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• W2002161089 abstract "Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGIα-(Δ349–670) and PKGIβ-(Δ364–685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 Å, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and KD values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGIα-isolated R domain and contraction of the PKGIβ-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGIβ holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIα R domain may partially account for the elongation of wild type PKGIα associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation. Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGIα-(Δ349–670) and PKGIβ-(Δ364–685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 Å, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and KD values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGIα-isolated R domain and contraction of the PKGIβ-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGIβ holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIα R domain may partially account for the elongation of wild type PKGIα associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation. Mammalian PKGs 4The abbreviations used are: PKG, cGMP-dependent protein kinase; R domain, regulatory domain; C domain, catalytic domain; IBMX, 3-isobutyl-1-methylxanthine; AA, amino acids; WT, wild type.4The abbreviations used are: PKG, cGMP-dependent protein kinase; R domain, regulatory domain; C domain, catalytic domain; IBMX, 3-isobutyl-1-methylxanthine; AA, amino acids; WT, wild type. (PKGIα, PKGIβ, and PKGII) are major intracellular receptors for cGMP (1Francis S.H. Corbin J.D. Murad F. Cyclic GMP: Synthesis, Metabolism, and Function. Academic Press, Orlando1993: 115-170Google Scholar, 2Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (260) Google Scholar). PKGIα and PKGIβ, products of alternative splicing, differ only in the first ∼100 AA (3Francis S.H. Woodford T.A. Wolfe L. Corbin J.D. Second Messengers and Phosphoproteins. 1988; 12: 301-310PubMed Google Scholar, 4Wernet W. Flockerzi V. Hofmann F. FEBS Lett. 1989; 251: 191-196Crossref PubMed Scopus (161) Google Scholar, 5Sandberg M. Natarajan V. Ronander I. Kalderon D. Walter U. Lohmann S.M. Jahnsen T. FEBS Lett. 1989; 255: 321-329Crossref PubMed Scopus (115) Google Scholar, 6Wolfe L. Corbin J.D. Francis S.H. J. Biol. Chem. 1989; 264: 7734-7741Abstract Full Text PDF PubMed Google Scholar), but PKGII is a separate gene product (7Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Abstract Full Text PDF PubMed Google Scholar, 8Gamm D.M. Francis S.H. Angelotti T.P. Corbin J.D. Uhler M.D. J. Biol. Chem. 1995; 270: 27380-27388Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Cyclic GMP-mediated activation of PKGs in response to nitric oxide, natriuretic peptides, or guanylins is integrally involved in myriad physiological processes, including modulation of vascular smooth muscle tone, water, and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation, and bone formation. Despite the central role of these enzymes in both physiological and pathophysiological processes such as erectile dysfunction, pulmonary hypertension, or systemic hypertension, little is known about the molecular mechanism whereby these kinases are activated. PKGs are chimeric proteins composed of regulatory (R) and catalytic (C) domains (9Takio K. Wade R.D. Smith S.B. Krebs E.G. Walsh K.A. Titani K. Biochemistry. 1984; 23: 4207-4218Crossref PubMed Scopus (191) Google Scholar). The C domain, located in the carboxyl-terminal region of the polypeptide, contains an ATP-binding subdomain and a protein substrate-binding subdomain. The C domain is the most highly conserved region among the PKGs and catalyzes transfer of the γ-phosphate from ATP to specific serines or threonines in consensus sequences in protein substrates. The R domain, located in the amino-terminal segment of PKG, also contains a number of critical functional subdomains; the amino-terminal 100 AA, which have only 35% identity in PKGIα and PKGIβ, include a dimerization subdomain, autoinhibitory subdomain, and autophosphorylation sites. The remainder of the R domain sequence, which is identical in PKGIα and PKGIβ, contains the two allosteric cGMP-binding sites A and B (Fig. 1A). Despite identical AA sequences of the cGMP-binding sites and the C domains in PKGIα and PKGIβ, the isoforms differ substantially in cyclic nucleotide analog specificities, KD for cGMP binding, and Ka for cGMP activation of catalytic activity (9Takio K. Wade R.D. Smith S.B. Krebs E.G. Walsh K.A. Titani K. Biochemistry. 