Sharron H. Francis

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge.

Cyclic GMP Phosphodiesterase-5: Target of Sildenafil

The advent of the medication, sildenafil, for treatment of male impotence has attracted widespread attention. This agent potently inhibits a cGMP-binding cGMP-specific phosphodiesterase (PDE5). PDE5 is particularly abundant in smooth muscle, which is enriched in other components of the cGMP signaling cascade. The characteristics of PDE5, its relationship to other PDEs, its role in cGMP signaling, and its involvement in the efficacious action of sildenafil on corpus cavernosum and vascular smooth muscle resulting in penile erection are the subjects of this review. Cyclic GMP has emerged recently as a principal focus in signal transduction. Much of this attention has derived from the fact that most of the non-lytic physiological effects of nitric oxide (Fig. 1) and all of the characterized effects of natriuretic peptides and guanylins are mediated by cGMP. In addition to the classical regulatory roles ascribed to cGMP such as stimulation of smooth muscle relaxation, neutrophil degranulation, inhibition of platelet aggregation, and initiation of visual signal transduction, numerous other physiological roles have recently been uncovered (1–10). Intracellular receptors for cGMP include cGMP-dependent protein kinases (PKG), cyclic nucleotide-gated channels, and cGMP-binding PDEs; cGMP may also cross-activate cAMP pathways by binding to cAMP-binding sites on cAMP receptors such as cAMP-dependent protein kinases (PKA) (11). Tissue cGMP levels are determined by a balance between the activities of the guanylyl cyclases that catalyze formation of cGMP from GTP and the cyclic nucleotide PDEs that catalyze the breakdown of cGMP (Fig. 1). The combination of a stimulator of guanylyl cyclase and a cGMP PDE inhibitor such as sildenafil produces synergistic enhancement of tissue cGMP levels (12). PDEs were first detected by Sutherland and co-workers (13, 14). The superfamily of PDEs is subdivided into two major classes, class I and class II (15), which have no recognizable sequence similarity. Class I includes all known mammalian PDEs and is comprised of at least 10 identified families that are products of separate genes (16–26). Some PDEs are highly specific for hydrolysis of cAMP (PDE4, PDE7, PDE8), some are highly cGMP-specific (PDE5, PDE6, PDE9), and some have mixed specificity (PDE1, PDE2, PDE3, PDE10). All of the characterized mammalian PDEs are dimeric, but the importance of the dimeric structure for function in each of the PDEs is unknown. Each PDE has a conserved catalytic domain of ;270 amino acids with a high degree of conservation (25–30%) of amino acid sequence among PDE families, which is located carboxyl-terminal to its regulatory domain. Activators of certain PDEs appear to relieve the influence of autoinhibitory domains located within the enzyme structures (27, 28). PDEs cleave the cyclic nucleotide phosphodiester bond between the phosphorus and oxygen atoms at the 39-position with inversion of configuration at the phosphorus atom (29, 30). This apparently results from an in-line nucleophilic attack by the OH of ionized H2O. It has been proposed that metals bound in the conserved metal binding motifs within PDEs facilitate the production of the attacking OH (31). The kinetic properties of catalysis are consistent with a random order mechanism with respect to cyclic nucleotide and the divalent cation(s) that are required for catalysis (32). The catalytic domains of all known mammalian PDEs contain two sequences (HX3HXn(E/D)) arranged in tandem, each of which resembles the single Zn-binding site of metalloendoproteases such as thermolysin (31). PDE5 specifically binds Zn, and the catalytic activities of PDE4, PDE5, and PDE6 are supported by submicromolar concentrations of Zn (31, 33). Whether each of the Zn binding motifs binds Zn independently or whether the two motifs interact to form a novel Zn-binding site is not known. The catalytic mechanism for cleaving phosphodiester bonds of cyclic nucleotides by PDEs may be similar to that of certain proteases for cleaving the amide ester of peptides, but the presence of two Zn motifs arranged in tandem in PDEs is unprecedented. The group of Sutherland and Rall (34), in the late 1950s, was the first to realize that at least part of the mechanism(s) whereby caffeine enhanced the effect of glucagon, a stimulator of adenylyl cyclase, on cAMP accumulation and glycogenolysis in liver involved inhibition of cAMP PDE activity. Since that time chemists have synthesized thousands of PDE inhibitors, including the widely used 3-isobutyl-1-methylxanthine (IBMX). Many of these compounds, as well as caffeine, are non-selective and inhibit many of the PDE families. One important advance in PDE research has been the discovery/design of family-specific inhibitors such as the PDE4 inhibitor rolipram and the PDE5 inhibitor sildenafil. Precise modulation of PDE function in cells is critical for maintaining cyclic nucleotide levels within a narrow rate-limiting range of concentrations. Increases in cGMP of 2–4-fold above the basal level will usually produce a maximum physiological response. There are three general schemes by which PDEs are regulated: (a) regulation by substrate availability, such as by stimulation of PDE activity by mass action after elevation of cyclic nucleotide levels or by alteration in the rate of hydrolysis of one cyclic nucleotide because of competition by another, which can occur with any of the dual specificity PDEs (e.g. PDE1, PDE2, PDE3); (b) regulation by extracellular signals that alter intracellular signaling (e.g. phosphorylation events, Ca, phosphatidic acid, inositol phosphates, protein-protein interactions, etc.) resulting, for example, in stimulation of PDE3 activity by insulin (18), stimulation of PDE6 activity by photons through the transducin system (35), which alters PDE6 interaction with this enzyme, or stimulation of PDE1 activity by increased interaction with Ca/calmodulin; (c) feedback regulation, such as by phosphorylation of PDE1, PDE3, or PDE4 catalyzed by PKA after cAMP elevation (17, 18, 36, 37), by allosteric cGMP binding to PDE2 to promote breakdown of cAMP or cGMP after cGMP elevation, or by modulation of PDE protein levels, such as the desensitization that occurs by increased concentrations of PDE3 or PDE4 following chronic exposure of cells to cAMP-elevating agents (17, 38) or by developmentally related changes in PDE5 content. Other factors that could influence any of the three schemes outlined above are cellular compartmentalization of PDEs (19) effected by covalent modifications such as prenylation or by specific targeting sequences in the PDE primary structure and perhaps translocation of PDEs between compartments within a cell. Within the PDE superfamily, four (PDE2, PDE5, PDE6, and PDE10) of the 10 families contain highly cGMP-specific allosteric (non-catalytic) cGMP-binding sites in addition to a catalytic site of varying substrate specificity. Each of the monomers of these di* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This work was supported by National Institutes of Health Grants GM41269 and DK40029 and American Heart Association Southeast Affiliate. ‡ To whom correspondence and reprint requests should be addressed: Dept. of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt University School of Medicine, 21st and Garland Aves., Nashville, TN 372320615. Tel.: 615-322-4384; Fax: 615-343-3794; E-mail: jackie.corbin@ mcmail.vanderbilt.edu. 1 Tradename VIAGRATM. 2 The abbreviations used are: PDE, 39:59-cyclic nucleotide phosphodiesterase; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; IBMX, 3-isobutyl-1-methylxanthine. 3 J. A. Beavo and K. Loughney, personal communication. 4 S. Francis, unpublished results. Minireview THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 20, Issue of May 14, pp. 13729–13732, 1999

