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Molecular Cloning, cDNA Structure, and Chromosomal Localization of the Human Type II cGMP-Dependent Protein Kinase
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 759–765 (1996)
0477
Molecular Cloning, cDNA Structure, and Chromosomal Localization of the Human Type II cGMP-Dependent Protein Kinase1 S. Ørstavik,*,2 R. Solberg,* K. Taskén,* M. Nordahl,* M. R. Altherr,† V. Hansson,* T. Jahnsen,* and M. Sandberg*,‡ *Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, Oslo, Norway; †Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico; and ‡Department of Anesthesiology, Ullevål Hospital, Oslo, Norway Received February 23, 1996 The type II cGMP-dependent protein kinase is an enzyme originally isolated from the small intestine, and is thought to be involved in the regulation of intestinal ion transport and fluid secretion. A complementary DNA clone encoding a part of the human type II cGMP-dependent protein kinase was isolated from a cerebellum library. Based on sequence information from this complementary DNA, the 59-end of the type II cGMP-dependent protein kinase was amplified from human brain messenger RNA using polymerase chain reaction. The composite complementary DNA encoded a 762 amino acid protein with a calculated molecular mass of 87.4 kDa. Messenger RNAs encoding the type II cGMP-dependent protein kinase were detected in small intestine, colon and prostate. By using polymerase chain reaction and Southern blotting on somatic cell hybrids, the gene encoding the type II cGMP-dependent protein kinase was mapped to human chromosome 4 q13.1q21.1. © 1996 Academic Press, Inc.
Cyclic guanosine monophosphate (cGMP) is a second messenger found in most mammalian cells, and mediates its effects through regulation of certain ion channels, phosphodiesterases and protein kinases (1, 2). Two main forms of cGMP-dependent protein kinases (cGKs) have been identified. The type I cGK is a soluble homodimer where each subunit is 76 kDa in size and is predominantly found in smooth muscle cells, platelets and cerebellum (reviewed in 3). There are two isoforms of type I cGK, designated Ia and Ib, and these differ only in the N-terminal part of the enzymes (4). Complementary DNAs (cDNAs) encoding type I cGK have been isolated from libraries prepared from bovine trachea (4) and from human placenta (5). The type II cGK protein was originally purified from rat intestine (6). Although type II cGK was first thought to exist as a monomer, recent data indicate that this isoform is also a dimer (7). The type I and type II cGKs are distinct proteins encoded by different genes. The proteins are distinguished by their isoelectric points, phosphopeptide maps, immunoreactivities and affinities for cyclic nucleotide analogs (6, 8). A cDNA encoding the type II cGK was recently isolated from a mouse brain cDNA library (9). Later, a type II cGK cDNA was successfully isolated from rat intestine (10). In mouse, mRNA for type II cGK was detected in brain and lung, and at low levels in kidney (9). In rat, type II cGK mRNA was detected in kidney, brain, and intestinal mucosa, but not in lung (10). The physiological role of the type II cGK in man is not fully understood, although it has been shown to be involved in the regulation of intestinal fluid secretion (11). This study presents the cDNA and amino acid
1