1984; 23: 4207-4218Crossref PubMed Scopus (191) Google Scholar, 10Reed R.B. Sandberg M. Jahnsen T. Lohmann S.M. Francis S.H. Corbin J.D. J. Biol. Chem. 1996; 271: 17570-17575Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 11Corbin J.D. Doskeland S.O. J. Biol. Chem. 1983; 258: 11391-11397Abstract Full Text PDF PubMed Google Scholar, 12Corbin J.D. Ogreid D. Miller J.P. Suva R.H. Jastorff B. Doskeland S.O. J. Biol. Chem. 1986; 261: 1208-1214Abstract Full Text PDF PubMed Google Scholar). This indicates that elements in the amino-terminal ∼100 AA (Fig. 1A) allosterically affect functions of the cGMP-binding sites and activation of kinase activity, but the molecular basis of this is not understood (6Wolfe L. Corbin J.D. Francis S.H. J. Biol. Chem. 1989; 264: 7734-7741Abstract Full Text PDF PubMed Google Scholar, 13Ruth P. Landgraf W. Keilbach A. May B. Egleme C. Hofmann F. Eur. J. Biochem. 1991; 202: 1339-1344Crossref PubMed Scopus (87) Google Scholar). Dimerization of PKGs in higher eukaryotes occurs in large part through a conserved leucine zipper motif near the amino terminus (14Atkinson R.A. Saudek V. Huggins J.P. Pelton J.T. Biochemistry. 1991; 30: 9387-9395Crossref PubMed Scopus (60) Google Scholar, 15Richie-Jannetta R. Francis S.H. Corbin J.D. J. Biol. Chem. 2003; 278: 50070-50079Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), and the autoinhibitory subdomain and autophosphorylation sites are located just carboxyl-terminal to this motif (Fig. 1A). The two allosteric cGMP-binding sites are homologous to each other and are arranged in tandem immediately amino-terminal to the C domain; this comprises the cGMP-binding subdomain. Regions outside the cGMP-binding sites in both PKGIα and PKGIβ modulate affinity of the enzymes for cGMP; Mg2+/ATP increases the dissociation rate of cGMP from site B of PKGIα by 10-fold, thereby weakening cGMP binding (11Corbin J.D. Doskeland S.O. J. Biol. Chem. 1983; 258: 11391-11397Abstract Full Text PDF PubMed Google Scholar, 16Kotera J. Grimes K.A. Corbin J.D. Francis S.H. Biochem. J. 2003; 372: 419-426Crossref PubMed Scopus (32) Google Scholar, 17Doskeland S.O. Vintermyr O.K. Corbin J.D. Ogreid D. J. Biol. Chem. 1987; 262: 3534-3540Abstract Full Text PDF PubMed Google Scholar), and the amino-terminal 100 AA alter cGMP affinity and cyclic nucleotide specificity of sites with identical primary AA sequence. Most of this influence on the cGMP-binding properties of PKGs has been presumed to involve interaction of the autoinhibitory domain with the C domain, but there is no clear documentation of this. In addition, the Mg2+/ATP effect observed in PKGIα is largely lacking in PKGIβ (16Kotera J. Grimes K.A. Corbin J.D. Francis S.H. Biochem. J. 2003; 372: 419-426Crossref PubMed Scopus (32) Google Scholar). Understanding the functional properties of the R and C domains is required in order to define the molecular mechanism by which cGMP binding brings about activation of PKG. We have previously used a number of approaches in an effort to better understand the molecular events associated with activation of PKG kinase activity. Cyclic GMP binding to PKGIα or PKGIβ induces a molecular elongation that is associated with activation of catalytic activity and increased surface electronegativity (18Chu D.M. Corbin J.D. Grimes K.A. Francis S.H. J. Biol. Chem. 1997; 272: 31922-31928Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 19Zhao J. Trewhella J. Corbin J. Francis S. Mitchell R. Brushia R. Walsh D. J. Biol. Chem. 1997; 272: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 20Wall M.E. Francis S.H. Corbin J.D. Grimes K. Richie-Jannetta R. Kotera J. Macdonald