Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activities.

In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971-14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50-70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2-0.5 microM PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 microM. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.     

Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells.

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.     

The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.     

Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity

Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively). [(3)H]cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.     

Structural and functional features in human PDE5A1 regulatory domain that provide for allosteric cGMP binding, dimerization, and regulation

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.     

Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression.

cGMP is a second messenger that produces its effects by interacting with intracellular receptor proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.     

Inhibition of cyclic GMP hydrolysis in human corpus cavernosum smooth muscle cells by vardenafil, a novel, selective phosphodiesterase type 5 inhibitor.

One of the key mediators of penile erectile function is nitric oxide (NO), which activates soluble guanylyl cyclase within the smooth muscle of erectile tissue and stimulates the production of cGMP. In addition to synthesis by cyclases, intracellular cGMP concentrations are tightly regulated by phosphodiesterases, which hydrolyze and inactivate cyclic nucleotides. In this study, we compared the inhibition of cGMP hydrolysis by vardenafil and sildenafil; two inhibitors selective for phosphodiesterase type 5 (PDE5). Vardenafil is a novel, high affinity PDE5 inhibitor currently under clinical development. In soluble extracts of human corpus cavernosum smooth muscle cells, vardenafil and sildenafil effectively inhibited cGMP hydrolysis at substrate concentrations of 1, 5 and 10 microM cGMP. The IC50 values for vardenafil were approximately 5-fold lower than for sildenafil at the substrate concentrations tested. Dixon plot analyses of the inhibition data demonstrated that vardenafil had a smaller inhibition constant (Ki = 4.5 nM) than sildenafil (Ki = 14.7 nM) in the same cellular extracts. In intact cells, 10 microM of the nitric oxide donor sodium nitroprusside resulted in a minimal (17%) increase in cGMP, relative to basal levels (321 +/- 65 fmol/mg prot). Treatment of cells with 10, 50 or 100 nM vardenafil, in the presence of 10 microM sodium nitroprusside, elevated cGMP levels in a dose dependent fashion, from 63% to 137% of basal levels. Equimolar concentrations of sildenafil also caused dose dependent increases in intracellular cGMP, but to a lesser extent (27-60%). These observations suggest that vardenafil is a more potent PDE5 inhibitor, than sildenafil in vitro. The more pronounced increase of cGMP in the presence of NO in intact cells suggests that vardenafil will be effective at lower doses than sildenafil under clinical conditions.     

PDE5 is converted to an activated state upon cGMP binding to the GAF A domain

cGMP-specific, cGMP-binding phosphodiesterase (PDE5) regulates such physiological processes as smooth muscle relaxation and neuronal survival. PDE5 contains two N-terminal domains (GAF A and GAF B), but the functional roles of these domains have not been determined. Here we show that recombinant PDE5 is activated directly upon cGMP binding to the GAF A domain, and this effect does not require PDE5 phosphorylation. PDE5 exhibited time- and concentration-dependent reversible activation in response to cGMP, with the highest activation (9- to 11-fold) observed at low substrate concentrations (0.1 micro M cGMP). A monoclonal antibody directed against GAF A blocked cGMP binding, prevented PDE5 activation and decreased basal activity, revealing that PDE5 in its non-activated state has low intrinsic catalytic activity. Activated PDE5 showed higher sensitivity towards sildenafil than non-activated PDE5. The stimulatory effect of cGMP binding on the catalytic activity of PDE5 suggests that this mechanism of enzyme activation may be common among other GAF domain-containing proteins. The data also suggest that development of agonists and antagonists of PDE5 activity based on binding to this site might be possible.     

Nitric oxide-evoked transient kinetics of cyclic GMP in vascular smooth muscle cells