The sequence data of human type II cGMP-dependent protein kinase cDNA have been deposited in the EMBL database under the accession number X94612. 2 Corresponding author. Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, N-0317 Oslo, Norway. Fax: +47-22851497. E-mail: [email protected]. Abbreviations: cGMP, cyclic guanosine 39 59 monophosphate; cGK, cGMP-dependent protein kinase; kDa, kilodalton; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SDS, sodium dodecyl sulphate; SSC, standard saline citrate. 759 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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sequences of a human type II cGK, mRNA expression in various human tissues and the localization of the gene on chromosome 4q13.1-q21.1. EXPERIMENTAL PROCEDURES Radiolabeling of DNA. cDNAs were radiolabeled with [a-32P]dCTP using the Megaprime DNA labeling system (Amersham, Buckinghamshire, UK) to a specific activity of approximately 5 × 108 cpm/mg. Oligonucleotides were synthesized by National Biosciences (Plymouth, MN) or the Biotechnology Centre of Oslo (University of Oslo, Oslo, Norway) and radiolabeled with [g-32P] ATP using T4 polynucleotide kinase (Bethesda Research Laboratories, Bethesda, MD) to a specific activity of approximately 1 × 108 cpm/mg. Screening and isolation of cDNA clones. A total of 1 × 106 recombinant clones from a human cerebellum lgt11 cDNA library (Clontech, Palo Alto, CA) were screened by high stringency hybridization as earlier described (12), using mouse type II cGK cDNA (9) as a probe (kindly provided by Dr. M. Uhler, University of Michigan, Ann Arbor, MI). Phage DNA was purified and the insert was subcloned into pUC19 as described previously (12). Rapid amplification of cDNA ends (RACE). Human brain mRNA and the marathon RACE kit were purchased from Clontech. Double stranded cDNA synthesis and anchor ligation were performed as described in the protocol of the manufacturer. The first polymerase chain reaction (PCR) (50 ml) contained 10 ng anchor-ligated double stranded cDNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 mM each of dATP, dCTP, dGTP, and dTTP, 200 nM oligo 1 (59-CAGTGTTTTTATCAGCTGTGGTTG-39), 200 nM anchor primer 1 (an oligonucleotide complementary to the anchor sequence), 1.25 units Taq DNA polymerase (Perkin Elmer, Foster City, CA) and 0.075 units Pfu polymerase (Stratagene, La Jolla, CA). The amplification mixture was preheated to 82°C for 2 min prior to addition of oligonucleotides and template DNA, subsequently denatured for 30 sec at 94°C, and annealed/extended for 4 min at 68°C for a total of 30 cycles in a DNA thermal cycler (Perkin Elmer). Amplification was repeated as above, using 1 ml of the first amplification mixture as the template DNA, and anchor primer 2, an oligonucleotide complementary to the anchor sequence and nested to anchor primer 1. The PCR products were resolved on a 1% agarose gel and transferred to a nylon membrane (Amersham) (13). The filter was prehybridized in 6 × SSC, 1 × Denhardt’s solution, 0.5% SDS, 0.1 mg/ml single stranded salmon sperm DNA and 0.05% sodium pyrophosphate at 42°C for 15 min. The filter was then hybridized in a solution containing 6 × SSC, 1 × Denhardt’s solution, 0.5% SDS, 25 mg/ml yeast tRNA, 0.05% sodium pyrophosphate, and radioactively labeled oligo 2 (59-GTAAATTCTTCAGCAAGGATACAC-39) at 42°C for 2 h. The filter was then washed three times in 6 X SSC, 0.05% sodium pyrophosphate for 5 min at room temperature, and finally washed twice in the same solution for 30 min at 60°C. Amplified fragments were subcloned into the plasmid vector pCRII using the Invitrogen TA cloning kit (San Diego, CA). DNA sequencing. The plasmids containing the inserts were sequenced by the dideoxy termination method (14) using a combination of vector and insert-specific primers using a modified T7 DNA polymerase (Sequenase, United States Biochemicals, CL). The clones were sequenced in both directions, and a total of three clones from different PCR reactions were sequenced. Nucleotide and amino acid sequence data were analyzed using the GCG program package (15). Chromosomal localization. DNA from 44 somatic cell hybrids (BIOS Laboratories Inc., New Haven, CT) were used for chromosomal assignment. In