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• W2171314350 endingPage "50079" @default.
• W2171314350 startingPage "50070" @default.
• W2171314350 abstract "All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Iβ this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats ((L3A/L10A/L17A/I24A), (L31A/I38A/L45A/I52A), (L17A/I24A/L31A/I38A/L45A/I52A), and (L3A/L10A/L45A/I52A)) of the motif produces a monomeric PKG Iβ. Mutation of two Leu/Ile heptad repeats can produce either a dimeric (L3A/L10A) or monomeric (L17A/I24A and L31A/I38A) PKG. Point mutation of Leu-17 or Ile-24 (L17A or I24A) does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Iβ. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions (Leu-17, Ile-24, Leu-31, and Ile-38) appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants (PKG Iβ (Δ1–52) and PKG Iβ L17A/I24A/L31A/I38A/L45A/I52A) is 2- to 3-fold greater than that for wild type dimeric PKG Iβ, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site (site A) of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme. All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Iβ this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats ((L3A/L10A/L17A/I24A), (L31A/I38A/L45A/I52A), (L17A/I24A/L31A/I38A/L45A/I52A), and (L3A/L10A/L45A/I52A)) of the motif produces a monomeric PKG Iβ. Mutation of two Leu/Ile heptad repeats can produce either a dimeric (L3A/L10A) or monomeric (L17A/I24A and L31A/I38A) PKG. Point mutation of Leu-17 or Ile-24 (L17A or I24A) does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Iβ. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions (Leu-17, Ile-24, Leu-31, and Ile-38) appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants (PKG Iβ (Δ1–52) and PKG Iβ L17A/I24A/L31A/I38A/L45A/I52A) is 2- to 3-fold greater than that for wild type dimeric PKG Iβ, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site (site A) of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme. Cyclic GMP-dependent protein kinase (PKG) 1The abbreviations used are: PKG(s)cGMP-dependent protein kinase(s)hcGKIβhuman PKG Iβ. is one of the major intracellular receptors for cGMP (1.Francis S.H. Corbin J.D. Murad F. Cyclic GMP: Synthesis, Metabolism and Function. Academic Press, Orlando1993: 115-170Google Scholar, 2.Francis S.H. Corbin J.D. Crit. Rev. Clin. Lab. Sci. 1999; 36: 275-328Crossref PubMed Scopus (260) Google Scholar). In higher eukaryotes, it is composed of a dimer of two identical polypeptide chains. There are three different isoforms of mammalian PKG, which are termed Iα, Iβ, and II. PKG Iα and Iβ are products of alternative splicing and differ only in approximately the first 100 amino acids with only 36% identity in this region (3.Francis S.H. Woodford T.A. Wolfe L. Corbin J.D. Second Messengers & Phosphoproteins. 1988; 12: 301-310PubMed Google Scholar, 4.Wernet W. Flockerzi V. Hofmann F. FEBS Lett. 1989; 251: 191-196Crossref PubMed Scopus (161) Google Scholar, 5.Sandberg 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, 6.Wolfe L. Corbin J.D. Francis S.H. J. Biol. Chem. 1989; 264: 7734-7741Abstract Full Text PDF PubMed Google Scholar). PKG II is a separate gene product, and the amino terminus has little overall similarity to the amino terminus of PKG I isoforms (7.Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Abstract Full Text PDF PubMed Google Scholar, 8.Gamm 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). PKGs are chimeric proteins that comprise multiple functional domains; these are present in all three isoforms (9.Takio 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 carboxyl-terminal region of each polypeptide chain contains the catalytic domain that includes the ATP and protein substrate-binding sites, and this domain is the most highly conserved region among the PKGs. This domain catalyzes transfer of the γ-phosphate from ATP to specific serines or threonines in protein substrates. Immediately amino-terminal to the catalytic domain are two allosteric cGMP-binding sites (site A and site B) that are arranged in tandem. These two sites are homologous to each other, although they differ in cGMP-binding kinetics and cyclic nucleotide analog specificities (7.Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Abstract Full Text PDF PubMed Google Scholar, 9.Takio 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, 10.Reed 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 (51) Google Scholar, 11.Corbin J.D. Doskeland S.O. J. Biol. Chem. 1983; 258: 11391-11397Abstract Full Text PDF PubMed Google Scholar, 12.Corbin 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). Binding of cGMP causes a conformational change that is associated with activation of the kinase (13.Chu 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, 14.Zhao 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, 15.Wall 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). Immediately amino-terminal to the cGMP-binding sites is the autoinhibitory/autophosphorylation domain; the autophosphorylation sites lie within and near this domain (16.Francis S.H. Smith J.A. Colbran J.L. Grimes K. Walsh K.A. Kumar S. Corbin J.D. J. Biol. Chem. 1996; 271: 20748-20755Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 17.Smith J.A. Francis S.H. Walsh K.A. Kumar S. Corbin J.D. J. Biol. Chem. 1996; 271: 20756-20762Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18.Aitken A. Hemmings B.A. Hofmann F. Biochim. Biophys. Acta. 1984; 790: 219-225Crossref PubMed Scopus (59) Google Scholar). The autoinhibitory domains of each of the PKGs contain a pseudosubstrate sequence that interacts with the catalytic site to block substrate access and thus maintain the kinase in an inactive state (19.Lincoln T.M. Flockhart D.A. Corbin J.D. J. Biol. Chem. 1978; 253: 6002-6009Abstract Full Text PDF PubMed Google Scholar, 20.Landgraf W. Hofmann F. Eur. J. Biochem. 1989; 181: 643-650Crossref PubMed Scopus (35) Google Scholar). Autoinhibition is relieved by cGMP binding and/or autophosphorylation in PKG I (11.Corbin J.D. Doskeland S.O. J. Biol. Chem. 1983; 258: 11391-11397Abstract Full Text PDF PubMed Google Scholar, 17.Smith J.A. Francis S.H. Walsh K.A. Kumar S. Corbin J.D. J. Biol. Chem. 1996; 271: 20756-20762Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 20.Landgraf W. Hofmann F. Eur. J. Biochem. 1989; 181: 643-650Crossref PubMed Scopus (35) Google Scholar, 21.Foster J.L. Guttmann J. Rosen O.M. J. Biol. Chem. 1981; 256: 5029-5036Abstract Full Text PDF PubMed Google Scholar, 22.Busch J.L. Bessay E.P. Francis S.H. Corbin J.D. J. Biol. Chem. 2002; 277: 34048-34054Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and cGMP binding activates PKG II (8.Gamm 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). Finally, the extreme amino terminus contains the dimerization domain. Proteolysis just carboxyl-terminal of this domain produces a monomeric PKG with sequence that begins just amino-terminal of the autoinhibitory domain (16.Francis S.H. Smith J.A. Colbran J.L. Grimes K. Walsh K.A. Kumar S. Corbin J.D. J. Biol. Chem. 1996; 271: 20748-20755Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 23.Monken C.E. Gill G.N. J. Biol. Chem. 1980; 255: 7067-7070Abstract Full Text PDF PubMed Google Scholar, 24.Wolfe L. Francis S.H. Corbin J.D. J. Biol. Chem. 1989; 264: 4157-4162Abstract Full Text PDF PubMed Google Scholar). In vitro, the monomeric PKGs retain the salient properties of dimeric