Cyclic-3',5'-guanosine monophosphate (cGMP) mediates the intracellular signaling cascade responsible for the nitric oxide (NO) initiated relaxation of vascular smooth muscle (VSM). However, the temporal dynamics, including the regulation of cGMP turnover, are largely unknown. Here we report new mechanistic insights into the kinetics of cGMP synthesis and hydrolysis in primary VSM cells by utilizing FRET-based cGMP-indicators [A. Honda, S.R. Adams, C.L. Sawyer, V. Lev-Ram, R.Y. Tsien, W.R. Dostmann, Proc. Natl. Acad. Sci. U S A 98 (5) (2001) 2437.]. First, 2-(N,N-Diethylamino)-diazenolate 2-oxide (DEA/NO) and 2,2'-(Hydroxynitrosohydrazono)-bis-ethanimine (DETA/NO) induced NO-concentration dependent, transient cGMP responses ("peaks") irrespective of their rates of NO release. The kinetic characteristics of these cGMP peaks were governed by the concerted action of the NO-sensitive guanylyl cyclase (GC) and phosphodiesterase type V (PDE5) as shown by their respective inhibition using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and Sildenafil. These responses occurred in the presence of moderately elevated cGMP (5-15% FRET ratio), and thus activated PKG and phosphorylated PDE5, suggesting a prominent role for GC in the maintenance and termination of cGMP peaks. Furthermore, cGMP transients could be elicited repeatedly without apparent desensitization of GC or by suppression of cGMP via long-term PDE5 activity. These results demonstrate a continuous sensitivity of the NO/cGMP signaling system, inherent to the phasic nature of smooth muscle physiology.     

Allosteric activation of cGMP-specific,cGMP-binding phosphodiesterase (PDE5) by cGMP

The effects of cGMP binding on the catalytic activity of cGMP-specific, cGMP-binding phosphodiesterase (PDE5) are unclear because cGMP interacts with both allosteric and catalytic sites specifically. We studied the effects of cGMP on the hydrolysis of a fluorescent substrate analogue, 2'-O-anthraniloyl cGMP, by PDE5 partially purified from rat cerebella. The preparation contained PDE5 as the major cGMP-PDE activity and was not contaminated with cAMP- or cGMP-dependent protein kinases. The Hill coefficients for hydrolysis of the analogue substrate were around 1.0 in the presence of cGMP at concentrations <0.3 microM, while they increased to 1.5 at cGMP concentrations >1 microM, suggesting allosteric activation by cGMP at concentrations close to the bulk binding constant of the enzyme. Consistent with an allosteric activation, increasing concentrations of cGMP enhanced the hydrolysis rate of fixed concentrations of 2'-O-anthraniloyl cGMP, which overcame competition between the two substrates. Such activation was not observed with cAMP, cyclic inosine 3',5'-monophosphate, or 2'-O-monobutyl cGMP, indicating specificity of cGMP. These results demonstrate that cGMP is a specific and allosteric activator of PDE5, and suggest that in cells containing PDE5, such as cerebellar Purkinje cells, intracellular cGMP concentrations may be regulated autonomously through effects of cGMP on PDE5.     

Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells.

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 microM cAMP and 3.1 microM catalytic subunit of PKA, which is equivalent to 6.2 microM cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 microM cGMP and 7.3 microM PKG monomer, equivalent to 14.6 microM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 microM. Treatment of washed human platelets with 10 microM (or 10 mM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 microM (10 microM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100 microM (or 1 microM) sodium nitroprusside elevated the platelet cGMP level to 4 microM (0.9 microM with 1 microM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 microM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increase in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of soluble guanylate cyclase activity by phosphorylation

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.     

Distinguishing the roles of the two different cGMP-binding sites for modulating phosphorylation of exogenous substrate (heterophosphorylation) and autophosphorylation of cGMP-dependent protein kinase

The role of each of the two different cGMP-binding sites (referred to as slow and fast sites) of type I cGMP-dependent protein kinase (PKG) in altering the rate of catalysis of phosphorylation of exogenous substrates (heterophosphorylation) or the rate of autophosphorylation has not been resolved. In the present study, the cGMP concentration required for half-maximal activation (A(50)) of wild-type PKG type Ibeta (WT) was 5-fold higher for heterophosphorylation than for autophosphorylation. cGMP occupation of the slow site was associated with an increase in the autophosphorylation rate, whereas occupation of the fast and slow site together was associated with a decrease in the autophosphorylation rate compared with the rate observed with occupation of the slow site alone. The contributions of each cGMP-binding site were investigated using PKG mutants containing substitutions of an invariant threonine residue that is critical for high affinity cGMP-binding in each site. Site-directed mutagenesis of Thr-317 of the fast site (T317A) increased the cGMP A(50) for heterophosphorylation 4-fold at 30 degrees C, with nominal effect on cGMP A(50) for autophosphorylation compared with WT. The analogous slow site mutation (T193A) increased the cGMP A(50) for heterophosphorylation and autophosphorylation 32- and 64-fold, respectively. Compared with WT, the cGMP A(50) of the double mutant (T193A/T317A) for heterophosphorylation was increased 300-fold, whereas the cGMP A(50) for autophosphorylation was similar to that of T193A. Thus, occupation of both cGMP-binding sites of PKG is required for maximal stimulation of heterophosphorylation, whereas occupation of the slow site alone is sufficient for stimulation of the rate of autophosphorylation, and additional occupation of the fast site reduces this rate.     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.     

