Cyclic Nucleotide Phosphodiesterase 3 Signaling ...

776 Review

Cyclic Nucleotide Phosphodiesterase 3 Signaling Complexes

Affiliations

Key words

▶ phosphodiesterase ● ▶ protein kinase A ● ▶ signalosomes ●

received 06.01.2012 accepted 19.04.2012 Bibliography DOI http://dx.doi.org/ 10.1055/s-0032-1312646 Published online: June 12, 2012 Horm Metab Res 2012; 44: 776–785 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence F. Ahmad PhD Cardiovascular Pulmonary Branch NHLBI/NIH Bethesda, MD 20892 USA Tel.: +1/301/496 3057 Fax: +1/301/402 1610 [email protected]

F. Ahmad1, E. Degerman2, V. C. Manganiello1 1 2

Cardiovascular Pulmonary Branch, National Heart, Lung and Blood Institute, Bethesda, MD, USA Department of Experimental Medical Science, Division for Diabetes, Metabolism and Endocrinology, Lund University, Lund, Sweden

Abstract

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The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic β-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemi-

Abbreviations

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AKAP A-kinase anchoring protein βAR, β1AR, β2AR, β3AR β1, 2, 3-adrenergic receptor ARF ADP-ribosylation factor AC adenylyl cyclase BIG brefeldin-inhibited guanine nucleotide binding protein Cav-1 caveolin-1 cAMP cyclic AMP cGMP cyclic GMP CL CL316243 CFTR cystic fibrosis transmembrane conductance regulator Epac guanine nucleotide exchange protein FRET fluorescence resonance energy transfer HMWC high-molecular-weight macromolecular complex

Ahmad F et al. PDE3 Signaling Complexes. Horm Metab Res 2012; 44: 776–785

cal basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes.

HAEC HSL HSP-90 IRS IGF MβCD NHR PDE PLK1 PKA PKA-RII PP2A PP1 PLB RyR PI3-Kγ siRNA SR SERCA VSMC

human aortic endothelial cells hormone sensitive lipase heat-shock protein 90 insulin receptor substrate insulin-like growth factor methyl-β-cyclodextrin N-terminal hydrophobic region phosphodiesterase polo-like kinase-1 cAMP-dependent protein kinase PKA-regulatory subunit protein phosphatase 2 A protein phosphatase 1 phospholamban ryanodine receptor phosphoinositide-3-kinaseγ small interfering RNA sarcoplasmic reticulum Ca2+ ATPase vascular smooth muscle cell

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Fig. 1 Role of cyclic nucleotide phosphodiesterases (PDEs) in regulation of signaling by cyclic nucleotides.

PDEs cAMP/cGMP

PDE4 PDE7 PDE8

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PDE1 PDE2 PDE3 PDE10 PDE11

5ÁMP

5´GMP

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cGMP specific PDE5 PDE6 PDE9

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Catalytic Domain PDE3 Insert

PDE3A–135

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Fig. 2 PDE3 isoforms: Schematic illustration of the PDE3A isoforms and PDE3B showing N terminal region hydrophobic regions NHR1 and NHR2. P1, P2: distinct phosphorylation sites for PKA and PKB on PDE3; Catalytic domain: conserved catalytic region; PDE3 insert (□): 44 aa insert in the catalytic domain.

PDE3A–118

P2 PDE3A–95

PDE3B (140 k) ~85% identical P1 P2

Introduction

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Cyclic nucleotide phosphodiesterases (PDEs) are critical in regulating intracellular concentrations of cAMP and cGMP, and, thereby, the physiological effects of these second messengers ▶ Fig. 1). The PDE superfamily contains 11 structurally related, (● but functionally distinct, gene families (PDE1-11), which differ in their primary structures, affinities for cAMP and cGMP, responses to specific effectors and inhibitors, and mechanisms through which they are regulated [1]. All PDE families are composed of more than one member and, in many cases, the members are products of more than one gene. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B are encoded by 2 highly related and similarly organized genes on human chromosomes 12p12 and 11p15, respectively [2–6], and both hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner [1, 5]. PDE3 isoforms are regulators of a number of important cAMP-mediated processes, including myocardial contractility, platelet aggregation, vascular and airway

muscle relaxation, insulin secretion, lipolysis, oocyte maturation, inflammation, cell proliferation, etc. [7–10].