PKGs (autoinhibition, autophosphorylation, cGMP binding, and kinase activity). cGMP-dependent protein kinase(s) human PKG Iβ. In mammalian tissues, cyclic nucleotide-dependent kinases are dimers, and dimerization primarily occurs through interaction at the amino terminus. The regulatory subunits of cyclic AMP-dependent protein kinase are dimerized in an anti-parallel alignment, and the dimerization contacts occur through hydrophobic interactions near the amino terminus of these subunits (25.Li Y. Rubin C.S. J. Biol. Chem. 1995; 270: 1935-1944Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 26.Leon D.A. Herberg F.W. Banky P. Taylor S.S. J. Biol. Chem. 1997; 272: 28431-28437Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). It has been proposed that dimerization of PKG occurs through a leucine zipper motif near the amino terminus, and experimental work with a synthetic peptide based on residues 1–39 of PKG Iα supports this interpretation (27.Atkinson R.A. Saudek V. Huggins J.P. Pelton J.T. Biochemistry. 1991; 30: 9387-9395Crossref PubMed Scopus (60) Google Scholar). However, the role and extent of the leucine zipper motif in dimerization of PKGs has not been experimentally established. All three mammalian PKG isoforms (Iα, Iβ, and II) contain a leucine zipper motif in this sequence, although the remainder of the residues in the sequence have limited sequence identity. Other eukaryotic PKGs such as the two Drosophila PKGs (28.Kalderon D. Rubin G.M. J. Biol. Chem. 1989; 264: 10738-10748Abstract Full Text PDF PubMed Google Scholar) and the PKGs found in Caenorhabditis elegans (29.Stansberry J. Baude E.J. Taylor M.K. Chen P. Jin S. Ellis R.E. Uhler M.D. J. Neurochem. 2001; 76: 1177-1178Crossref PubMed Scopus (26) Google Scholar), Hydra oligactis, and Apis mellifera (30.Ben-Shahar Y. Robichon A. Sokolowski M.B. Robinson G.E. Science. 2002; 296: 741-744Crossref PubMed Scopus (393) Google Scholar) also contain a leucine zipper motif in this region (see Fig. 1). The number of heptad repeats in the leucine zipper motif of these proteins varies from five to eight. Lower eukaryotes have monomeric PKGs that lack the leucine zipper motif; PKG from Paramecium has been shown to be a monomer (31.Miglietta L.A.P. Nelson D.L. J. Biol. Chem. 1988; 263: 16096-16105Abstract Full Text PDF PubMed Google Scholar), but this kinase retains functional activities of PKG seen in PKGs of higher eukaryotes, although careful studies of cGMP binding and activation of this PKG compared with the mammalian isoforms are lacking. In addition to being the proposed mechanism of dimerization for PKG, the leucine zipper motif has also been shown to be involved in the interaction of PKG with a number of targeting proteins. PKG Iα specifically associates with skeletal muscle troponin T (32.Yuasa K. Michibata H. Omori K. Yanaka N. J. Biol. Chem. 1999; 274: 37429-37434Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), GKAP 42 in germ cells (33.Yuasa K. Omori K. Yanaka N. J. Biol. Chem. 2000; 275: 4897-4905Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and the myosin-binding subunit of myosin phosphatase (34.Surks H.K. Mochizuki N. Kasai Y. Georgescu S.P. Tang K.M. Ito M. Lincoln T.M. Mendelsohn M.E. Science. 1999; 286: 1583-1587Crossref PubMed Scopus (440) Google Scholar). PKG Iβ specifically binds to skeletal muscle troponin T and inositol 3-phosphate receptor-associated PKG substrate (35.Ammendola A. Geiselhoringer A. Hofmann F. Schlossmann J. J. Biol. Chem. 2001; 276: 24153-24159Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Much of the interaction with the targeting proteins occurs through the region containing the dimerization domain, and mutations within the leucine zipper motif abolish most of these interactions. However, it has not been shown that these substitutions actually disrupt dimerization. PKG has also been shown to phosphorylate these targeting proteins, and localizing PKG to substrates through the leucine zipper motif provides for efficient phosphorylation. The leucine zipper motif was first characterized in the transcription factor field (36.Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2536) Google Scholar). It was shown to mediate the heterodimerization of transcription factors, Fos and Jun, and the homodimerization of the yeast transcription factor GCN4 (37.Kouzarides T. Ziff E. Nature. 1989; 340: 568-571Crossref PubMed Scopus (184) Google Scholar). The majority of leucine zipper motifs do not contain more than five heptad repeats. The leucine zipper motif of each of the mammalian PKGs contains several more heptad repeats than does the same motif in transcription factors. Mammalian PKGs contain six to nine heptad repeats, depending on isoform (six heptad repeats in PKG Iα, seven to eight repeats in PKG Iβ, and eight to nine repeats in PKG II), whereas transcription factors studied so far contain four to five heptad repeats. Because three to four heptad repeats are sufficient to mediate dimerization of these transcription factors (38.Van Heeckeren W.J. Sellers J.W. Struhl K. Nucleic Acids Res. 1992; 20: 3721-3724Crossref PubMed Scopus (28) Google Scholar, 39.Ransone L.J. Visvader J. Sassone-Corsi P. Verman I. Genes Dev. 1989; 3: 770-781Crossref PubMed Scopus (104) Google Scholar, 40.Kouzarides T. Ziff E. Nature. 1988; 336: 646-651Crossref PubMed Scopus (551) Google Scholar, 41.Turner R. Tjian R. Science. 1989; 243: 1689-1694Crossref PubMed Scopus (425) Google Scholar, 42.Gentz R. Rauscher III, F.J. Abate C. Curran T. Science. 1989; 243: 1695-1699Crossref PubMed Scopus (399) Google Scholar), it is not presently possible to predict the contacts required for dimerization of the PKGs. However, other proteins in addition to PKG contain six or more heptad repeats (43.Gassama-Diagne A. Hullin-Matsuda F. Li R.Y. Nauze M. Ragab A. Pons V. Delagebeaudeuf C. Simon M.F. Fauvel J. Chap H. J. Biol. Chem. 2001; 276: 18352-18360Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 44.Rook F. Weisbeek P. Smeekens S. Plant Mol. Biol. 1998; 37: 171-178Crossref PubMed Scopus (37) Google Scholar, 45.Burbelo P.D. Gabriel G.C. Kibbey M.C. Yoshihiko Y. Kleinman H.K. Weeks B.S. Gene. 1994; 139: 241-245Crossref PubMed Scopus (27) Google Scholar, 46.Marini M.G. Chan K. Casula L. Kan Y.W. Cao A. Moi P. J. Biol. Chem. 1997; 272: 16490-16497Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 47.Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar, 48.Bikle D.D. Munson S. Morrison N. Eisman J. J. Biol. Chem. 1993; 268: 620-626Abstract Full Text PDF PubMed Google Scholar, 49.Kubota R. Noda S. Wang Y. Minoshima S. Asakawa S. Kodoh J. Mashima Y. Oguchi Y. Shimizu N. Genomics. 1997; 41: 360-369Crossref PubMed Scopus (275) Google Scholar); two of these proteins contain more than 20 heptad repeats (43.Gassama-Diagne A. Hullin-Matsuda F. Li R.Y. Nauze M. Ragab A. Pons V. Delagebeaudeuf C. Simon M.F. Fauvel J. Chap H. J. Biol. Chem. 2001; 276: 18352-18360Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 48.Bikle D.D. Munson S. Morrison N. Eisman J. J. Biol. Chem. 1993; 268: 620-626Abstract Full Text PDF PubMed Google Scholar). In PKGs and other extended leucine zipper motif proteins, it is not known if the entire leucine zipper motif is required for dimerization or if three to four repeats are sufficient as is the case with transcription factors. This report examines the role of each of the residues within the mammalian PKG Iβ leucine zipper motif