addition, a human/hamster somatic cell hybrid panel was available to sublocalize the gene to a defined region on chromosome 4 (16). The PCR amplifications were performed in 25 ml using 100 ng DNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 mM each of dATP, dCTP, dGTP, and dTTP, 400 nM oligo 3 (59-TCTTTGGAAGACCATTAGGG-39), 400 nM oligo 4 (59-CCAGCACGTTTTCTACTCTG-39), and 1.25 units Taq DNA polymerase (Perkin Elmer). The amplification mixture was denatured for 30 sec at 94°C, annealed for 30 sec at 60°C and extended for 30 sec at 72°C for 30 cycles, with a final extension of 10 min at 72°C. The amplification product was analyzed on a 10% polyacrylamide gel, subcloned into pCRII and sequenced to verify identity. A chromosomal panel filter containing BgIII-digested DNA from human (5 mg), hamster (5 mg), mouse (5 mg) and somatic cell hybrids (15 mg) was a generous gift from Dr. Grzeschik (Institut für Humangenetik und Humangenetische Poliklinik, Universität Marburg, Germany). The methods of cell fusion, preparation and chromosomal characterization of cell hybrid clones with cytogenetic and biochemical techniques have been described elsewhere (17–21). The filter was hybridized (13) using an EcoRI fragment of clone 1, corresponding to nucleotides 1911 to 3314 of the human type II cGK cDNA, as a probe. Northern blots. Northern blots containing 2 mg poly A + RNA from various human tissues were purchased from Clontech. The filters were prehybridized in 5 × Denhardt’s solution, 5 × SSC, 50 mM sodium phosphate buffer, pH 6.8, 0.1% SDS, 250 mg/ml single stranded salmon sperm DNA, and 50% (v/v) formamide at 42°C for 3 h, and hybridized for 16 h in a similar solution containing radiolabeled human type II cGK cDNA corresponding to nucleotides 799–1910. The membranes were washed four times in 2 × SSC, 0.1% SDS for 5 min at room temperature, followed by two washes using 0.1 × SSC, 0.1% SDS at 50°C for 30 min. Autoradiography was performed at −70°C using Amersham Hyperfilm MP and intensifying screens.
RESULTS AND DISCUSSION Radioactively labeled mouse type II cGK cDNA was used as a probe in screening a human cerebellum lgt11 library. One million clones were screened, and one positive clone (clone 1) was 760
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isolated (Fig. 1A). The cDNA insert of clone 1 was 2516 nucleotides in length, and the first 1491 nucleotides showed 90% identity to nucleotides 799 to 2289 of the mouse sequence (9) including an in-frame stop codon. In addition, the clone contained 1025 nucleotides of the 39-untranslated region, showing no homology to any previously known sequence. In order to isolate the 59 end of the human type II cGK cDNA, an oligonucleotide (oligo 1) complementary to nucleotides 1024 to 1047 of the open reading frame of the human type II cGK cDNA was used in 59-RACE. PCR was performed with a mixture of Taq and Pfu DNA polymerases on anchor-ligated double-stranded cDNA prepared from human total brain polyA + RNA (see Materials and Methods). When analyzed by gel electrophoresis, several products differing in length from 400 bp to 1.5 kb were observed. A major product of 1 kb was shown by Southern blotting to hybridize to oligo 2, an oligonucleotide corresponding to nucleotides 848 to 871 of the human type II cGK cDNA (data not shown). The 39 end of this product (clone 2) contained a region of 249 bp that was identical to the 59 region of clone 1, making it very likely that these cDNA clones were derived from the same mRNA. To ensure the correctness of the cDNA sequence, PCR followed by DNA sequencing were repeated three times. The composite sequence, based on clones 1 and 2, contained an open reading frame of 2289 nucleotides including a start and a stop codon, 14 nucleotides of the 59-untranslated region, and 1025 nucleotides of