Structural Organization of PDE3A and PDE3B

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The structural organization of PDE3A and PDE3B proteins is identical, with highly conserved catalytic domains in the C-terminal region of the molecules separating divergent N-terminal ▶ Fig. 2) [11, 12]. regulatory regions and hydrophilic C-terminii (● The N-terminal portion of PDE3B and PDE3A includes 2 domains, NHR1 [amino acids (aa) 1–300], which contains a large hydrophobic domain with 5–6 predicted transmembrane helices, and NHR2 (aa 300–500), with a smaller hydrophobic region of ̴ 50 aa. NHR1 and NHR2 in PDE3A and PDE3B differ considerably. Downstream of NHR1 are consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) and protein kinase B (PKB) [9, 10]. Subcellular fractionation and immunofluorescence localization of PDE3B in 3T3-L1 adipocytes and Flag-tagged Ahmad F et al. PDE3 Signaling Complexes. Horm Metab Res 2012; 44: 776–785


cAMP specific

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Interactions with Ligands and Other Proteins

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Functional compartmentation of PDEs Scaffold, anchoring, and adaptor proteins contribute to the specificity of signal transduction by recruiting enzymes into compartmentalized signaling networks and/or by placing enzymes close to their substrates. In early studies, family specific PDE inhibitors were utilized to define, in individual cells, signaling pathways and biological responses regulated by specific PDEs. In cultured 3T3-L1 murine adipocytes and primary rodent adipocytes, for example, spatial segregation of PDE3B and the lipolytic machinery, resulting in PDE3B regulation of a pool of cAMP related specifically to modulation of PKA and hormone sensitive lipase (HSL), could explain the observed inhibition of the antilipolytic effect of insulin by PDE3 inhibitors but not by PDE4 inhibitors [26, 27]. Current studies using fluorescence resonance energy transfer (FRET) imaging or biosensors, such as Epacbased cAMP sensors and cyclic-nucleotide-sensitive ion channels, have demonstrated, in real time, temporal, spatial, and functional compartmentation of cyclic nucleotides [28, 29]. At the molecular level, PDEs contribute to this compartmentation by virtue of their targeting to different subcellular locations and their interactions with cellular structural elements, with molecular scaffolds such as AKAPs or β-arrestin, or with other regulatory partners, including Epacs [28, 30–34]. AKAPs serve dual functions, as tethers for PKA at different subcellular locations in close proximity to PKA substrates for their selective phosphorylation, and as scaffolds for macromolecular complexes or “signalosomes” with different proportions of PKA, adenylyl cyclases, other kinases, phosphatases, Epacs, PDEs, and other effector molecules [32–34].


Roles of compartmentalized PDE4 in cardiac function In cardiomyocytes, stimulation of β-adrenergic receptors (βAR) induces strong inotropic and lusitropic responses by increasing cAMP, which in turn activates PKA, resulting in phosphorylation of key regulators of the excitation/coupling process. In adult rodent cardiomyocytes, β1AR-mediated cAMP signals were almost completely regulated by PDE4 isoforms, whereas β2ARmediated signals were regulated by multiple PDEs, including PDE3 and PDE4 [35]. In adult mouse ventriculomyocytes, PDE4B was tethered to L-type Ca++ channels (LTCC) and co-localized with the LTCC complex along T-tubules. As a component of this LTCC complex, PDE4B may regulate Ca++-induced Ca++ release, and may protect the murine heart against ventricular arrythmias [36]. In addition, in rodent cardiomyocytes, PDE4D5 preferentially associates with β-arrestin, and the complex is recruited to the βAR following agonist stimulation. Recruitment of the complex initiates a dual desensitization to cAMP/PKA signaling and ERK1 signaling by physically interfering with βAR/ Gα-coupled activation of adenylyl cyclase and by increasing cAMP hydrolysis in the vicinity of the βAR. This latter effect downregulates βAR-associated PKA activity and thus inhibits βAR/Gi activation of ERK1 signaling [34, 37]. In rodents, chronic stimulation of βAR, especially β1AR, is associated with cardiac remodeling and development of hypertrophy [38], and, in rodent cardiomyocytes, a PDE4D/ HSP20 complex, by regulation of PKA-induced phosphorylation of HSP20, modulates βAR-induced hypertrophic responses [39, 40]. In addition, in cardiomyocytes, mAKAP provides the scaffold for a signalosome that is located at the nuclear envelope and modulates cAMP-mediated hypertrophic responses [34, 37, 41]. PDE4D3 is a central component of this signalosome, which contains PKA, Epac1, PP2A, adenylyl cyclase, and an ERK1 signaling module. In this macromolecular complex, PKA-induced phosphorylation/activation [42] and PP2A-induced dephosphorylation/inactivation of PDE4D3 [43] have important roles in the coordinated regulation and integration of PKA and Epac1/ERK1 signaling [44]. PDE4D-knockout (KO) mice also exhibit an age-dependent progressive cardiomyopathy, with increased incidence of arrhythmias [45]. Cardiomyopathy in PDE4D-KO mice was associated with cAMP/PKA-induced hyperphosphorylation of the ryanodine receptor 2 (RyR2), which was likely due to the absence of PDE4D3 from a regulatory mAKAP/PKA/PDE4D/ RyR2 complex and which resulted in dysregulation of Ca2+ transients [45, 46]. The possible relevance of these phenomena to human cardiac disease is unknown.