B.A. Gibson R.R. Trewhella J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2380-2385Crossref PubMed Scopus (61) Google Scholar). The molecular basis for the conformational change associated with activation of each isozyme is unknown. The Stokes radii of the cGMP-bound and cGMP-free forms of PKGIα and PKGIβ differ by ∼3–4 Å as measured by gel filtration chromatography (18Chu D.M. Corbin J.D. Grimes K.A. Francis S.H. J. Biol. Chem. 1997; 272: 31922-31928Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Small angle x-ray scattering studies have shown that cGMP binding to dimeric PKGIα increases its radius of gyration (Rg) and maximum linear dimension (dmax) 25–30%, indicating that upon cGMP binding the average protein mass moves farther from the center of mass, resulting in a more asymmetric structure (19Zhao J. Trewhella J. Corbin J. Francis S. Mitchell R. Brushia R. Walsh D. J. Biol. Chem. 1997; 272: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Cyclic GMP also induces increased elongation and asymmetry in a monomeric cGMP-dependent PKG mutant, PKGIβ-(Δ1–52), that retains the salient biochemical features of wild type (WT) PKG (20Wall M.E. Francis S.H. Corbin J.D. Grimes K. Richie-Jannetta R. Kotera J. Macdonald B.A. Gibson R.R. Trewhella J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2380-2385Crossref PubMed Scopus (61) Google Scholar); the magnitude of this change can fully account for the elongation observed in dimeric PKG. These results demonstrate that elongation of type I PKGs correlates with activation and is independent of dimerization. However, the molecular basis for the elongation is not understood and could be the result of structural changes initiated by the R domain, C domain, or both. The novel amino-terminal AA sequences of PKGIα and PKGIβ provide for different cGMP-binding characteristics. Whether this is because of contacts between the respective amino termini and C domain or between the amino termini and elements within the R domain itself has not been experimentally tested. Understanding the role of the respective functional domains in the activation process is key to dissecting the activation mechanism of PKG and interpreting results of biophysical studies of this process. We document the following for the first time: (a) dimerization of PKG R domains occurs in the absence of contributions from the C domain; (b) counter to currently held beliefs, determinants of differences in cGMP binding affinities of PKGIα and PKGIβ reside exclusively within the R domain; and (c) cGMP binding induces isoform-specific conformational changes in the isolated R domains that can be related to the mechanism for activation of kinase catalytic activity. Mutagenesis of PKGIα and PKGIβ to Produce the Isolated Regulatory Domains—PKGIβ cDNA encodes full-length human PKGIβ. A stop codon was introduced after AA 364 of PKGIβ to create the isolated R domain. PKGIβ cDNA was ligated into the EcoRI and BamHI unique sites of the baculovirus expression vector pVL1392. Oligonucleotides (Operon, Inc.) with the following sequences were used to construct the deletion mutant. The underlined nucleotides incorporate a 3′ stop codon and a 3′ BamHI site. For deletion mutagenesis of PKGIβ-(Δ364–685), the coding strand oligonucleotide was 5′-GCCATGTGACCCT-3′ and the noncoding strand oligonucleotide was 5′-GCCGGGATCCTTACGCTTCAGCTTCATATTT-3′. This region was amplified by PCR for 35 cycles with the melting, annealing, and elongating temperatures of 95, 50, and 72 °C, respectively. The 800-bp PCR product was digested with BamHI and Tth111I (New England Biolabs), purified, and cloned into BamHI/Tth111I-digested wild type PKGIβ clone in the pVL1392 baculovirus expression vector. Because PKGIα and PKGIβ differ only in their amino termini, PKGIα-(Δ349–670) was produced by digesting PKGIβ-(Δ364–685) in pVL1392 with NcoI/BamHI and subcloning the small digestion product into NcoI/BamHI-digested wild type PKGIα cDNA in the pVL1392 baculovirus expression vector. Escherichia coli DH5α was used for transformations with pVL1392. DNA fragments were purified using a Clontech gel extraction kit according