the 39-untranslated region (Fig. 1B). The cDNA encoded a protein with a calculated molecular mass of 87.4 kDa, and the deduced amino acid sequence was 96% identical to mouse and rat type II cGK (9, 10), indicating that the isolated cDNA clones represented a human isoform of type II cGK. A Northern filter containing polyA + RNA from different human tissues was hybridized with a 1.1 kb EcoRI fragment of clone 1, corresponding to nucleotides 799 to 1910 of the open reading frame. The probe detected a major 6 kb mRNA in prostate, small intestine and colon, in addition to a 4.4 kb mRNA in thymus and prostate (Fig. 2). Low levels of the 6 kb mRNA was also detected in brain and placenta (data not shown). The type II cGK has previously been shown to be present in large amounts in the rat intestinal brush border (10) and is thought to be involved in the regulation of chloride secretion (11). The high levels of type II cGK found in human prostate could very well be related to the secretory function of this organ. However, this has to be verified. Polymerase chain reaction using oligo 3 and oligo 4, corresponding to nucleotides 2384 to 2403 and 3080 to 3099, respectively, of the human type II cGK cDNA, resulted in a 716 base pair product from human genomic DNA (data not shown). In contrast, no detectable product was amplified using hamster or mouse genomic DNA. The human product was subcloned, sequenced and found to be identical to the human type II cGK cDNA sequence. A cell hybrid panel, consisting of 44 different human/mouse and human/hamster hybrids, was tested using these primers in an amplification reaction (Table 1). The PCR product mainly segregated with chromosome 4. Low amounts of product were amplified from a cell line that had not been shown to contain chromosome 4. However, karyotyping had only been performed on 20 cells from each cell culture. A small fraction of the cells containing chromosome 4 could be responsible for the weaker signal observed in this cell line. Furthermore, one cell line had karyotypic evidence of chromosome 4 while testing negative for the PCR product. This could be due to a deletion of chromosome 4 in the region containing type II cGK, undetected by karyotyping. To be able to unambiguously determine the chromosomal localization of type II cGK, a Southern blot consisting of BgIII-digested DNA from another 15 somatic cell hybrids was hybridized with a 1.4-kb EcoRI fragment from clone 1. A 6.0-kb human specific fragment was detected and found to be present in ten of the cell hybrids, segregating with chromosome 4 (Table 1). To determine the subchromosomal localization, a somatic cell hybrid panel with defined regions of chromosome 4 was tested using PCR and the type II cGK specific oligonucleotides as described earlier. The PCR product was amplified only from the cell lines containing the region 4q13.1-21.1 (Fig. 3), showing this to be the area where the type 761
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FIG. 1. (A) Cloning strategy of the human type II cGK cDNA. Filled bars represent the open reading frame, while open bars represent the 59- and 39-untranslated regions. Numbers correspond to the nucleotide sequence, where positions +1 to +3 indicate the start codon. Clone 1 was isolated using the mouse type II cGK as a probe, and clone 2 was generated using 59-RACE (see text for details). (B) Nucleotide sequence and deduced amino acid sequence of the human type II cGK. 762
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FIG. 2. Northern blot analysis of type II cGK in various human tissues. Hybridization was performed using a 1.1 kb EcoRI fragment from clone 1 as a probe. Migration of RNA markers is indicated on the right. TABLE 1 Chromosomal Localization of Human Type II cGK; Segregation of the Type II cGK PCR Product (P) or a Specific 6.0-kb Southern Fragment (S) with Human Chromosomes in DNA from Somatic Cell Hybrids (See Text for Details) Concordancy P Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Discordancy S