Roles of compartmentalized PDE3A and PDE3B in cardiac function PDE3A and PDE3B are also components of important signaling pathways and signalosomes in cardiomyocytes. Our laboratory generated PDE3A-KO and PDE3B-KO mice to dissect out cAMPsignaling pathways that regulate myocardial contractility as well as mitogenesis and remodeling in cultured vascular smooth muscle myocytes (VSMCs). With respect to the latter, we reported that, in VSMCs grown from aortae of PDE3A-KO mice, not PDE3B-KO mice, mitogen-induced proliferative responses were inhibited, due to cell cycle arrest at the G0–G1 phase [47]. PDE3 inhibitors, such as milrinone, are known to induce myocardial contractility, most likely via PKA-catalyzed phosphorylation of phospholamban (PLB) and consequent activation of the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2) and Ca++ uptake


N-terminal truncated PDE3B suggested that structural determinants responsible for membrane association are localized in NHR1 domain while NHR2 seemed to be important for efficient membrane association and targeting [12–17]. In human PDE3B, the catalytic core spans aa 654–1 073 [2, 18, 19], similar to the findings in rat PDE3B [20]. The conserved catalytic core contains a histidine-containing signature motif (HD(X2)H(X4)N) common to all mammalian PDEs, as well as 2 consensus metal binding domains [HX3HX24–26E] [21–24]. The PDE3 catalytic domain also contains a 44 aa insert, which is unique to the PDE3 family but differs in PDE3A and PDE3B. This insert not only distinguishes PDE3 from the other PDE gene families, but also differentiates ▶ Fig. 2) the catalytic domains of PDE3A and PDE3B subfamilies (● [9–12]. There are 3 different variants of PDE3A (PDE3A1-3), which are products of the single PDE3A gene [4, 5, 25]. The 3 PDE3A isoforms have identical sequences except for N-terminal ▶ Fig. 2). These N-tersequence deletions of different lengths (● minal differences predict differential phosphorylation by PKA and PKB, and affect their intracellular distribution. PDE3A1 is found exclusively in cardiac membranes, and includes consensus sites for phosphorylation by PKA and PKB. PDE3A2 is found in both cardiac cytosol and membrane fractions and includes a PKA site, but lacks the PKB phosphorylation site. PDE3A3 is primarily cytosolic and lacks both PKA and PKB phosphorylation sites. All 3 PDE3A isoforms have very similar catalytic activities and sensitivities to PDE3 inhibitors [5].

