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• W2009408911 abstract "The ability of cGMP-dependent protein kinases (cGKs) to activate cAMP response element (CRE)-dependent gene transcription was compared with that of cAMP-dependent protein kinases (cAKs). Although both the type Iβ cGMP-dependent protein kinase (cGKIβ) and the type II cAMP-dependent protein kinase (cAKII) phosphorylated the cytoplasmic substrate VASP (vasodilator- and A kinase-stimulatedphosphoprotein) to a similar extent, cyclic nucleotide regulation of CRE-dependent transcription was at least 10-fold higher in cAKII-transfected cells than in cGKIβ-transfected cells. Overexpression of each kinase in mammalian cells resulted in a cytoplasmic localization of the unactivated enzyme. As reported previously, the catalytic (C) subunit of cAKII translocated to the nucleus following activation by 8-bromo-cyclic AMP. However, cGKIβ did not translocate to the nucleus upon activation by 8-bromo-cyclic GMP. Replacement of an autophosphorylated serine (Ser79) of cGKIβ with an aspartic acid resulted in a mutant kinase with constitutive kinase activity in vitroand in vivo. The cGKIβS79D mutant localized to the cytoplasm and was only a weak activator of CRE-dependent gene transcription. However, an amino-terminal deletion mutant of cGKIβ was found in the nucleus as well as the cytoplasm and was a strong activator of CRE-dependent gene transcription. These data suggest that the inability of cGKs to translocate to the nucleus is responsible for the differential ability of cAKs and cGKs to activate CRE-dependent gene transcription and that nuclear redistribution of cGKs is not required for NO/cGMP regulation of gene transcription. The ability of cGMP-dependent protein kinases (cGKs) to activate cAMP response element (CRE)-dependent gene transcription was compared with that of cAMP-dependent protein kinases (cAKs). Although both the type Iβ cGMP-dependent protein kinase (cGKIβ) and the type II cAMP-dependent protein kinase (cAKII) phosphorylated the cytoplasmic substrate VASP (vasodilator- and A kinase-stimulatedphosphoprotein) to a similar extent, cyclic nucleotide regulation of CRE-dependent transcription was at least 10-fold higher in cAKII-transfected cells than in cGKIβ-transfected cells. Overexpression of each kinase in mammalian cells resulted in a cytoplasmic localization of the unactivated enzyme. As reported previously, the catalytic (C) subunit of cAKII translocated to the nucleus following activation by 8-bromo-cyclic AMP. However, cGKIβ did not translocate to the nucleus upon activation by 8-bromo-cyclic GMP. Replacement of an autophosphorylated serine (Ser79) of cGKIβ with an aspartic acid resulted in a mutant kinase with constitutive kinase activity in vitroand in vivo. The cGKIβS79D mutant localized to the cytoplasm and was only a weak activator of CRE-dependent gene transcription. However, an amino-terminal deletion mutant of cGKIβ was found in the nucleus as well as the cytoplasm and was a strong activator of CRE-dependent gene transcription. These data suggest that the inability of cGKs to translocate to the nucleus is responsible for the differential ability of cAKs and cGKs to activate CRE-dependent gene transcription and that nuclear redistribution of cGKs is not required for NO/cGMP regulation of gene transcription. The cyclic nucleotides, cAMP and cGMP, are intracellular second messengers mediating the actions of a large number of hormones and neurotransmitters. These cyclic nucleotides act to allosterically regulate the action of a small number of important proteins. Unlike cAMP, which acts mainly through cAMP-dependent protein kinases (cAKs), 1The abbreviations used are:cAK, cAMP-dependent protein kinase; bp, base pair(s); C subunit, catalytic subunit; cGK, cGMP-dependent protein kinase; CMV, cytomegalovirus; CRE, cAMP response element; CREB, cAMP response element binding protein; DMEM, Dulbecco's modified Eagle's medium; HCG, human chorionic gonadotropin; NLS, nuclear localization signal; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PKI, protein kinase inhibitor; R subunit, regulatory subunit; VASP, vasodilator- and A kinase-stimulated phosphoprotein; 8-Br-cAMP, 8-bromo-cyclic AMP; 8-Br-cGMP, 8-bromo-cyclic GMP; IBMX, 3-isobutyl-1-methylxanthine.1The abbreviations used are:cAK, cAMP-dependent protein kinase; bp, base pair(s); C subunit, catalytic subunit; cGK, cGMP-dependent protein kinase; CMV, cytomegalovirus; CRE, cAMP response element; CREB, cAMP response element binding protein; DMEM, Dulbecco's modified Eagle's medium; HCG, human chorionic gonadotropin; NLS, nuclear localization signal; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PKI, protein kinase inhibitor; R subunit, regulatory subunit; VASP, vasodilator- and A kinase-stimulated phosphoprotein; 8-Br-cAMP, 8-bromo-cyclic AMP; 8-Br-cGMP, 8-bromo-cyclic GMP; IBMX, 3-isobutyl-1-methylxanthine. cGMP is able to activate three classes of proteins: ion channels, phosphodiesterases, and cGMP-dependent protein kinases (cGKs). The cAKs and the cGKs are highly homologous protein kinase families with similar substrate specificities. Phosphorylation of cellular proteins by both families of kinases leads to alterations in calcium mobilization, protein phosphatase activity, ion channel function, gene transcription, smooth muscle contractility, and platelet aggregation (1Francis S.H. Corbin J.D. Adv. Pharmacol. 1994; 26: 115-170Crossref PubMed Scopus (77) Google Scholar, 2McKnight G.S. Curr. Opin. Cell Biol. 1991; 3: 213-217Crossref PubMed Scopus (153) Google Scholar, 3Walsh D.A. Van Patten S.M. FASEB J. 1994; 8: 1227-1236Crossref PubMed Scopus (213) Google Scholar, 4Lohmann S.M. Vaandrager A.B. Smolenski A. Walter U. De Jonge H.R. Trends Biochem. Sci. 1997; 22: 307-312Abstract Full Text PDF PubMed Scopus (348) Google Scholar). The cGKs are classified into two types based on their historical order of characterization. The type I enzymes (cGKIs) are highly expressed in lung (5Kuo J.F. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2256-2259Crossref PubMed Scopus (45) Google Scholar), cerebellum (6Lohmann S.M. Walter U. Miller P.E. Greengard P. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 653-657Crossref PubMed Scopus (194) Google Scholar), platelets (7Walter U. Rev. Physiol. Biochem. Pharmacol. 1989; 113: 41-88Crossref PubMed Google Scholar), and smooth muscle (8Francis S.H. Noblett B.D. Todd B.W. Wells J.N. Corbin J.D. Mol. Pharmacol. 1988; 34: 506-517PubMed Google Scholar). Two type I isoforms, Iα and Iβ, arise from the alternative splicing of a single gene (9Sandberg 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, 10Wernet W. Flockerzi V. Hofmann F. FEBS Lett. 1989; 251: 191-196Crossref PubMed Scopus (161) Google Scholar, 11Tamura N. Itoh H. Ogawa Y. Nakagawa O. Harada M. Chun T.H. Suga S. Yoshimasa T. Nakao K. Hypertension. 1996; 27: 552Crossref PubMed Google Scholar, 12Orstavik S. Natarajan V. Tasken K. Jahnsen T. Sandberg M. Genomics. 1997; 42: 311-318Crossref PubMed Scopus (99) Google Scholar). These two forms differ in their amino-terminal autoinhibitory domains but share the same cGMP-binding sites and catalytic domains (4Lohmann S.M. Vaandrager A.B. Smolenski A. Walter U. De Jonge H.R. Trends Biochem. Sci. 1997; 22: 307-312Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 13Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (403) Google Scholar). In Purkinje cells (6Lohmann S.M. Walter U. Miller P.E. Greengard P. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 653-657Crossref PubMed Scopus (194) Google Scholar), smooth muscle cells (14Lincoln T.M. Pryzwansky K.B. Cornwell T.L. Wyatt T.A. MacMillan L.A. Adv. Second Messenger Phosphoprotein Res. 1993; 28: 121-132PubMed Google Scholar, 15Cornwell T.L. Pryzwansky K.B. Wyatt T.A. Lincoln T.M. Mol. Pharmacol. 1991; 40: 923-931PubMed Google Scholar), monocytes (16Pryzwansky K.B. Kidao S. Wyatt T.A. Reed W. Lincoln T.M. J. Leukocyte Biol. 1995; 57: 670-678Crossref PubMed Scopus (31) Google Scholar), and neutrophils (17Pryzwansky K.B. Wyatt T.A. Lincoln T.M. Blood. 1995; 85: 222-230Crossref PubMed Google Scholar, 18Wyatt T.A. Lincoln T.M. Pryzwansky K.B. J. Biol. Chem. 1991; 266: 21274-21280Abstract Full Text PDF PubMed Google Scholar), the majority of the cGKI immunoreactivity is soluble and localized to the cytoplasm. A second type of cGK, termed the type II cGK (cGKII), is highly expressed in intestinal microvilli (19de Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333PubMed Google Scholar) and is encoded by a gene distinct from that encoding cGKI proteins (20Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Abstract Full Text PDF PubMed Google Scholar, 21Jarchau T. Hausler C. Markert T. Pohler D. Vanderkerckhove J. De Jonge H.R. Lohmann S.M. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9426-9430Crossref PubMed Scopus (137) Google Scholar). While cGKI isoforms are soluble proteins, cGKII is particulate and associated with cellular membranes (19de Jonge H.R. Adv. Cyclic Nucleotide Res. 1981; 14: 315-333PubMed Google Scholar). Both types possess amino-terminal leucine zipper motifs and exist as homodimers in native tissues (13Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (403) Google Scholar). In contrast to the cAKs, which have separate catalytic and regulatory subunits, each monomer of the cGKs consists of both a regulatory domain and a catalytic domain contained in the same polypeptide (13Francis S.H. Corbin J.D. Annu. Rev. Physiol. 1994; 56: 237-272Crossref PubMed Scopus (403) Google Scholar). Many mammalian tissues coexpress isoforms of cAK and cGK, where the cAK and cGK proteins are thought to play distinct roles in cellular regulation. The in vitro substrate specificities of the cAKs and the cGKs are very similar, although a number of proteins have been identified as specific substrates for either cAKs or cGKs. For example, the type I regulatory (R) subunit of cAK (22Geahlen R.L. Krebs E.G. J. Biol. Chem. 1980; 255: 1164-1169Abstract Full Text PDF PubMed Google Scholar), G-substrate (23Aswad D.W. Greengard P. J. Biol. Chem. 1981; 256: 3487-3493Abstract Full Text PDF PubMed Google Scholar), histone H2B (24Glass D.B. Krebs E.G. J. Biol. Chem. 1982; 257: 1196-1200Abstract Full Text PDF PubMed Google Scholar), and the bovine lung cGMP-binding cGMP-specific phosphodiesterase (25Colbran J.L. Francis S.H. Leach A.B. Thomas M.K. Jiang H. McAllister L.M. Corbin J.D. J. Biol. Chem. 1992; 267: 9589-9594Abstract Full Text PDF PubMed Google Scholar, 26Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14971-14978Abstract Full Text PDF PubMed Google Scholar) are specific substrates of cGKI in vitro, while the cAMP response element-binding protein (CREB) has been shown to be a specific in vitro substrate of cAK (76Colbran J.