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

The superfamily of cyclic nucleotide (cN) phosphodiesterases (PDEs) is comprised of 11 families of enzymes. PDEs break down cAMP and/or cGMP and are major determinants of cellular cN levels and, consequently, the actions of cN-signaling pathways. PDEs exhibit a range of catalytic efficiencies for breakdown of cAMP and/or cGMP and are regulated by myriad processes including phosphorylation, cN binding to allosteric GAF domains, changes in expression levels, interaction with regulatory or anchoring proteins, and reversible translocation among subcellular compartments. Selective PDE inhibitors are currently in clinical use for treatment of erectile dysfunction, pulmonary hypertension, intermittent claudication, and chronic pulmonary obstructive disease; many new inhibitors are being developed for treatment of these and other maladies. Recently reported x-ray crystallographic structures have defined features that provide for specificity for cAMP or cGMP in PDE catalytic sites or their GAF domains, as well as mechanisms involved in catalysis, oligomerization, autoinhibition, and interactions with inhibitors. In addition, major advances have been made in understanding the physiological impact and the biochemical basis for selective localization and/or recruitment of specific PDE isoenzymes to particular subcellular compartments. The many recent advances in understanding PDE structures, functions, and physiological actions are discussed in this review.

Cyclic Nucleotide-Dependent Protein Kinases: Intracellular Receptors for cAMP and cGMP Action

Intracellular cAMP and cGMP levels are increased in response to a variety of hormonal and chemical stimuli; these nucleotides play key roles as second messenger signals in modulating myriad physiological processes. The cAMP-dependent protein kinase and cGMP-dependent protein kinase are major intracellular receptors for these nucleotides, and the actions of these enzymes account for much of the cellular responses to increased levels of cAMP or cGMP. This review summarizes many studies that have contributed significantly to an improved understanding of the catalytic, regulatory, and structural properties of these protein kinases. These accumulated findings provide insights into the mechanisms by which these enzymes produce their specific physiological effects and are helpful in considering the actions of other protein kinases as well.

Crystal Structures of Phosphodiesterases 4 and 5 in Complex with Inhibitor 3-Isobutyl-1-methylxanthine Suggest a Conformation Determinant of Inhibitor Selectivity

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. Crystal structures of the catalytic domains of cGMP-specific PDE5A1 and cAMP-specific PDE4D2 in complex with the nonselective inhibitor 3-isobutyl-1-methylxanthine have been determined at medium resolution. The catalytic domain of PDE5A1 has the same topological folding as that of PDE4D2, but three regions show different tertiary structures, including residues 79-113, 208-224 (H-loop), and 341-364 (M-loop) in PDE4D2 or 535-566, 661-676, and 787-812 in PDE5A1, respectively. Because H- and M-loops are involved in binding of the selective inhibitors, the different conformations of the loops, thus the distinct shapes of the active sites, will be a determinant of inhibitor selectivity in PDEs. IBMX binds to a subpocket that comprises key residues Ile-336, Phe-340, Gln-369, and Phe-372 of PDE4D2 or Val-782, Phe-786, Gln-817, and Phe-820 of PDE5A1. This subpocket may be a common site for binding nonselective inhibitors of PDEs.