into the SR [48, 49]. The observation that inotropic responses to PDE3 inhibitors were preserved in PDE3B-KO, but not in PDE3AKO, mice suggested that PDE3A isoforms, not PDE3B isoforms, are specifically involved in the regulation of myocardial contractility [50]. Recent studies using PDE3A-KO mice also indicate that PDE3A, not PDE3B, is the milrinone-sensitive PDE3 isoform that regulates basal Ca++ transients and contractility (Shen, W., Becca, S., et. al., Unpublished). In mice, a multi-protein AKAP18 complex that includes SERCA2, PLB and PKA appears to regulate uptake of cytoplasmic Ca2+ into the SR [51]. Our unpublished data support the notion that phosphorylated PDE3A is recruited into these SERCA2-containing signalosomes, which contain AKAP18, PLB, PDE3A, PP2A, and PP1 (Shen, W., Becca, S., et. al., Unpublished). Our unpublished data in human cardiomyocytes also suggest that phosphorylated cardiac PDE3A isoforms are incorporated into macromolecular complexes containing SERCA2, PLB, PKARII, PP2A, and AKAP18, and that PDE3A may regulate a discrete cAMP pool that controls contractility by modulating Ca2+ uptake into the SR (Ahmad, F., et. al., Unpublished). These and other findings suggest that PDE3A is an important regulator of cAMP-signaling in signalosomes involved in cAMPdependent modulation of SERCA2 activity and intracellular Ca2+ transients in cardiomyocytes.

Role of PDE3B in the compartmentalized regulation of phosphatidylinositol 3-kinaseγ (PI3-Kγ) functions in the cardiovascular system Adrenergic stimulation of the heart involves cAMP and phosphoinositide second messenger signaling. Phosphoinositide-3kinaseγ-deficient (PI3-Kγ–/–) mice show enhanced cardiac contractility due to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca++ content and release, demonstrating that PI3-Kγ regulates cAMP levels and contractility in cardiomyocytes [52]. In murine heart, PDE3B has been reported to associate with PI3-Kp110γ and its regulatory subunit p87PIKAP [53–55]. PI3-Kp110γ anchors PKA, which activates PDE3B to enhance cAMP degradation and inhibit PIP(3) production, providing a local feedback control of PIP(3) and cAMP signaling events [55]. This signalosome may be especially important in the regulation of cAMP levels and, consequently, cardiac function in the presence of chronic pressure overload induced by trans-aortic constriction [53–55]. Recent studies also suggest that PDE3Btethered Epac1 regulates PI3-Kγ activity in HAEC (human aortic endothelial cells), and that this allows dynamic, cAMP-dependent regulation of HAEC adhesion, spreading, as well as tubule formation and angiogenesis [56]. The N-terminal domain of PDE3B was required for incorporation of PDE3B into these Epacbased signaling complexes [31]. In addition, PI3-Kγ also attenuates the cAMP/PKA pathway through activation of PDE4 and regulates Ca2+ transients in compartments containing SERCA2 [57].

Incorporation of PDE3A and PDE3B into Noncardiac Signalosomes

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In pig trachea, PDE3A physically and functionally interacts with the cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cAMP, which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function [58]. Brefeldin-inhibited guanine nucleotide binding proteins 1

and 2 (BIG1 and BIG2) catalyze the activation of class I ADP-ribosylation factors (ARFs) by accelerating replacement of bound GDP with GTP. BIG1 and BIG2 contain AKAP domains, and PKAinduced phosphorylation inhibits BIG activity. In Hela cells, PDE3A was found to be a component of a signaling complex containing BIG1/BIG2 and proteins involved in cAMP signaling, including PP2A, 14-3-3, PKA-RII. PDE3A in these BIG1 and BIG2 signaling complexes may contribute to the regulation of ARF function via regulation of cAMP/PKA-induced inhibition of BIG activity, with spatial and temporal specificity [59]. PDE3A was also found to be a component of a multi-protein complex containing Polo-like kinase1 (PLK1), PKA, PP2A, and Cdc25c, which may regulate cAMP/PKA-induced phosphorylation/inactivation of PLK1 and thus modulate maturation of murine oocytes [60]. In platelets, PDE3A is constitutively associated with the leptin receptor. Leptin-induced activation of platelet function is associated with activation of a signaling cascade that includes the long form of the leptin receptor, 3 kinases [Janus kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K), and PKB], insulin receptor substrate-1 (IRS-1), and phosphorylation/activation of PDE3A [61]. PDE3B has also been reported to interact with insulin receptor in human adipocytes [62]. Phosphorylation of PDE3A and PDE3B promotes binding of 14-3-3 proteins [63–66]; association of phospho-PDE3A with 14-3-3 proteins may protect PDE3A from proteolytic cleavage [66]. PDE3B has been reported to associate with PKB and a 50 kD protein in rodent and in 3T3L1 adipocytes [67, 68], and with caveolin-1 in primary rat adipocytes [13].