-L. Roach P.J. Foil C.J. Dixon J.E. Andrisani O.M. Corbin J.D. Biochem. Cell Biol. 1992; 70: 1277-1282Crossref PubMed Scopus (39) Google Scholar). Although in vitro substrate specificity may be an important indicator of in vivo substrate specificity, recent evidence suggests that colocalization of kinase and substrate in the cell is at least an equally important factor (27Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The cAMP signaling pathway is used to regulate the transcription of many genes and involves the phosphorylation of specific transcription factors by the C subunit of cAK (28Montminy M. Annu. Rev. Biochem. 1997; 66: 807-822Crossref PubMed Scopus (851) Google Scholar). In the absence of cAMP, cAK exists predominantly as an inactive tetrameric holoenzyme composed of two R subunits and two C subunits. The inactive holoenzyme may be localized diffusely in the cytoplasm or localized to specific subcellular compartments by interaction of the R subunits with protein kinase A anchoring proteins (27Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Upon cytoplasmic elevation of cAMP, cAMP binds to each R subunit, causing the holoenzyme complex to dissociate into a homodimer of R subunits and two catalytically active C subunits. Once released, the C subunit can phosphorylate cytoplasmic substrates, and because of its small size (40 kDa), it can also passively diffuse through the nuclear pores into the nucleus (29Harootunian A.T. Adams S.R. Wen W. Meinkoth J.L. Taylor S.S. Tsien R.Y. Mol. Biol. Cell. 1993; 4: 993-1002Crossref PubMed Scopus (144) Google Scholar). In the nucleus, the C subunit can phosphorylate nuclear transcription factors, such as members of the CREB/ATF family, which bind directly to specific enhancer sequences and alter levels of gene transcription (30Nigg E.A. Hilz H. Eppenberger H.M. Dutly F. EMBO J. 1985; 4: 2801-2806Crossref PubMed Scopus (197) Google Scholar,31Hagiwara M. Brindle P. Harootunian A. Armstrong R. Rivier J. Vale W. Tsien R. Montminy M.R. Mol. Cell. Biol. 1993; 13: 4852-4859Crossref PubMed Scopus (375) Google Scholar). Substrates of cAK therefore include both cytoplasmic and nuclear proteins. Like the cAKs, the cGKs are capable of activating gene transcription in a cyclic nucleotide-dependent manner (32Pilz R.B. Suhasini M. Idriss S. Meinkoth J.L. Boss G.R. FASEB J. 1995; 9: 552-558Crossref PubMed Scopus (178) Google Scholar, 33Gudi T. Huvar I. Meinecke M. Lohmann S.M. Boss G.R. Pilz R.B. J. Biol. Chem. 1996; 271: 4597-4600Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 34Gudi T. Lohmann S.M. Pilz R.B. Mol. Cell. Biol. 1997; 17: 5244-5254Crossref PubMed Scopus (110) Google Scholar, 35Haby C. Lisovoski F. Aunis D. Zwiller J. J. Neurochem. 1994; 62: 496-501Crossref PubMed Scopus (147) Google Scholar). For example, in PC-12 cells, stimulation of the NO/cGMP pathway leads to increased expression of the immediate early genes c-fos andjunB. Importantly, this induction can be blocked by the selective cGK inhibitor KT5823, suggesting that this induction is dependent on cGK activity (35Haby C. Lisovoski F. Aunis D. Zwiller J. J. Neurochem. 1994; 62: 496-501Crossref PubMed Scopus (147) Google Scholar). While the mechanisms by which the cAKs activate gene transcription have been well characterized, little is known about cGK regulation of gene transcription. In this report, we demonstrate that both transiently expressed and endogenous cGKs are localized to the cytoplasm in mammalian cells. Treatment of cells with cyclic nucleotide does not cause nuclear accumulation of the cGKs, while the C subunit of cAKs does translocate to the nucleus. As a result of its cytoplasmic restriction, cGKIβ is a weaker activator of CRE-dependent gene transcription than cAKII. These data suggest that regulation of gene transcription by cGK involves phosphorylation of