Subcellular localization and regulation of type-1C and type-5 phosphodiesterases

We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for the first time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype.     

Cyclic nucleotide phosphodiesterase 3 signaling complexes

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 biochemical 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.     

Phosphorylation and regulation of G-protein-activated phospholipase C-beta 3 by cGMP-dependent protein kinases

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, beta-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca(2+)] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca(2+) by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-beta) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca(2+). In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-beta2 and PLC-beta3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-activated PLC-beta3 activity by 50-70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-beta3 by PKG (Ser(26) and Ser(1105)). Mutation at these two sites (S26A and S1105A) of PLC-beta3 completely blocked the phosphorylation of PLC-beta3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-beta3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca(2+)] by the NO-cGMP-dependent signaling pathway.     

Expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) in human colonic epithelial cells: role in the induction of cellular refractoriness to the heat-stable enterotoxin peptide

Stable toxin (ST) peptides are the causative agents for a severe form of watery diarrhea. These peptides bind to a membrane-associated form of guanylyl cyclase, guanylyl cyclase C. The result is an accumulation of cyclic guanosine monophosphate (cGMP) in the intestinal cell, regulating protein kinase activity and the phosphorylation of a number of proteins involved in ion transport across the intestine. Using the human T84 colonic cell line as a model system, we show that cGMP accumulation in these cells after ST application is regulated by the activity of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5). The presence of human PDE5 in this cell line was confirmed by Western blot analysis, using an antibody raised to the bovine enzyme, and by the observation that cGMP hydrolytic activity detected in T84 cell lysates was almost completely inhibited by low concentrations of zaprinast, a specific inhibitor of PDE5. An increase in activity of PDE5 was observed in T84 cell lysates on exposure to the ST peptide and prolonged exposure of T84 cells to the ST peptide led to the induction of cellular refractoriness in these cells, which was largely contributed in terms of an increased rate of degradation of cGMP in desensitized cells as a result of PDE5 activation. This activation was correlated with an increase in the affinity of the enzyme for the substrate cGMP, as well as an increased affinity for zaprinast. We provide evidence for the first time that cGMP levels in the human colonocyte are regulated by the cGMP-hydrolytic activity of PDE5 and suggest that the expression and regulation of PDE5 in the intestine could therefore be important in controlling cGMP-mediated signaling in this tissue.     

IRAG and novel PKG targeting in the cardiovascular system

Signaling by nitric oxide (NO) determines several cardiovascular functions including blood pressure regulation, cardiac and smooth muscle hypertrophy, and platelet function. NO stimulates the synthesis of cGMP by soluble guanylyl cyclases and thereby activates cGMP-dependent protein kinases (PKGs), mediating most of the cGMP functions. Hence, an elucidation of the PKG signaling cascade is essential for the understanding of the (patho)physiological aspects of NO. Several PKG signaling pathways were identified, meanwhile regulating the intracellular calcium concentration, mediating calcium desensitization or cytoskeletal rearrangement. During the last decade it emerged that the inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG), an endoplasmic reticulum-anchored 125-kDa membrane protein, is a main signal transducer of PKG activity in the cardiovascular system. IRAG interacts specifically in a trimeric complex with the PKG1β isoform and the inositol 1,4,5-trisphosphate receptor I and, upon phosphorylation, reduces the intracellular calcium release from the intracellular stores. IRAG motifs for phosphorylation and for targeting to PKG1β and 1,4,5-trisphosphate receptor I were identified by several approaches. The (patho)physiological functions for the regulation of smooth muscle contractility and the inhibition of platelet activation were perceived. In this review, the IRAG recognition, targeting, and function are summarized compared with PKG and several PKG substrates in the cardiovascular system.