to the manufacturer's protocol (BD Biosciences). DNA was purified from large scale vector preparations using a BD Biosciences Plasmid Midi kit according to the manufacturer's protocol. All DNA segments subjected to mutagenesis and subcloning reactions were sequenced in their entirety to ensure the presence of the desired mutation and proper in-frame subcloning. Plasmids were sequenced on a 373A DNA sequencer (Automated Biosystems, Inc.) at the Vanderbilt University Cancer Center DNA Core. Expression and Purification of WT and Mutant PKGI R Domain Constructs—Sf9 cells (Pharmingen) were cotransfected with BaculoGold linear baculovirus DNA (Pharmingen) and either WT PKGIα, WT PKGIβ or one of the mutated PKGIα or PKGIβ clones in the pVL1392 baculovirus expression vector by the calcium phosphate method according to the protocol from Pharmingen. At 5 days post-infection, the cotransfection supernatant was collected, amplified three times in Sf9 cells, and then used directly as viral stock for expression without additional purification of recombinant viruses. Sf9 cells grown at 27 °C in complete Grace's insect medium with 10% fetal bovine serum and 10 μg/ml gentamycin (Sigma) in T-175 flasks (Corning) were typically infected with 10–100 μl of viral stock/flask. The optimum volume of viral stock used per T-175 flask was experimentally determined. The Sf9 cell pellet was harvested at 72–96 h post-infection. The Sf9 cell pellet for each T-175 flask (∼2 × 107 cells) was resuspended in 3 ml of ice-cold 10 mm potassium phosphate, pH 6.8, 1 mm EDTA, and 25 mm β-mercaptoethanol (KPEM) containing the concentration of protease inhibitor mixture tablets (Roche Applied Science) as recommended by the manufacturer. Cell suspension was homogenized in 10- to 20-ml aliquots two times with 4-s bursts in an Ultra Turrex microhomogenizer with a 20-s recovery between bursts. The cell homogenate was centrifuged at 12,000 rpm in a Beckman JA-20 rotor for 30 min at 4 °C. The supernatant was loaded onto an 8-aminohexylamino-cAMP-Sepharose (Sigma) column (1 × 1.5 cm) equilibrated with KPEM. The supernatant volume varied depending on number of T-175 flasks infected. The column was washed with 5 ml of KPEM containing protease inhibitors followed by 10 ml of 0.5 m NaCl in KPEM with protease inhibitors. 10 mm cAMP in KPEM containing 1 m NaCl and protease inhibitors was added to the column and allowed to soak into the cAMP-Sepharose. The column was stopped for 20 min at 4 °C, after which five 0.3-ml elutions were collected. The column was again incubated for 20 min at 4 °C, and an additional five 0.3-ml fractions were collected. Elutions containing significant protein as measured by the Bradford method were pooled and concentrated on Centricon-30 (Amicon). The concentrated sample was chromatographed on a Sephacryl S-200 column (0.9 × 35 cm) equilibrated in KPEM, 0.15 m NaCl with protease inhibitors, and 0.5-ml fractions were collected. This step removed the free cAMP from the affinity column elution. All purification steps were performed at 4 °C; enzyme was flash-frozen in KPEM containing 0.3 m NaCl and 10% sucrose and stored at –70 °C until use. Two preparations of the isolated R domains of PKGIα and PKGIβ were expressed, purified, and used in the experiments. [3H]cGMP Binding—To further reduce cAMP that might be contained in the protein samples following the gel filtration step of the purification procedure, WT PKGs and R domains of each (3–6 μm) were diluted 500× with KPEM, 0.15 m NaCl containing protease inhibitors and 1 mg/ml bovine serum albumin. Even if the PKG proteins were still saturated with cAMP after gel filtration, this dilution would reduce cAMP concentration to ∼1.2–2.5 nm, which is well below the KD values of 6 and 10 μm for intact PKGIα and PKGIβ, respectively (6Wolfe L. Corbin J.D. Francis S.H. J. Biol. Chem. 1989; 264: 7734-7741Abstract Full Text PDF PubMed Google Scholar). To saturate the PKGs with cGMP, 50-μl aliquots of the diluted PKG constructs were incubated for 30–60 min at room temperature with 50 μl of [3H]cGMP and 150 μl of cGMP-binding assay mixture (25 mm K2HPO4, 25 mm KH2PO4, 1 mm