P
Discordancy %
+/+
−/−
+/+
−/−
+/−
−/+
+/−
S −/+
P %
S %
1 0 0 3 3 0 2 1 0 0 0 0 2 1 1 0 0 1 2 0 1 1 1 1
36 38 37 39 28 37 38 36 38 38 38 37 36 34 37 38 37 37 35 37 35 37 38 37
6 2 9 10 4 5 6 6 2 5 6 6 4 7 4 5 5 5 5 3 8 7 10 3
4 3 2 5 3 2 3 4 4 5 2 4 2 2 3 5 4 3 4 5 3 4 3 5
3 4 4 1 1 4 2 3 4 4 4 4 2 3 3 4 4 3 2 4 3 3 3 3
4 2 3 1 12 3 2 4 2 2 2 3 4 6 3 2 3 3 5 3 5 3 2 3
4 8 1 0 6 5 4 4 8 5 4 4 6 3 6 5 5 5 5 7 2 3 0 7
1 2 3 0 2 3 2 1 1 0 3 1 3 3 2 0 1 2 1 0 2 1 2 0
16 14 16 5 30 16 9 16 14 14 14 16 14 20 14 14 16 14 16 16 18 14 11 14
33 67 27 0 53 53 40 33 60 33 47 33 60 40 53 33 40 47 40 47 27 27 13 47
Note. Concordance indicates the presence of both the PCR product/6.0-kb Souther band and the chromosome (+/+) or the absence of both (−/−). Discordance indicates the presence of the PCR-product/6.0-kb Southern band in the absence of the chromosome (+/−) or vice versa (−/+). 763
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FIG. 3. Chromosomal sublocalization of the gene encoding type II cGK. The sections of chromosome 4 present in the various cell lines are indicated schematically. The type II cGK specific PCR product was amplified from cell lines indicated by “+,” and no product was amplified from cell lines indicated by “−.” The arrow points to the region where the gene encoding type II cGK is located. The cell lines used (HHW 416-1659) have been described in (16).
II cGK locus is located. This region also contains the locus for the polycystic kidney disease type II, an autosomal dominant disease for which the gene has not yet been identified (22). ACKNOWLEDGMENTS This work was supported by The Norwegian Cancer Society, the Research Council of Norway (NFR), Anders Jahre’s Foundation for the Promotion of Science, Abbott Norway, and Nordisk Insulin Foundation Committee.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Schmidt, H. H., Lohmann, S. M., and Walter, U. (1993) Biochim. Biophys. Acta 1178, 153–175. Hofmann, F., Dostmann, W., Keilbach, A., Landgraf, W., and Ruth, P. (1992) Biochim. Biophys. Acta 1135, 51–60. Walter, U. (1989) Rev. Physiol. Biochem. Pharmacol. 113, 41–88. Wernet, W., Flockerzi, V., and Hofmann, F. (1989) FEBS Lett. 251, 191–196. Sandberg, M., Natarajan, V., Ronander, I., Kalderon, D., Walter, U., Lohmann, S. M., and Jahnsen, T. (1989) FEBS Lett. 255, 321–329. de Jonge, H. R. (1981) Adv. Cyclic Nucleotide Res. 14, 315–333. Gamm, D. M., Francis, S. H., Angelotti, T. P., Corbin, J. D., and Uhler, M. D. (1995) J. Biol. Chem. 270, 27380–27388. Vaandrager, A. B., and de Jonge, H. R. (1994) Adv. Pharmacol. 26, 253–283. Uhler, M. D. (1993) J. Biol. Chem. 268, 13586–13591. Jarchau, T., Häusler, C., Markert, T., Pöhler, D., Vandekerckhove, J., de Jonge, H. R., Lohmann, S. M., and Walter, U. (1994) Proc. Natl. Acad. Sci. USA 91, 9426–9430. Markert, T., Vaandrager, A. B., Gambaryan, S., Pöhler, D., Häusler, C., Walter, U., de Jonge, H. R., Jarchau, T., and Lohmann, S. M. (1995) J. Clin. Invest. 96, 822–830. 764
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12. Sandberg, M., Taskén, K., Øyen, O., Hansson, V., and Jahnsen, T. (1987) Biochem. Biophys. Res. Commun. 149, 939–945. 13. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467. 15. Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, WI 53711. 16. Wijmenga, C., Winokur, S. T., Padberg, G. W., Skraastad, M. I., Altherr, M. R., Wasmuth, J. J., Murray, J. C., Hofker, M. H., and Frants, R. R. (1993) Hum. Genet. 92, 198–203. 17. Grzeschik, K. H., Mayerova, A., Bender, K., and Johannsmann, R. (1978) Cytogenet. Cell. Genet. 22, 412–414. 18. Grzeschik, K. H. (1976) Somatic Cell Genet. 2, 401–410. 19. Boyle, J. M., Hey, Y., and Fox, M. (1989) Biochem. Genet. 27, 655–671. 20. Boyle, J. M., Hey, Y., Guerts van Kessel, A., and Fox, M. (1988) Hum. Genet. 81, 88–92. 21. Sakaguchi, A. Y., Zabel, B. U., Grzeschik, K. H., Law, M. L., Ellis, R. W., Scolnick, E. M., and Naylor, S. L. (1984) Mol. Cell. Biol. 4, 989–993. 22. San Millan, J. L., Viribay, M., Peral, B., Martinez, I., Weissenbach, J., and Moreno, F. (1995) Am. J. Hum. Genet. 56, 248–253.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 759–765 (1996)