in the mechanism of dimerization of mammalian PKG Iβ. In addition, the influence of dimerization on enzyme functions has been determined. PKG Iβ Site-directed Mutagenesis—Human PKG Iβ (hcGKIβ) clone encodes a full-length human placental PKG Iβ. The QuikChange site-directed mutagenesis kit (Stratagene) was used to synthesize all site-directed mutations in the hcGKIβ clone in pKSII+ vector according to the protocol from Stratagene. The following mutants were made: PKG Iβ L17A, I24A, L3A/L10A, L17A/I24A, L31A/I38A, L3A/L10A/L17A/I24A, L31A/I38A/L45A/I52A, L3A/L10A/L45A/I52A, and L17A/I24A/L31A/I38A/L45A/I52A. The oligonucleotides used in mutagenesis are all listed in Table I. The point mutations for PKG Iβ L17A/I24A/L31A/ I38A/L45A/I52A were made in three steps. The first step mutated Leu-17 and Ile-24. The next step utilized PKG Iβ L17A/I24A as a template and mutated Leu-31 and Ile-38. PKG Iβ L17A/I24A/L31A/I38A was used as a template to mutate Leu-45 and Ile-52: The point mutations for PKG Iβ L3A/L10A/L17A/I24A, PKG Iβ L31A/I38A/L45A/I52A, and PKG Iβ L3A/L10A/L45A/I52A were made in two steps in the same manner as discussed for L17A/I24A/L31A/I38A/L45A/I52A. A 580-bp fragment containing the desired mutations was excised from hcGKIβ using EcoRI/NcoI (New England Biolabs) digestion, and subcloned in EcoRI/NcoI-digested wild type hcGKIβ clone in the pVL 1392 baculovirus expression vector.Table IOligonucleotide pairs utilized in site-directed mutagenesis of PKG IβPKG Iβ L17A/I24A/L31A/I38A/L45A/I52A Step 15′-CTCCAGGAGAAGATCGAGGAGGCGAGGCAGCGGGATGCTCTCGCCGACGAGCTGGAGCTGGAGTTG-3′5′-CAACTCCAGCTCCAGCTCGTCGGCGAGAGCATCCCGCTGCCTCGCCTCCTCGATCTTCTCCTGGAG-3′ Step 25′-GCCGACGAGCTGGAGCTGGAGGCGGATCAGAAGGACGAACTGGCCCAGAAGCTGCAGAACGAGCTG-3′5′-CAGCTCGTTCTGCAGCTTCTGGCCCAGTTCGTCCTTCTGATCCGCCTCCAGCTCCAGCTCGTCGGC-3′ Step 35′-GCCCAGAAGCTGCAGAACGAGGCGGACAAGTACCGCTCGGTGGCCCGACCAGCCACCCAGCAGGCG-3′5′-CGCCTGCTGGGTGGCTGGTCGGGCCACCGAGCGGTACTTGTCCGCCTCGTTCTGCAGCTTCTGGGC-3′PKG Iβ L3A/L10A/L17A/I24A Step 15′-GCCCGGAGGAGCATGGGCACCGCGCGGGATTTACAGTACGCGGCCCAGGAGAAGATCGAGGAGCTG-3′5′-CAGCTCCTCGATCTTCTCCTGGGCCGCGTACTGTAAATCCCGCGCGGTGCCCATGCTCCTCCGGGC-3′ Step 25′-GCCCAGGAGAAGATCGAGGAGGCGAGGCAGCGGGATGCTFCTCGCCGACGAGCTGGAGCTGGAGTTG-3′5′-CAACTCCAGCTCCAGCTCGTCGGCGAGAGCATCCCGCTGCCTCGCCTCCTCGATCTTCTCCTGGGC-3′PKG Iβ L31/I38A/L45A/I52A Step 15′-ATCGACGAGCTGGAGCTGGAGGCGGATCAGAAGGACGAACTGGCCCAGAAGCTGCAGAACGAGCTG-3′5′-CAGCTCGTTCTGCAGCTTCTGGCCCAGTTCGTCCTTCTGATCCGCCTCCAGCTCCAGCTCGTCGAT-3′ tep 25′-GCCCAGAAGCTGCAGAACGAGGCGGACAAGTACCGCTCGGTGGCCCGACCAGCCACCCAGCAGGCG-3′5′-CGCCTGCTGGGTGGCTGGTCGGGCCACCGAGCGGTACTTGTCCGCCTCGTTCTGCAGCTTCTGGGC-3′PKG Iβ L3A/L10A/L45A/I52A Step 15′-GCCCGGAGGAGCATGGGCACCGCGCGGGATTTACAGTACGCGGCCCAGGAGAAGATCGAGGAGCTG-3′5′-CAGCTCCTCGATCTTCTCCTGGGCCGCGTACTGTAAATCCCGCGCGGTGCCCATGCTCCTCCGGGC-3′ Step 25′-ATCCAGAAGCTGCAGAACGAGGCGGACAAGTACCGCTCGGTGGCCCGACCAGCCACCCAGCAGGCG-3′5′-CGCCTGCTGGGTGGCTGGTCGGGCCACCGAGCGGTACTTGTCCGCCTCGTTCTGCAGCTTCTGGAT-3′PKG Iβ L3A/L10A5′-GCCCGGAGGAGCATGGGCACCGCGCGGGATTTACAGTACGCGGCCCAGGAGAAGATCGAGGAGCTG-3′5′-CAGCTCCTCGATCTTCTCCTGGGCCGCGTACTGTAAATCCCGCGCGGTGCCCATGCTCCTCCGGGC-3′PKG Iβ L17A/I24A5′-CTCCAGGAGAAGATCGAGGAGGCGAGGCAGCGGGATGCTCTCGCCGACGAGCTGGAGCTGGAGTTG-3′5′-CAACTCCAGCTCCAGCTCGTCGGCGAGAGCATCCCGCTGCCTCGCCTCCTCGATCTTCTCCTGGAG-3′PKG Iβ L31A/I38A5′-ATCGACGAGCTGGAGCTGGAGGCGGATCAGAAGGACGAACTGGCCCAGAAGCTGCAGAACGAGCTG-3′5′-CAGCTCGTTCTGCAGCTTCTGGCCCAGTTCGTCCTTCTGATCCGCCTCCAGCTCCAGCTCGTCGAT-3′PKG Iβ L17A5′-CTCCAGGAGAAGATCGAGGAGGCGAGGCAGCGGGATGCTCTCATC-3′5′-GATGAGAGCATCCCGCTGCCTCGCCTCCTCGATCTTCTCCTGGAG-3′PKG Iβ I24A5′-CTGAGGCAGCGGGATGCTCTCGCCGACGAGCTGGAGCTGGAGTTG-3′5′-CAACTCCAGCTCCAGCTCGTCGGCGAGAGCATCCCGCTGCCTCAG-3′ Open table in a new tab Escherichia coli XL1-blue cells were used for