cytoplasmic substrate protein(s) that remain to be characterized. cDNA library screening was performed essentially as described previously (20Uhler M.D. J. Biol. Chem. 1993; 268: 13586-13591Abstract Full Text PDF PubMed Google Scholar). A 1.0-kilobase pairEcoRI–SalI restriction fragment from pCGKI.6 was isolated and labeled by random primer extension in the presence of [α-32P]dATP. The resulting radiolabeled DNA fragments were used to screen a mouse brain cDNA library. Clones mcGKIβ3D and mcGKI2.2 were isolated, and their inserts were restriction-mapped. mcGKIβ3D, which contains the entire open reading frame (ORF) of murine cGKIβ, was fully sequenced in both directions using Sequenase DNA polymerase (U.S. Biochemical Corp.). mcGKI2.2, which lacks the first 1130 bp of the cGKIβ ORF, was partially sequenced to confirm a 2-bp deletion in the ORF of mcGKIβ3D. The murine cGKIβ cDNA sequence derived from the sequencing of both clones has been submitted to the GenBankTM data base. Sequence analyses were performed using DNASTAR software. The pCMV.mcGKIβ mammalian expression vector was constructed by the polymerase chain reaction (PCR) method using the oligonucleotides 5′-GGA GAT CTC CAC CAT GGG CAC CCT GCG GGA TTT AC-3′ and 5′-GGA TCC AAG CTT ACA TTA GAA GTC TAT GTC-3′ with cDNA clone mcGKIβ3D as a template. The resulting 2100-bp PCR fragment containing the coding region of murine cGKIβ flanked by a BglII site and a BamHI site was digested with BglII andBamHI and ligated into the BglII site of pCMV.Neo (36Huggenvik J.I. Collard M.W. Stofko R.E. Seasholtz A.F. Uhler M.D. Mol. Endocrinol. 1991; 5: 921-930Crossref PubMed Scopus (85) Google Scholar) to create pCMV.mcGKIβ. During sequencing of cDNA clone mcGKIβ3D, a two-nucleotide deletion (Fig. 1) was discovered in the ORF sequence. To correct the deletion in pCMV.mcGKIβ, a 600-bpBsrGI–AflII restriction fragment from cDNA clone mcGKI2.2, containing the correct sequence, was ligated intoBsrGI- and AflII-digested pCMV.mcGKIβ. pCMV.mcGKIβ was sequenced to confirm the entire coding region sequence. 10-cm plates of CV-1 or HEK293 cells were transiently transfected using a calcium phosphate coprecipitation method (37Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar). 48 h after application of DNA precipitates, plates were washed twice with ice-cold phosphate-buffered saline (PBS). Following the addition of 200 μl of homogenization buffer (10 mm sodium phosphate (pH 7.0), 1 mm EDTA, 1 mm dithiothreitol, 250 mm sucrose) containing 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, and 1 μg/ml leupeptin (Sigma), cells were scraped into separate tubes and sonicated twice for 10 s. For kinase activity determinations, cyclic nucleotide (50 μm) was added to or omitted from separate tubes containing a phosphotransferase assay mixture consisting of 20 mm Tris (pH 7.5), 10 mm MgAc, 500 μm IBMX, 200 μm ATP, 11 nm[γ-32P]ATP (ICN) (specific activity = 200–300 cpm/pmol), 10 mm NaF, 10 mm dithiothreitol, and the synthetic phosphate acceptor peptide Kemptide (LRRASLG; 150 μm) or H2Btide (RKRSRAE; 110 μm). When assaying cGKIβ activity, protein kinase inhibitor (PKI) peptide (1 μm) was added to all tubes. The assay was initiated by the addition of cell extracts (0.2 mg/ml), and the phosphotransfer reaction was allowed to proceed for 10 min at 30 °C. The assay was terminated by spotting aliquots onto P81 phosphocellulose (Whatman). The P81 phosphocellulose was washed in 10 mm phosphoric acid and counted. The mutations in pCMV.mcGKIβR75A, pCMV.mcGKIβS79D, pCMV.mcGKIβK404R, pCMV.mcGKIβK407R/R409Q, pCMV.mcGKIβH419Q, and pCMV.mcGKIβW530R were generated by a two-step PCR method using mutagenic