Insulin- and cAMP-induced Formation of Individual Signalosomes in Adipocytes

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Differential activation of PDE3B in caveolae and ER/ Golgi fractions by CL 316243 (CL) (β3-agonist) and insulin, respectively A very important characteristic of PDE3s is their capacity to be rapidly activated in response to agents that increase cAMP, and to insulin, IGF-1, leptin, and IL-4 in different cells [69]. Phosphorylation/activation of PDE3 (and PDE4) by cAMP/PKA represents a feedback-type regulation that may be important in regulating acute changes in cAMP and in attenuating the magnitude and duration of cAMP signals [69]. Our studies in 3T3-L1 adipocytes, primary adipocytes, and FDCP2 myeloid cells [13, 14, 70–73], and those of others [67], demonstrated that insulin and IGF-1 activated PDE3B via wortmannin-sensitive, PI3K-dependent signals that resulted in activation of PKB, which directly phosphorylated/activated PDE3B [72]. This PI3-K/PKB paradigm for growth factor/cytokine activation of PDE3 also provides a mechanism for counter-regulatory effects of insulin, IGF-1, and leptin in inhibiting certain cAMP-mediated processes. For example, activation of PDE3B by insulin in adipocytes [69], by IGF-1 and leptin in pancreatic β-cells [74, 75], and by leptin in hepatocytes [76] is important in reducing cAMP and mediating the antilipolytic action of insulin [69], the inhibitory effects of IGF1 and leptin on cAMP-mediated insulin secretion [74, 75], and the inhibitory effects of leptin on glycogenolysis [76]. Activation of PDE3A by leptin in platelets [61] and of PDE3 (most likely a PDE3A homolog) by insulin/IGF-1 in Xenopus oocytes [77, 78] is important in reducing cAMP and mediating the stimulatory effects of insulin and IGF-1 on oocyte maturation [77, 78] and of leptin on platelet activation [61]. Ahmad F et al. PDE3 Signaling Complexes. Horm Metab Res 2012; 44: 776–785


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Apply solubilized proteins on S6 column. Collect fractions. HMW-ins and HMW-CL (high stoke’s radius) macromolecules will elute first. Determine proteins in eluate using Western and PDE assay. Estimate mol weight.

Fig. 3 Separation of membrane fractions and macromolecular complexes by sucrose gradient centrifugation and Superose 6 chromatography.

Fig. 4 Activation of PDE3B in PM/Caveolae and ER/Golgi fractions by CL316243 or insulin, respectively in 3T3-L1 adipocytes involves formation of distinct signalosomes.

IR

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In adipocytes, mechanisms for activation of PDE3B by insulin or cAMP-elevating agents involve formation of distinct signalosomes. Subcellular fractionation of membranes from primary rat adipocytes and 3T3-L1 adipocytes on 10–45 % continuous sucrose gradients indicated that membrane-associated PDE3B was distributed in PM/caveolae and ER/Golgi fractions, and that in 3T3-L1 adipocytes ̴ 35 % of total membrane PDE3 activity was ▶ Fig. 3) found in PM/caveolae and ̴ 65 % in ER/Golgi fractions (● [13, 14, 73]. In 3T3-L1 adipocytes, the β3-AR agonist CL and insulin diffrerentially regulate PDE3B in PM/caveolae and ER/Golgi fractions, respectively. CL preferentially phosphorylated/activated PDE3B in PM/caveolae fractions, whereas insulin preferentially phosphorylated/activated PDE3B in ER/Golgi fractions ▶ Fig. 4), suggesting different signaling roles for PDE3B in dif(● ferent microdomains of the cell [73]. PDE3B did not seem to be translocated significantly between membranes after stimulation of adipocytes with insulin or CL.