The Type II Isoform of cGMP-dependent Protein Kinase Is Dimeric and Possesses Regulatory and Catalytic Properties Distinct from the Type I Isoforms

The type I cGMP-dependent protein kinases (cGK Iα and Iβ) form homodimers (subunit Mr ∼ 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (Mr ∼ 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography were used to determine Mr values for holoenzymes of cGK Iα (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK Iα possessed significantly lower Ka values for cGMP (8-fold) and β-phenyl-1,N2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp- and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3′,5′-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f2B had a 20-fold greater Vmax/Km ratio for cGK Iα than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater Vmax/Km ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities.

Multiple Conformations of Phosphodiesterase-5 IMPLICATIONS FOR ENZYME FUNCTION AND DRUG DEVELOPMENT

Phosphodiesterase-5 (PDE5) is the target for sildenafil, vardenafil, and tadalafil, which are drugs for treatment of erectile dysfunction and pulmonary hypertension. We report here the crystal structures of a fully active catalytic domain of unliganded PDE5A1 and its complexes with sildenafil or icarisid II. These structures together with the PDE5A1-isobutyl-1-methylxanthine complex show that the H-loop (residues 660-683) at the active site of PDE5A1 has four different conformations and migrates 7-35Å upon inhibitor binding. In addition, the conformation of sildenafil reported herein differs significantly from those in the previous structures of chimerically hybridized or almost inactive PDE5. Mutagenesis and kinetic analyses confirm that the H-loop is particularly important for substrate recognition and that invariant Gly659, which immediately precedes the H-loop, is critical for optimal substrate affinity and catalytic activity.

Autophosphorylation of Type Iβ cGMP-dependent Protein Kinase Increases Basal Catalytic Activity and Enhances Allosteric Activation by cGMP or cAMP

Autophosphorylation of purified bovine Iβ isozyme of cGMP-dependent protein kinase (Iβ cGK) in the presence of cGMP or cAMP increased basal kinase activity (−cGMP) as much as 4-fold and reduced the Ka for both cGMP and cAMP; maximum catalytic activity (+cGMP) was not altered. Autophosphorylation proceeded with at least two rate components. The faster rate correlated with phosphorylation of Ser-63. The slower rate, as well as the increase in basal kinase activity and decrease in Ka for cyclic nucleotides, correlated with phosphorylation of Ser-79. Autophosphorylation of either residue was an intramolecular reaction. Autophosphorylation of a proteolytically generated Iβ cGK monomer lacking amino-terminal residues 1-64 increased basal activity (3-fold) and decreased Ka for cAMP (15-fold). This indicated that autophosphorylation of Ser-79 did not require dimeric cGK and that the phosphorylation of Ser-79 in the monomer was sufficient to alter enzymatic characteristics of Iβ cGK. These studies suggested that increases in intracellular cGMP or cAMP could result in autophosphorylation of Iβ cGK, which would increase basal kinase activity as well as the sensitivity of cGK to activation by cGMP or to cross-activation by cAMP. Autophosphorylation could also prolong the increased kinase activity after decline of the second messenger.

Autophosphorylation: a salient feature of protein kinases

Most protein kinases catalyze autophosphorylation, a process which is generally intramolecular and is modulated by regulatory ligands. Either serine/threonine or tyrosine serves as the phosphoacceptor, and several sites on the same kinase subunit are usually autophosphorylated. Autophosphorylation affects the functional properties of most protein kinases. Members of the protein kinase family exhibit diversity in the characteristics and functions of autophosphorylation, but certain common themes are emerging.

POTENTIAL ROLES OF CONSERVED AMINO ACIDS IN THE CATALYTIC DOMAIN OF THE CGMP-BINDING CGMP-SPECIFIC PHOSPHODIESTERASE (PDE5)

The known mammalian 3′:5′-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain. To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5). Wild-type and mutant proteins were compared with respect toK m for cGMP, k cat, and IC50 for zaprinast. The most dramatic decrease ink cat was seen with H643A and D754A mutants with the decrease in free energy of binding (ΔΔG T ) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue. His643 and Asp754 are conserved in all known PDEs and are strong candidates to be directly involved in catalysis. Substitutions of His603, His607, His647, Glu672, and Asp714 also produced marked changes in k cat, and these residues are likely to be important for efficient catalysis. The Y602A and E775A mutants exhibited the most dramatic increases inK m for cGMP, with calculated ΔΔG T of 2.9 and 2.8 kcal/mol, respectively, that these two residues are important for cGMP binding in the catalytic site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.