primers as described previously (38Gamm D.M. Uhler M.D. J. Biol. Chem. 1995; 270: 7227-7232Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The resulting PCR fragments were subcloned into pCMV.mcGKIβ using convenient restriction sites, and each construct was verified by DNA sequencing of the PCR-amplified region. pCMV.FC/CD, the expression plasmid for the chimeric cGKI deletion mutant, was constructed by replacing the amino-terminal regulatory domain (amino acids 1–355) of cGKIβ with an amino-terminal Flag epitope (DYKDDDDK) followed by the amino-terminal (amino acids 1–20) of murine Cα (39Uhler M.D. Carmichael D.F. Lee D.C. Chrivia J.C. Krebs E.G. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1300-1304Crossref PubMed Scopus (199) Google Scholar). The two-step PCR method was used to create this chimeric cDNA. Initially, a Cα PCR fragment containing aBamHI site and an amino-terminal Flag epitope was generated using the primers 5′-GGG GGA TCC ACC ATG GAC TAC AAG GAC GAC GAT GAC AAG GGC AAC GCC GCG GCC GCC AAG AA-3′ and 5′-AAG TAC TCC GGA GTC CCA C-3′ with pGEM-4.Cα (40Brown N.A. Stofko R.E. Uhler M.D. J. Biol. Chem. 1990; 265: 13181-13189Abstract Full Text PDF PubMed Google Scholar) as a template. The resulting PCR fragment was ligated into pGEM-T (Promega) to create pGEM-T.Flag-Cα1. To generate the cAK/cGK chimera, PCR fragments coding for the Flag-tagged amino terminus of murine Cα and the carboxyl terminus of murine cGKIβ were amplified in separate PCRs. A PCR fragment coding for the amino terminus of murine Cα was generated using primer 5′-CAG GAA ACA GCT ATG AC-3′ and the chimeric primer 5′-GGC TTC ATA TTT TGC TTT TGC TAG GAA CTC TTT CAC GCT-3′ with pGEM-T.Flag-Cα1 as a template. A PCR fragment coding for the carboxyl terminus of murine cGKIβ was generated using primer 5′-AGG CAC GCT TCC ATC AAC-3′ and the chimeric primer 5′-AGC GTG AAA GAG TTC CTA GCA AAA GCA AAA TAT GAA GCC-3′ with pCMV.mcGKIβ as a template. The partially overlapping PCR fragments were isolated, combined with flanking primers 5′-GGG GGA TCC ACC ATG GAC TAC AAG GAC GAC GAT GAC AAG GGC AAC GCC GCG GCC GCC AAG AA-3′ and 5′-AGG CAC GCT TCC ATC AAC-3′, and amplified. The resulting fragment was cut with BamHI and BsrGI, isolated, and ligated into the BglII- and BsrGI-digested pCMV.mcGKIβ to create pCMV.FC/CD. pCMV.FC/CD was restriction-mapped and sequenced to confirm the amplified sequence. pCMV.mcGKII and pCMV.mcGKIIG2A were constructed by PCR. PCR fragments were generated using the following oligonucleotides as forward primers: 5′-GAG ATC TGC TAG CCC ACC ATG GGA AAT GGT TCA GTG-3′ (cGKII) and 5′-GAG ATC TGC TAG CCC ACC ATG GCA AAT GGT TCA GTG-3′ (cGKIIG2A). The oligonucleotide 5′-CTC TAT CGA GGG CCC AAG-3′ was used as the reverse primer, and pCMV.His6cGKII (41Gamm 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) was used as the template in the PCRs to amplify 780-bp DNA fragments coding for the amino termini of mcGKII and mcGKIIG2A. pCMV.His6cGKII was digested with BglII. The full-length cGKII insert was subcloned into the BglII site of pSP73 (Promega) to create pSP73.His6cGKII. The PCR fragments described above were digested with BglII and NsiI, isolated, and ligated individually into pSP73.His6cGKII, which had been fully digested with NsiI and partially digested withBglII, generating pSP73.mcGKII and pSP73.cGKIIG2A. pSP73.mcGKII and pSP73.cGKIIG2A were individually cut withBglII, and 2.4-kilobase pair fragments were isolated and ligated into BglII-cut pCMV.Neo to generate pCMV.mcGKII and pCMV.mcGKIIG2A, respectively. Both pCMV.mcGKII and pCMV.mcGKIIG2A were restriction-mapped, and the amplified regions were fully sequenced. His6cGKII