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Molecular Cloning, cDNA Structure, and Chromosomal Localization of the Human Type II cGMP-Dependent Protein Kinase1 S. Ørstavik,*,2 R. Solberg,* K. Taskén,* M. Nordahl,* M. R. Altherr,† V. Hansson,* T. Jahnsen,* and M. Sandberg*,‡ *Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, Oslo, Norway; †Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico; and ‡Department of Anesthesiology, Ullevål Hospital, Oslo, Norway Received February 23, 1996 The type II cGMP-dependent protein kinase is an enzyme originally isolated from the small intestine, and is thought to be involved in the regulation of intestinal ion transport and fluid secretion. A complementary DNA clone encoding a part of the human type II cGMP-dependent protein kinase was isolated from a cerebellum library. Based on sequence information from this complementary DNA, the 59-end of the type II cGMP-dependent protein kinase was amplified from human brain messenger RNA using polymerase chain reaction. The composite complementary DNA encoded a 762 amino acid protein with a calculated molecular mass of 87.4 kDa. Messenger RNAs encoding the type II cGMP-dependent protein kinase were detected in small intestine, colon and prostate. By using polymerase chain reaction and Southern blotting on somatic cell hybrids, the gene encoding the type II cGMP-dependent protein kinase was mapped to human chromosome 4 q13.1q21.1. © 1996 Academic Press, Inc.
Cyclic guanosine monophosphate (cGMP) is a second messenger found in most mammalian cells, and mediates its effects through regulation of certain ion channels, phosphodiesterases and protein kinases (1, 2). Two main forms of cGMP-dependent protein kinases (cGKs) have been identified. The type I cGK is a soluble homodimer where each subunit is 76 kDa in size and is predominantly found in smooth muscle cells, platelets and cerebellum (reviewed in 3). There are two isoforms of type I cGK, designated Ia and Ib, and these differ only in the N-terminal part of the enzymes (4). Complementary DNAs (cDNAs) encoding type I cGK have been isolated from libraries prepared from bovine trachea (4) and from human placenta (5). The type II cGK protein was originally purified from rat intestine (6). Although type II cGK was first thought to exist as a monomer, recent data indicate that this isoform is also a dimer (7). The type I and type II cGKs are distinct proteins encoded by different genes. The proteins are distinguished by their isoelectric points, phosphopeptide maps, immunoreactivities and affinities for cyclic nucleotide analogs (6, 8). A cDNA encoding the type II cGK was recently isolated from a mouse brain cDNA library (9). Later, a type II cGK cDNA was successfully isolated from rat intestine (10). In mouse, mRNA for type II cGK was detected in brain and lung, and at low levels in kidney (9). In rat, type II cGK mRNA was detected in kidney, brain, and intestinal mucosa, but not in lung (10). The physiological role of the type II cGK in man is not fully understood, although it has been shown to be involved in the regulation of intestinal fluid secretion (11). This study presents the cDNA and amino acid
1