ER/Golgi

Isolation of distinct signalosomes by gel filtration and immunoprecipitation Stimulation of 3T3-L1 adipocytes with insulin or CL leads to recruitment of phosphorylated and activated PDE3B into distinct high molecular weight macromolecular complexes (HMWC-ins or HMWC-CL) or signalosomes, which can be separated and isolated during Superose 6 (S6) gel filtration chromatography of solubilized membranes [14, 73]. During chromatography on Superose 6 columns, solubilized membrane-associated PDE3B from unstimulated 3T3-L1 adipocytes exhibited a molecular mass (Mr) of ̴ 600–1 000 kD, much larger than its free monomeric Mr of ̴ 135 kD [13, 14]. After incubation of adipocytes with either insulin or CL, however, PDE3 activity increased and about 50 % of PDE3B eluted at a higher apparent Mr of ≥ 3 000 kD, consistent with its presence in HMWCs induced by insulin or CL ▶ Fig. 5) [73]. After treatment of 32P-labelled adipocytes with (● insulin or CL, Superose 6 chromtography of solubilized membranes and phosphoimager analysis of immunoprecipitated PDE3B confirmed that the HMWCs contained most of the


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Fig. 6 Interactions between PDE3B and pPKB recombinant proteins.

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P-phosphorylated and activated PDE3B. Recruitment of PDE3B into HMWCs seemed to require phosphorylation in that PDE3B not recruited into HMWC-ins or HMWC-CL exhibited little or no phosphorylation, and little or no PDE3B was recruited into HMWCs in control adipocytes not exposed to insulin or CL. These HMWCs contain signaling molecules potentially involved in activation of PDE3B by insulin or CL. Insulin-induced signalosomes included IRS-1, PI3K p85, HSP90, and PKB; CL-induced signalosomes included β3-AR, HSL and PKA-RII, while some signaling molecules were common to both signalosomes, including PP2A, 14-3-3, perilipin, and caveolin-1 (cav-1) [73]. These molecules co-immunoprecipitated with PDE3B in HMWC-ins or HMWC-CL fractions, not in fractions eluting at ̴ 600–900 kDa, suggesting that phosphorylation might be important in these ▶ Fig. 5). Wortmannin inhibited insulin-induced interactions (● formation of HMWC-ins, phosphorylation/activation of PKB and PDE3B [79, 80], as well as their interactions with other signaling molecules during Superpose 6 chromatography, and their coimmunoprecipitation. Thus, differential phosphorylation/activation of PDE3B in PM/caveolae and ER/Golgi fractions may be related to the formation of different signalosomes in response to ▶ Fig. 5) [73]. Both insulininsulin or to the β3-AR agonist CL (● and CL-induced signalosomes were enriched in cholesterol, and contained certain common signaling proteins (14-3-3, PP2A, cav-1).

Structural determinants for the interaction of PDE3B and PKB Insulin-induced activation of PKB may be critical in phosphorylation/activation of PDE3B and its incorporation into insulininduced signalosomes, which also contain PKB. The structural

determinants responsible for interaction between PDE3B and PKB seem to reside in the N-terminal regulatory region of PDE3B and the pleckstrin homology (PH) domain of PKB, respectively ▶ Fig. 6) [14]. During Superose 12 chromatography, recom(● binant mouse PDE3B (M3B) co-eluted to a greater extent with recombinant pPKB (phosphorylated/activated) than with dephospho recombinant PKB or p-ΔPKB [pPKB lacking the PH domain] [14]. Truncated recombinant M3BΔ196, M3BΔ302 and M3BΔ604 (N-terminal truncated M3B recombinant proteins in which the first 196, 302, or 604 amino acids were deleted, respectively), co-immunoprecipitated with recombinant pPKB to a much lower extent than WT-M3B. M3B-Δ604 did not coimmunoprecipitate, suggesting that the regulatory domain (RD) (1–302 aa) may be critically important for interaction of PDE3B with pPKB [14]. Thus, the PH domain of pPKB and the N-terminal portion of RD (1–302aa) may be responsible for the interac▶ Fig. 6). tion of PDE3B and pPKB (●

The role of PDE3B in signalosome formation Insulin- and CL-induced HMWCs contained highly phosphorylated/activated PDE3B and signaling molecules potentially involved in its activation. In adipocytes, recruitment of recombinant PDE3B into HMWC-ins required the presence of its N-terminal regulatory region, which contains sites phosphorylated in recombinant PDE3B expressed in adipocytes which were stimulated by insulin and cAMP-elevating agents [81]. Recombinant mouse PDE3B lacking the N-terminal 604 aa (MPDE3BΔ604), was not recruited into HMWC-ins by insulin. However, expression of MPDE3B∆604 did not prevent insulin-induced formation of HMWC-ins. siRNA (small interfering RNA)-induced PDE3B knockdown (KD) also did not prevent insulin-stimulated recruitAhmad F et al. PDE3 Signaling Complexes. Horm Metab Res 2012; 44: 776–785