was expressed in Spodoptera frugiperda (Sf9) cells and purified as described previously (41Gamm 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). Purified His6cGKII was used to immunize rabbits for polyclonal antibody production (Research Genetics Inc.). CV-1 and HEK293 cells were grown separately on 10-cm plates to 30 and 50% confluency, respectively, and transfected using a standard calcium phosphate method (37Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar) with 0.5 μg of the cAMP-responsive reporter construct human chorionic gonadotropin-luceriferase (HCG-luciferase) (42Mellon P.L. Clegg C.H. Correll L.A. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4887-4891Crossref PubMed Scopus (200) Google Scholar) as well as the indicated amounts of pRSV.βgal and expression vectors. The total amount of plasmid DNA was brought to 30 μg with the parental vector pCMV.Neo. 24 h after transfection, cells were incubated for an additional 24 h in Dulbecco's modified Eagle's medium (DMEM) with or without added cyclic nucleotides. Following treatment, cells were washed twice with ice-cold PBS, scraped into homogenization buffer, sonicated, and assayed for both luciferase and β-galactosidase activities as described (43Gamm D.M. Baude E.J. Uhler M.D. J. Biol. Chem. 1996; 271: 15736-15742Crossref PubMed Scopus (73) Google Scholar). A full-length cDNA sequence coding for murine VASP (I.M.A.G.E. Consortium clone identification no. 354921/GenBankTM accession no. W45954) (44Lennon G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1088) Google Scholar) was identified in a search of the expressed sequence tag data base for protein sequences homologous to human VASP using the BLAST algorithm (45Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar). This I.M.A.G.E. Consortium (LLNL) cDNA clone was obtained from Genome Systems Inc. It was sequenced in both directions using Sequenase DNA polymerase (U.S. Biochemical), and sequence analyses were performed using DNASTAR software. The nucleotide sequence of the murine cDNA derived from a mouse embryo (embryonic day 13.5–14.5) cDNA library differs from that of the published murine VASP genomic sequence (46Zimmer M. Fink T. Fischer L. Hauser W. Scherer K. Lichter P. Walter U. Genomics. 1996; 36: 227-233Crossref PubMed Scopus (11) Google Scholar), in that it contains a G instead of an A at nucleotide position 955, a three-nucleotide deletion from position 1192 to 1194, and an A instead of a G at nucleotide position 1867. The first difference changes threonine 209 to an alanine, the second difference eliminates glutamine 292, and the third difference is located in the 3′-untranslated region. The murine VASP cDNA sequence has been submitted to the GenBankTMdata base. A mammalian expression plasmid encoding amino-terminal Flag-tagged murine VASP protein was constructed using the PCR method. A PCR fragment containing an amino-terminal Flag epitope (DYKDDDDK) was generated using the primers 5′-GGA TCC GGT ACC TCC ACC ATG GAC TAC AAG GAC GAC GAT GAC AAG GGC GGA GGT ATG AGC GAG ACG GTC ATC TGT TCC-3′ and 5′-GGA TCC CTC GAG TCA AGG AGA ACC CCG CTT CCT CAG-3′ with clone 354921 as a template. The resulting PCR fragment was digested withBamHI, isolated, and ligated into BglII digested pCMV.Neo to create pCMV.Flag-VASP. pCMV.Flag-VASP was restriction-mapped, and the inserted DNA was fully sequenced. For phosphorylation studies, 10-cm plates of CV-1 cells at 30% confluency were transfected using a calcium phosphate transfection method as described previously (37Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar). Each plate was transfected with 20 μg of pCMV.Flag-VASP. 24 h after