The sequence data of human type II cGMP-dependent protein kinase cDNA have been deposited in the EMBL database under the accession number X94612. 2 Corresponding author. Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, N-0317 Oslo, Norway. Fax: +47-22851497. E-mail: [email protected]. Abbreviations: cGMP, cyclic guanosine 39 59 monophosphate; cGK, cGMP-dependent protein kinase; kDa, kilodalton; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SDS, sodium dodecyl sulphate; SSC, standard saline citrate. 759 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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sequences of a human type II cGK, mRNA expression in various human tissues and the localization of the gene on chromosome 4q13.1-q21.1. EXPERIMENTAL PROCEDURES Radiolabeling of DNA. cDNAs were radiolabeled with [a-32P]dCTP using the Megaprime DNA labeling system (Amersham, Buckinghamshire, UK) to a specific activity of approximately 5 × 108 cpm/mg. Oligonucleotides were synthesized by National Biosciences (Plymouth, MN) or the Biotechnology Centre of Oslo (University of Oslo, Oslo, Norway) and radiolabeled with [g-32P] ATP using T4 polynucleotide kinase (Bethesda Research Laboratories, Bethesda, MD) to a specific activity of approximately 1 × 108 cpm/mg. Screening and isolation of cDNA clones. A total of 1 × 106 recombinant clones from a human cerebellum lgt11 cDNA library (Clontech, Palo Alto, CA) were screened by high stringency hybridization as earlier described (12), using mouse type II cGK cDNA (9) as a probe (kindly provided by Dr. M. Uhler, University of Michigan, Ann Arbor, MI). Phage DNA was purified and the insert was subcloned into pUC19 as described previously (12). Rapid amplification of cDNA ends (RACE). Human brain mRNA and the marathon RACE kit were purchased from Clontech. Double stranded cDNA synthesis and anchor ligation were performed as described in the protocol of the manufacturer. The first polymerase chain reaction (PCR) (50 ml) contained 10 ng anchor-ligated double stranded cDNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 mM each of dATP, dCTP, dGTP, and dTTP, 200 nM oligo 1 (59-CAGTGTTTTTATCAGCTGTGGTTG-39), 200 nM anchor primer 1 (an oligonucleotide complementary to the anchor sequence), 1.25 units Taq DNA polymerase (Perkin Elmer, Foster City, CA) and 0.075 units Pfu polymerase (Stratagene, La Jolla, CA). The amplification mixture was preheated to 82°C for 2 min prior to addition of oligonucleotides and template DNA, subsequently denatured for 30 sec at 94°C, and annealed/extended for 4 min at 68°C for a total of 30 cycles in a DNA thermal cycler (Perkin Elmer). Amplification was repeated as above, using 1 ml of the first amplification mixture as the template DNA, and anchor primer 2, an oligonucleotide complementary to the anchor sequence and nested to anchor primer 1. The PCR products were resolved on a 1% agarose gel and transferred to a nylon membrane (Amersham) (13). The filter was prehybridized in 6 × SSC, 1 × Denhardt’s solution, 0.5% SDS, 0.1 mg/ml single stranded salmon sperm DNA and 0.05% sodium pyrophosphate at 42°C for 15 min. The filter was then hybridized in a solution containing 6 × SSC, 1 × Denhardt’s solution, 0.5% SDS, 25 mg/ml yeast tRNA, 0.05% sodium pyrophosphate, and radioactively labeled oligo 2 (59-GTAAATTCTTCAGCAAGGATACAC-39) at 42°C for 2 h. The filter was then washed three times in 6 X SSC, 0.05% sodium pyrophosphate for 5 min at room temperature, and finally washed twice in the same solution for 30 min at 60°C. Amplified fragments were subcloned into the plasmid vector pCRII using the Invitrogen TA cloning kit (San Diego, CA). DNA sequencing. The plasmids containing the inserts were sequenced by the dideoxy termination method (14) using a combination of vector and insert-specific primers using a modified T7 DNA polymerase (Sequenase, United States Biochemicals, CL). The