transformations with pKSII+, and E. coli DH5α were used for transformations with pVL 1392. DNA fragments were purified using a Qiagen gel extraction kit according to the manufacturer's protocol (Qiagen). DNA was purified from large scale vector preparations using a Qiagen 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 inframe subcloning. Mutagenesis of PKG Iβ (Δ1–52)—Full-length PKG Iβ cDNA was ligated into the EcoRI and SmaI unique sites of the baculovirus expression vector pVL 1392. A 1441-bp EcoRI/SacI fragment containing the regulatory domain of human PKG Iβ was ligated into pBluescript IIKS+ (Stratagene) for oligonucleotide-directed mutagenesis based on the method of Kunkel et al. (50.Kunkel T.A. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1994: 8.1.1-8.1.6Google Scholar). The oligonucleotide (Vanderbilt University DNA Core Facility) with the following sequence was constructed to be complementary to the cDNA-coding strand except for the underlined nucleotides that serve to construct the deletion mutation and incorporate a 5′ NdeI site. Δ1–52, 5′-TGGGTGGCTGGTCGCATATG-GAATTCGTACTTGTCCAGCTC-3′. pVL 1392-PKG Iβ (Δ1–52) vector was created by subcloning the EcoRI/SacI fragment for PKG Iβ (Δ1–52) into the large SacI/SmaI fragment of pVL 1392-PKG Iβ. pVL 1392 vectors and pBluescript IIKS+ were propagated in E. coli (DH5α and XL1 Blue, respectively). All plasmids were sequenced on an Automated Biosystems, Inc. DNA Sequencer 373A in the Cancer Center DNA Core facility of Vanderbilt University. Expression of Wild type and Mutant PKG Iβ—Sf9 cells (BD Pharmingen) were cotransfected with BaculoGold linear baculovirus DNA (BD Pharmingen) and one of the mutated hcGKIβ clones in the pVL 1392 baculovirus expression vector by calcium phosphate method according to the protocol from BD Pharmingen. At 5 days post-infection, the cotransfection supernatant was collected, amplified three times in Sf9 cells, and then used directly as virus 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 gentamicin (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. Purification of PKG Iβ—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 protease inhibitor mixture tablets (Roche Applied Science) of amount per volume recommended by manufacturer. Cell suspension was homogenized in 10- to 20-ml aliquots by 3 × 10-s bursts in an Ultra Turrex microhomogenizer with a 20-s recovery between bursts. The cell homogenate was centrifuged at 13,000 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 with 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 incubated for 20 min at 4 °C before six 0.3-ml elutions were collected. The column was incubated for 20 additional minutes at 4 °C, and six 0.3-ml fractions were collected a second time. Elutions containing kinase activity 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 with protease inhibitors. This step removed cAMP from the affinity column elution. 0.5-ml fractions were collected from the Sephacryl S-200 column. For full-length PKG Iβ, fractions containing PKG Iβ were applied to a 2-ml DEAE-Sephacel ion exchange column equilibrated in KPEM with protease inhibitors. The column was washed with 20 ml of KPEM with protease inhibitors followed by 20 ml of 0.12 m NaCl in KPEM with protease inhibitors to remove any breakdown products. The column was eluted with 4 ml of 0.3 m NaCl in KPEM with protease inhibitors. All purification steps were done at 4 °C, and enzyme was flash-frozen in 0.3 m NaCl and 10% sucrose and stored at –70 °C until use. Four preparations of PKG Iβ (Δ1–52) and two preparations of wild type PKG Iβ and PKG Iβ L17A/I24A/L31A/I38A/L45A/I52A were expressed, purified, and used in the experiments described in this