transfection, the media was changed, and the cells were incubated for 24 h in DMEM without serum. Following treatment with cyclic nucleotides, cells were quickly washed three times with 5 ml of ice-cold PBS, scraped into 400 μl of ice-cold homogenization buffer, and sonicated for 10 s. Extracts were quickly diluted with 500 μl of RIPA buffer (20 mmsodium phosphate (pH 7.0), 300 mm NaCl, 2% sodium deoxycholate, 2% Triton X-100, 2% SDS, 2 mm EDTA, 2 mm EGTA, 100 mm NaF, 10 mm sodium pyrophosphate) and vortexed. Fractions were denatured in SDS-PAGE buffer at 95 °C for 5 min, resolved on 10% SDS-PAGE gels, and transferred to 0.45-μm nitrocellulose membranes (BA-85; Schleicher and Schuell). Membranes were blocked for 1 h in TBST (50 mm Tris (pH 7.5), 150 mm NaCl, and 0.05% Tween 20) supplemented with 5% nonfat dried milk and subsequently incubated with a 1:1000 dilution of an anti-Flag epitope antibody (M2) (Eastman Kodak Co.) in TBST supplemented with 5% nonfat dried milk for 1 h. Filters were washed three times for 10 min with TBST and incubated with an 35S-labeled sheep anti-mouse antibody (0.5 μCi/ml) (Amersham Pharmacia Biotech) in PBS supplemented with 0.5% bovine serum albumin and 0.1% Triton X-100 as the secondary antibody for 1 h. Following the final set of three 10-min washes with TBST, the blots were dried and quantitated. PhosphorImager quantitation was performed in a PhosphorImager apparatus and analyzed with ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). CV-1 cells were grown in DMEM containing 10% fetal calf serum in eight-well tissue culture chambers on poly-l-lysine-coated glass slides (Lab-Tek) to 30% confluency. Cells were transiently transfected using a standard calcium phosphate method (37Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar) with 0.1 μg of pCMV.mcGKIβ; 0.01 μg of pCMV.Flag-Cα3; and 0.04 μg of pCMV.RIIα, 0.1 μg of pCMV.mcGKIβS79D, or 0.1 μg of pCMV.FC/CD. Total plasmid concentration was maintained at 0.25 μg by the addition of the parental vector, pCMV.Neo. Following a 12-h incubation with DNA precipitates, cells were washed once with DMEM containing 10% fetal calf serum and grown for 24 h. Indicated cells were stimulated with 8-Br-cAMP (1 mm) or 8-Br-cGMP (1 mm) and 3-isobutyl-1-methylxanthine (500 μm) in DMEM for various times at 37 °C. Following stimulation, cells were washed twice with ice-cold PBS and fixed with 4% formaldehyde in PBS for 10 min at room temperature followed by a 1:1 mixture of methanol and acetone for 5 min. After washing three times with PBS, cells were incubated with a rabbit polyclonal antibody generated against the carboxyl-terminal 15 amino acids of cGKI (anti-cGMP-PK CT) (Upstate Biotechnology, Inc.) at a 1:1000 dilution or an anti-Flag epitope antibody (M2) (Eastman Kodak) at a 1:2000 dilution in blocking buffer (PBS supplemented with 1% bovine serum albumin, 3% goat serum, and 0.1% saponin (Sigma)) for 1 h at room temperature. After four washes with wash buffer (PBS supplemented with 0.1% saponin), a 1:1000 dilution of Cy3-F(ab′)2 fragment goat anti-rabbit IgG (Jackson) or a 1:3000 dilution of Cy3-F(ab′)2 fragment goat anti-mouse IgG (Jackson) was incubated with the cells for 1 h in the dark in blocking buffer. Prior to examination by" @default.
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• W2009408911 title "Cyclic AMP- and Cyclic GMP-dependent Protein Kinases Differ in Their Regulation of Cyclic AMP Response Element-dependent Gene Transcription" @default.
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• W2009408911 doi "https://doi.org/10.1074/jbc.274.13.8391" @default.
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