, the cGI‐PDE and the hormone‐sensitive lipase were observed. These data suggest that activation of PI 3‐kinase upstream of the insulin‐stimulated cGI‐PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin‐induced cGI‐PDE activation/ phosphorylation and anti‐lipolysis.

Type III cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE3 gene family)

Seven different but related cyclic nucleotide phosphodiesterase (PDE) gene families have been identified. Type III cGMP-inhibited (cGI) PDEs, the PDE3 gene family, are found in many tissues. cGI PDEs exhibit a high affinity for both cAMP and cGMP, and are selectively and relatively specifically inhibited by certain agents which augment myocardial contractility, promote smooth muscle relaxation and inhibit platelet aggregation. Adipocyte, platelet, and hepatocyte cGI PDE activities are regulated by cAMP-dependent phosphorylation. Insulin-induced phosphorylation/activation of adipocyte and hepatocyte cGI PDEs is thought to be important in acute regulation of triglyceride and glycogen metabolism by insulin. Two distinct cGI PDE subfamilies, products of distinct but related genes, have been identified. They exhibit the domain structure common to PDEs with a carboxyterminal region, conserved catalytic domain and divergent regulatory domain. In their catalytic domains cGI PDEs contain a 44 amino acid insertion not found in other PDE families. The expression of cGIP1 and cGIP2 mRNAs differs in different rat tissues, suggesting distinct functions for the two cGI PDE subfamilies, i.e., cGIP1 in adipose tissue, liver, testis and cGIP2 in myocardium, platelets and smooth muscle.

Protein kinase B activity is required for the effects of insulin on lipid metabolism in adipocytes

Protein kinase B (PKB) is known to mediate a number of biological responses to insulin and growth factors, its role in glucose uptake being one of the most extensively studied. In this work, we have employed a recently described allosteric inhibitor of PKB, Akti, to clarify the role of PKB in lipid metabolism in adipocytes-a subject that has received less attention. Pretreatment of primary rat and 3T3L1 adipocytes with Akti resulted in dose-dependent inhibition of PKB phosphorylation and activation in response to insulin, without affecting upstream insulin signaling [insulin receptor (IR), insulin receptor substrate (IRS)] or the insulin-induced phosphoinositide 3-kinase (PI3K)-dependent activation of the ERK/p90 ribosomal kinase (RSK) pathway. PKB activity was required for the insulin-induced activation of phosphodiesterase 3B (PDE3B) and for the antilipolytic action of insulin. Moreover, inhibition of PKB activity resulted in a reduction in de novo lipid synthesis and in the ability of insulin to stimulate this process. The regulation of the rate-limiting lipogenic enzyme acetyl-CoA carboxylase (ACC) by insulin through dephosphorylation of S79, which is a target for AMP-activated protein kinase (AMPK), was dependent on the presence of active PKB. Finally, AMPK was shown to be phosphorylated by PKB on S485 in response to insulin, and this was associated with a reduction in AMPK activity. In summary, we propose that PKB is required for the positive effects of insulin on lipid storage and that regulation of PDE3B and AMPK by PKB is important for these effects.