EDTA, pH 6.8, 2 m NaCl, 200 μm 3-isobutyl-1-methylxanthine (IBMX), and 0.5 mg/ml histone IIAS (Sigma)). The final cGMP concentration varied from 0 to 7 μm for PKGIβ and 0 to 3 μm for PKGIα, and the final concentration of enzyme (monomer) was ∼2 nm. After incubation, 2 ml of cold aqueous saturated (NH4)2SO4 was added to each sample. Samples were filtered onto 0.45-μm pore nitrocellulose paper (Millipore) that had been premoistened with saturated (NH4)2SO4 and rinsed three times with 2 ml of cold saturated (NH4)2SO4. Papers were dried and shaken in vials containing 1.5 ml 2% SDS. Aqueous scintillant (10 ml) was added, and the vials were shaken again and then counted in a liquid scintillation counter. KD value was determined by GraphPad Prism graphics. [3H]cGMP Dissociation—WT PKGIα and PKGIβ and their respective isolated R domains (0.20–0.40 mg/ml initial concentration) were diluted 200× with KPEM, 0.15 m NaCl containing protease inhibitors and 1 mg/ml bovine serum albumin. 1 ml of diluted PKG was incubated for 30–60 min at room temperature with 3 ml of cGMP-binding assay mixture 100 μ and l of [3H]cGMP for a final concentration of 2.9 μm [H]cGMP. This incubation time was experimentally determined to be adequate for saturation of the cGMP-binding sites in these proteins. After incubation, samples were cooled to 4 °C and divided into 200-μl aliquots per tube. Addition of 100-fold excess unlabeled cGMP at time 0 (Bo) initiated the dissociation (exchange) of bound [3H]cGMP. The cGMP exchange in each tube was stopped at the appropriate time point by addition of 2 ml of cold aqueous saturated ammonium sulfate. Samples were filtered and washed as described previously for cGMP binding. The portion of [3H]cGMP bound at any time point was determined by the method of Rannels and Corbin (21Rannels S.R. Corbin J.D. Methods Enzymol. 1983; 99: 168-175Crossref PubMed Scopus (16) Google Scholar). Gel Filtration Chromatography and Determination of Stokes Radius—Each of the respective purified PKG proteins (∼10 μg) was combined with two internal standards, crystalline catalase (3 mg) and ovalbumin (4 mg), in a volume of 200 μl and applied to a Sephacryl S-200 gel filtration column (0.9 × 35 cm) equilibrated in KPEM, 0.15 m NaCl with protease inhibitors at 4 °C. The column was eluted with the same buffer, and fractions (0.5 ml) were collected and assayed for [3H]cGMP-binding activity to determine the elution position of PKG proteins. Catalase was located by absorbance at 280 and/or 400 nm, and ovalbumin was located by absorbance at 280 nm. The column was standardized with proteins of known Stokes radii as follows: cytochrome c (16.6 Å), ovalbumin (29 Å), bovine serum albumin (35 Å), and catalase (52 Å). The thyroglobulin elution volume was taken as the void volume, and the elution position of [3H]H2O was taken as the inclusion volume. Elution positions of the protein standards were used to generate a standard curve of (–log Kav)½ versus Stokes radius where Kav was determined as shown in Equation 1,Kav=elutionvolume-voidvolumeinclusionvolume-voidvolume(Eq. 1) The Stokes radii of PKG proteins were determined from the standard curve based on elution volumes. Sucrose Density Gradient Centrifugation and Determination of Sedimentation Coefficient—Purified PKG (∼10 μg) was combined with two internal standards, crystalline phosphorylase b (3 mg), and hemoglobin (0.5 mg), in a volume of 200 μl in KPEM containing 0.15 m NaCl with protease inhibitor tablets (1 tablet per 25 ml of buffer) and layered onto a 13-ml linear 5–20% sucrose gradient containing KPEM, 0.15 m NaCl. The gradients were centrifuged at 37,000 rpm in a Beckman SW 41 rotor for 40–44 h at 4 °C, and fractions (0.5 ml) were collected from the bottom of the tubes. The s20,w value of phosphorylase b was taken as 8.0 S and was located by absorbance at 280 nm. The s20,w value of hemoglobin was taken as 3.2 S and was located by absorbance at 280 nm and/or 411 nm. [3H]cGMP-binding activity was measured to determine the sedimentation position of the PKG proteins. Sedimentation coefficients of the PKG proteins were then determined by the distance migrated