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Fig. 5 Insulin- and CL-induced assembly of different signalosomes involved in phosphorylation/ activation of PDE3B. Solubilized total membranes from adipocytes were subjected to chromatography on Superose 6. PDE3 activities in fractions from control (○), insulin- (■) or CL- (▲) stimulated adipocytes were analyzed. Co-immunoprecipitation with PDE3B (BOX): stimulation with insulin or CL shows different signaling molecules co-immunoprecipitating with PDE3B.

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ment of signaling molecules into HMWC-ins, indicating that PDE3B is not necessary for formation of HMWC-ins [14]. Wortmannin did, however, inhibit insulin-induced phosphorylation/ activation of PDE3B and its incorporation into signalosomes [14]. Thus, although in virtually all of our studies, phosphorylation of PDE3B is associated with its recruitment into signalosomes, we can not be certain whether phosphorylation is required for recruitment, or whether recruitment of PDE3B into (or its association with) signalosomes is required for effective phosphorylation of PDE3B. Future studies with PDE3B phosphorylation site mutants, expressed in adipocytes, may shed light on the overall relationship between signaling and complex formation, as well as the role of phosphorylation of PDE3B in its activation and/or recruitment.

The Role of Caveolin-1 in Regulation of Signaling Pathways and Signalosome Formation in Adipocytes

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Caveolae are unique membrane domains, which are frequently found as small (50–100 nm) omega-shaped invaginations of plasma membranes [82]. These structures are sites for sequestration and organization of membrane-targeted signaling molecules, and are thought to be important in regulation of diverse signal transduction pathways. Caveolae, which are special types of lipid rafts, are highly enriched in cholesterol and sphingolipids, and are stabilized by caveolin isoforms, a family of scaffolding/regulatory proteins which bind cholesterol [83, 84]. In addition to their presence in caveolae, caveolins are present in different subcellular compartments (PM, golgi apparatus, ER) [85, 86], as well as in recycling endosomes and other cellular regions [87]. Recent studies suggest that caveolins can act independently of caveolae, both in cells that lack caveolae (e. g., neurons and leukocytes) and in noncaveolar regions of cells that contain caveolae (e. g., cardiac myocytes and fibroblasts). Caveolins may act as scaffold proteins in both caveolar and non caveolar regions in different membrane compartments [88]. In immune cells, caveolin is found in the Golgi-ER compartment but not on the plasma membrane, consistent with the absence of caveolae from these cells [89]. In response to increases in cAMP in myofibroblasts, scaffolding of adenylyl cyclase (AC) at focal adhesion sites by phosphorylated caveolin-1 in a caveolae-free microdomain, suggested that phosphorylated caveolins contribute to cell attachment and migration [90]. Studies with small Ahmad F et al. PDE3 Signaling Complexes. Horm Metab Res 2012; 44: 776–785

Fig. 7 Subcellular localization of PDE3B, caveolin in 3T3-L1 adipocytes.