report. SDS-PAGE and Western Blot of PKG Iβ—PKG was boiled for 4 min in the presence of 10% SDS, 2 m 2-mercaptoethanol, and 0.1% bromphenol blue and subjected to 8% SDS-polyacrylamide gel electrophoresis. Proteins were visualized by Coomassie Brilliant Blue staining. For Western blot, gel was transferred to polyvinylidene difluoride membrane (Millipore). Primary antibody was rabbit polyclonal anti-PKG, and secondary antibody was goat anti-rabbit horseradish peroxidase (BioSource International). The blot was developed using ECL chemiluminescence kit (Amersham Biosciences). PKG Iβ Kinase Assay—The kinase activity of PKG Iβ was determined by measuring 32Pi incorporation from [γ-32P]ATP into a synthetic heptapeptide substrate (Arg-Lys-Arg-Ser-Arg-Ala-Glu, Peninsula Laboratories, Inc.) as previously described. Ten μl of enzyme was added to 10 μl of 100 μm cGMP and 50 μl of a reaction mixture, which contained 20 mm Tris at pH 7.4, 20 mm magnesium acetate, 200 μm ATP, 100 μm 3-isobutyl-1-methylxanthine, 136 μg/ml heptapeptide, 5,000–20,000 cpm [γ-32P]ATP/μl, and 0.45 μm synthetic peptide inhibitor of the cAMP-dependent protein kinase (Peninsula Laboratories, Inc.). The reaction was terminated by applying 50-μl aliquots to phosphocellulose papers (Whatman P-81, 1.5 × 2 cm) and washing with four changes of ∼500 ml 75 mm phosphoric acid and one ethanol change. The papers were dried and counted by Cerenkov method. cGMP dissociation—PKG Iβ (0.030–0.050 mg/ml) was incubated for 10 min at 30 °C with an equal volume of cGMP-binding mix (25 mm K2HPO4, 25 mm KH2PO4, 1 mm EDTA (pH 6.8), 2 m NaCl, 200 μm 3-isobutyl-1-methylxanthine, 0.5 mg/ml histone IIAS (Sigma)) and 10 μl of [3H]cGMP (Amersham Biosciences) for a final concentration of 3 μm [3H]cGMP (∼5000 cpm/μl). An incubation time of 10 min at 30 °C was adequate for saturation of the cGMP-binding sites. After incubation, samples were cooled to 4 °C and aliquoted in 10-μl portions. The addition of 100-fold excess unlabeled cGMP at time 0 (Bo) initiated the dissociation (exchange) of bound [3H]cGMP. The cGMP exchange was stopped at various time points by the addition of 2 ml of cold aqueous saturated ammonium sulfate. The samples were filtered and washed as described previously (6.Wolfe L. Corbin J.D. Francis S.H. J. Biol. Chem. 1989; 264: 7734-7741Abstract Full Text PDF PubMed Google Scholar). The half-life of the bound cGMP was determined by the method of Rannels and Corbin (51.Rannels S.R. Corbin J.D. Methods Enzymol. 1983; 99: 168-175Crossref PubMed Scopus (16) Google Scholar). Determination of Stokes Radius—Purified PKG (∼7–10 μg) was combined with two internal standards, crystalline catalase (3 mg), and ovalbumin (4 mg), in a volume of 200 μl and loaded onto a Sephacryl S-200 gel filtration column (0.9 × 35 cm) equilibrated in KPEM and 150 mm NaCl at 4 °C. The column was eluted with the same buffer, and fractions (0.5 ml) were collected and assayed for PKG activity to determine the elution position of the enzyme. Catalase was located by absorbance at 280 nm and/or 400 nm, and ovalbumin was located by absorbance at 280 nm. The column was standardized with protein standards of known Stokes radii: 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. Elution positions of the protein standards were used to generate a standard curve of (–log Kav)1/2versus Stokes radius (52.Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (" @default.
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• W2171314350 title "Dimerization of cGMP-dependent Protein Kinase Iβ Is Mediated by an Extensive Amino-terminal Leucine Zipper Motif, and Dimerization Modulates Enzyme Function" @default.
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