into the gradients compared with the standards. The molecular weights were calculated from the Stokes radii and sedimentation coefficients, according to the method of Siegel and Monty (22Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1545) Google Scholar), using Equation 2,Mr=(6πnNas)/(1-νρ)(Eq. 2) where N is Avogadro's number; n is viscosity of medium, assumed to be 1; a is Stokes radius; s is sedimentation coefficient; ρ is density of medium, assumed to be 1; and ν is the partial specific volume, assumed to be 0.725 ml/g. Frictional coefficients were calculated using Equation 3,f/f0=a/(10(3νMr)/(4πN))1/3(Eq. 3) where ν is the partial specific volume, assumed to be 0.725 ml/g, and N is Avogadro's number. Preparations of Sf9 Cell Extracts Used for Studies of PKG Protein Conformation—One T-175 tissue culture flask at 50–70% confluence was infected with the optimal amount of baculovirus for the PKGIα or PKGIβ R domain. At 72 h post-infection, cells were dislodged with a cell scraper (Nunc) and centrifuged for 15 min at 1000–2000 rpm in a Damon table-top swinging bucket centrifuge at room temperature. Cell pellet was resuspended in 5 ml of KPEM with protease inhibitor tablets (1 tablet per 25 ml of buffer). Cell suspension was homogenized two times with 4-s bursts in an Ultra Turrex microhomogenizer with a 20-s recovery between bursts, and the homogenate was centrifuged at 10,000 rpm in a Beckman JA-20 rotor for 20 min at 4 °C. 200 μl of the supernatant was combined with 4 mg of catalase and applied to a Sephacryl S-200 column. For studies in the absence of cGMP, the column was equilibrated in KPEM containing 0.15 m NaCl and protease inhibitors. For studies in the presence of cGMP, the column was equilibrated in KPEM containing 0.15 m NaCl, 50 μm 3-isobutyl-1-methylxanthine (IBMX), 100 μm cGMP, and protease inhibitors. Elution position of catalase was determined by absorbance at 400 nm, and the elution positions of PKG proteins were determined by assaying [3H]cGMP binding activity. Calculation of Stokes radius was done as described above under “Gel Filtration Chromatography and Determination of Stokes Radius.” SDS-PAGE and Western Blot of PKGIβ—PKG was boiled for 4 min in the presence of 10% SDS, 2 m β-mercaptoethanol, and 0.1% bromphenol blue and subjected to 8% SDS-PAGE. Proteins were visualized by Coomassie Brilliant Blue staining. For Western blot, gel was transferred to polyvinylidene difluoride membrane (Millipore). Primary antibody was 1:7500 diluted rabbit polyclonal anti-PKG, and secondary antibody was 1:5000 diluted goat anti-rabbit horseradish peroxidase (BioSource International). The blot was developed using ECL chemiluminescence kit (Amersham Biosciences). Protein Quantification—Protein was determined by the method of Bradford using staining reagent from Bio-Rad and bovine serum albumin fraction V (Sigma) as standard. This method routinely overestimates the amount of PKG protein by 37%, and this correction factor was applied (6Wolfe L. Corbin J.D. Francis S.H. J. Biol. Chem. 1989; 264: 7734-7741Abstract Full Text PDF PubMed Google Scholar). Materials—Reagents for recombinant enzyme preparations were obtained via the Vanderbilt Diabetes Center Molecular Biology Core. The Vanderbilt Diabetes Center Tissue Culture Core facility provided culture media and competent cells. Baculovirus expression vector pVL 1392, BaculoGold DNA, and transfection reagents and protocols were obtained from Pharmingen. Sf9 log phase cells were purchased from Pharmingen. Protease inhibitor tablets were purchased from Roche Applied Science. Catalase, cytochrome c, phosphorylase b, ovalbumin, and bovine serum albumin were from Sigma. Hemoglobin was obtained from Nutritional Biochemicals Corp. T-175 tissue culture flasks were from Corning or Fisher. Expression of R Domains of PKGIα and PKGIβ—Deletion mutagenesis was used to remove the C domain from PKGIα and PKGIβ, creating PKGIα-(Δ349–670) and PKGIβ-(Δ364–685). These isolated R domains contained the dimerization subdomain, autoinhibitory subdomain, and the two cGMP-binding sites (Fig. 1A). The proteins were then expressed using the