interfering RNAs (siRNAs) and caveolin-KO mice have also demonstrated the importance of caveolin in cellular attachment, trafficking and survival [90–93]. Loss of caveolin-1 expression in heart drives p42/44 MAP kinase activation and cardiac hypertrophy, and levels of endothelial and inducible nitric oxide synthase are dramatically upregulated [92]. Caveolin-1-KO mice are lean [94], fail to undergo hepactocyte regeneration in response to liver injury [95], and show vascular dysfunction [96] and neurological abnormalities [97]. Myocardial cAMP content was significantly increased by 42 % in caveolin-3-KO mice [98]. Immunofluorescence and immunoelectron microscopic studies in adult cardiac myocytes suggested that caveolin-3 was coexpressed with signaling molecules in intercalated discs (between cells) and in transverse tubules/sarcoplasmic reticulum regions [99]. Cardiomyocytes treated with the cholesteroldepleting agent methyl-beta-cyclodextrin (MβCD) have a 60–70 % increase in phosphorylation of PKA targets, suggesting that disruption of caveolae results in dysregulation of cAMP generation [100]. In 3T3-L1 preadipocytes catenin, a plasma membrane (PM) marker, was detected primarily at the periphery of the cell, and PDE3B was not detected. This configuration changed with differentiation, as PDE3B was markedly upregulated and 3T3-L1 adipocytes developed membranous invaginations of the PM, which stained for cat▶ Fig. 7). In rodent adipocytes and 3T3-L1 enin [14] and caveolin-1 (● adipocytes, membrane-associated PDE3B is found in both PM/caveolae and ER/Golgi fractions [13, 14, 16, 73]. Localization of human PDE3B to caveolae was established via immunogold labeling and electron microscopy of PM sheets from primary human adipocytes [73]. Similar localizations of PDE3B, caveolin-1 and Bodipy (neutral lipid stained structures) in different membrane compartments of ▶ Fig. 7) are of special interest, considering the role of adipocytes (● PDE3B in the regulation of cAMP- and insulin-regulated pathways [10, 69]. Studies with caveolin-1-KO mice [94, 101] suggested a critical role for caveolin-1 in regulation of catecholamine-stimulated lipolysis. In 3T3-L1 adipocytes, PDE3B is preferentially phosphorylated/activated in caveolae/PM by CL [73], indicating that caveolae/PM platforms may be very important for cAMP-regulated pathways. Similar to caveolin-1-KO mice [94, 101], siRNA-mediated knockdown (KD) of caveolin-1 in 3T3-L1 adipocytes resulted in inhibition of CL-stimulated phosphorylation of HSL and perilipin A, and of lipolysis [73]. siRNA mediated KD of caveolin-1 in 3T3-L1 adipocytes also resulted in downregulation of expression of membrane-associated PDE3B. In addition, although insulin-induced activation of PDE3B was reduced, CL-mediated activation was


Bodipy

almost totally abolished; similar results were obtained in adipocytes from caveolin-1-KO mice [73]. Cholesterol and caveolin-1 are components of HMWC-ins and HMWC-CL, and insulin- and CLmediated formation of HMWCs was significantly attenuated in caveolin-1 KD adipocytes, suggesting that caveolin-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for signalosome formation and for the proper stabilization and phosphorylation/activation of PDE3B in response to CL and insulin [73]. In adipocytes, caveolin-1 may also be a scaffolding factor that is normally required for PKA-mediated signaling, including phosphorylation/activation of PDE3B as well as phosphorylation of perilipin and HSL, and activation of lipolysis.

Summary and Conclusion

▼

Formation of PDE-containing signalosomes through adaptor, anchoring, and scaffold proteins contribute to the specificity of cAMP-mediated signal transduction by recruiting specific signaling molecules into compartmentalized signaling networks, in which spatial constraints and organization allow tight local control of cAMP signals and their temporal transduction along specific pathways. For example, analysis of signalosomes in cardiomyocytes suggest that PDE3A isoforms, when phosphorylated, are incorporated into macromolecular regulatory complexes containing PDE3A/AKAP18/SERCA2, and that, as a component of this signalosome, PDE3A may regulate a discrete cAMP pool that controls contractility by modulating Ca2+ uptake into the SR. On the other hand, PDE3B was found to associate with PI3-Kγ-containing scaffolds that also regulate cAMP levels and cardiac function. Similarly, in adipocytes PDE3B is an important regulatory effector in signaling pathways controlled by insulin and cAMP-increasing hormones. Subcellular localization of membrane-associated PDE3B in distinct compartments of adipocytes plays an important role in differential regulation of PDE3B via PKA and PKB signaling pathways, and provide an important example for formation of distinct signalosomes in a single cell. These studies also demonstrated important chaperone and scaffolding roles of caveolin-1 in adipocytes, suggesting that although caveolin-1 is preferentially required for PKA-mediated signaling and lipolysis, it is also critical in the maintenance/formation of insulin- and CL-induced signalosomes, and in phosphorylation/activation of PDE3B in these cholesterol rich lipid domains.

Acknowledgements

▼

F. A. and V. M. are supported by the NHLBI Intramural Research Program; E. D. is supported in part by Swedish Research Council (Project 2010-3362).

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Notice: This article was changed according to the following erratum on September 26th 2012: The Acknowledgements were changed.



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