baculovirus system in Sf9 cells and purified as described under “Experimental Procedures.” The amount of PKG proteins typically displayed at least 100-fold greater [3H]cGMP-binding activity over that of mock-infected cells. The SDS-PAGE migration positions of purified R domains of PKGIα and PKGIβ on SDS-PAGE were consistent with the molecular weights predicted from AA sequences of 40 kDa for PKGIα and 41 kDa for PKGIβ (Fig. 1B). In the most purified samples, one main protein band was observed by Coomassie Brilliant Blue staining after SDS-PAGE; a minor contaminant of slightly higher molecular weight was present in the PKGIβ preparation. Western blot analysis confirmed that the 40- and 41-kDa bands were derived from PKG (not shown). Structural and Functional Properties of the Isolated R Domains of PKGIα and PKGIβ—Using an exhaustive mutational analytic approach, we recently demonstrated the critical importance of a leucine zipper motif near the amino terminus for dimerization of PKGIβ (15Richie-Jannetta R. Francis S.H. Corbin J.D. J. Biol. Chem. 2003; 278: 50070-50079Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Based on strong conservation of this motif among PKGs, it seemed likely that it is also crucial for dimerization of PKGIα and PKGII as well. This was supported by the fact that removal of the amino-terminal ∼35 amino acids of PKGIα by limited proteolysis produced a monomeric and cGMP-dependent kinase (23Monken C.E. Gill G.N. J. Biol. Chem. 1980; 255: 7067-7070Abstract Full Text PDF PubMed Google Scholar). However, one study concluded that an isolated R domain construct of PKGIα was monomeric (24Dostmann W.R. Koep N. Endres R. FEBS Lett. 1996; 398: 206-210Crossref PubMed Scopus (13) Google Scholar). We further examined this issue by determining the quaternary structure of the PKGIα and PKGIβ isolated R domains using the method of Siegel and Monty (22Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1545) Google Scholar) and comparing these values with those derived from the respective recombinant WT PKGs. Gel filtration chromatography was used to determine the Stokes radius, which is a reflection of both the shape and mass of a molecule. As shown in Table 1, the Stokes radii of the isolated PKG R domains and the respective WT PKGs were quite similar despite the fact that the R domains of PKGIα and PKGIβ were approximately half the mass of the full-length PKGs; the Stokes radii were 44 and 51 Å for the isolated R domains of PKGIα and PKGIβ, respectively, compared with 47 and 48 Å for the respective full-length enzymes.TABLE 1Structural and functional properties of WT PKGIα and PKGIβ and their respective isolated R domainsStokes radiusSedimentation coefficientCalculated masscGMP KDSlow site t½cGMP BmaxAssigned quaternary structureÅs20,wkDanmminmol/molWT PKGIαaData are from Chu et al. (18).47 ± 0.1aData are from Chu et al. (18).7.4 ± 0.1aData are from Chu et al. (18).180aData are from Chu et al. (18).20 ± 56.3 ± 1.31.7 ± 0.67DimerPKGIα R domain44 ± 0.85.0 ± 0.1" @default.
• W2002161089 created "2016-06-24" @default.
• W2002161089 creator A5012382947 @default.
• W2002161089 creator A5034675601 @default.
• W2002161089 creator A5037878505 @default.
• W2002161089 creator A5053138482 @default.
• W2002161089 creator A5080256711 @default.
• W2002161089 date "2006-03-01" @default.
• W2002161089 modified "2023-09-27" @default.
• W2002161089 title "Isolated Regulatory Domains of cGMP-dependent Protein Kinase Iα and Iβ Retain Dimerization and Native cGMP-binding Properties and Undergo Isoform-specific Conformational Changes" @default.
• W2002161089 cites W1004292963 @default.
• W2002161089 cites W1486377867 @default.
• W2002161089 cites W1518970589 @default.
• W2002161089 cites W1549319495 @default.
• W2002161089 cites W1553359417 @default.
• W2002161089 cites W1570624812 @default.
• W2002161089 cites W1589398776 @default.
• W2002161089 cites W1654031744 @default.
• W2002161089 cites W1969505923 @default.
• W2002161089 cites W1976752102 @default.
• W2002161089 cites W1981528113 @default.
• W2002161089 cites W1987183975 @default.
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