clones were sequenced in both directions, and a total of three clones from different PCR reactions were sequenced. Nucleotide and amino acid sequence data were analyzed using the GCG program package (15). Chromosomal localization. DNA from 44 somatic cell hybrids (BIOS Laboratories Inc., New Haven, CT) were used for chromosomal assignment. In addition, a human/hamster somatic cell hybrid panel was available to sublocalize the gene to a defined region on chromosome 4 (16). The PCR amplifications were performed in 25 ml using 100 ng DNA, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 mM each of dATP, dCTP, dGTP, and dTTP, 400 nM oligo 3 (59-TCTTTGGAAGACCATTAGGG-39), 400 nM oligo 4 (59-CCAGCACGTTTTCTACTCTG-39), and 1.25 units Taq DNA polymerase (Perkin Elmer). The amplification mixture was denatured for 30 sec at 94°C, annealed for 30 sec at 60°C and extended for 30 sec at 72°C for 30 cycles, with a final extension of 10 min at 72°C. The amplification product was analyzed on a 10% polyacrylamide gel, subcloned into pCRII and sequenced to verify identity. A chromosomal panel filter containing BgIII-digested DNA from human (5 mg), hamster (5 mg), mouse (5 mg) and somatic cell hybrids (15 mg) was a generous gift from Dr. Grzeschik (Institut für Humangenetik und Humangenetische Poliklinik, Universität Marburg, Germany). The methods of cell fusion, preparation and chromosomal characterization of cell hybrid clones with cytogenetic and biochemical techniques have been described elsewhere (17–21). The filter was hybridized (13) using an EcoRI fragment of clone 1, corresponding to nucleotides 1911 to 3314 of the human type II cGK cDNA, as a probe. Northern blots. Northern blots containing 2 mg poly A + RNA from various human tissues were purchased from Clontech. The filters were prehybridized in 5 × Denhardt’s solution, 5 × SSC, 50 mM sodium phosphate buffer, pH 6.8, 0.1% SDS, 250 mg/ml single stranded salmon sperm DNA, and 50% (v/v) formamide at 42°C for 3 h, and hybridized for 16 h in a similar solution containing radiolabeled human type II cGK cDNA corresponding to nucleotides 799–1910. The membranes were washed four times in 2 × SSC, 0.1% SDS for 5 min at room temperature, followed by two washes using 0.1 × SSC, 0.1% SDS at 50°C for 30 min. Autoradiography was performed at −70°C using Amersham Hyperfilm MP and intensifying screens.
RESULTS AND DISCUSSION Radioactively labeled mouse type II cGK cDNA was used as a probe in screening a human cerebellum lgt11 library. One million clones were screened, and one positive clone (clone 1) was 760
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isolated (Fig. 1A). The cDNA insert of clone 1 was 2516 nucleotides in length, and the first 1491 nucleotides showed 90% identity to nucleotides 799 to 2289 of the mouse sequence (9) including an in-frame stop codon. In addition, the clone contained 1025 nucleotides of the 39-untranslated region, showing no homology to any previously known sequence. In order to isolate the 59 end of the human type II cGK cDNA, an oligonucleotide (oligo 1) complementary to nucleotides 1024 to 1047 of the open reading frame of the human type II cGK cDNA was used in 59-RACE. PCR was performed with a mixture of Taq and Pfu DNA polymerases on anchor-ligated double-stranded cDNA prepared from human total brain polyA + RNA (see Materials and Methods). When analyzed by gel electrophoresis, several products differing in length from 400 bp to 1.5 kb were observed. A major product of 1 kb was shown by Southern blotting to hybridize to oligo 2, an oligonucleotide corresponding to nucleotides 848 to 871 of the human type II cGK cDNA (data not shown). The 39 end of this product (clone 2) contained a region of 249 bp that was identical to the 59 region of clone 1, making it very likely that these cDNA clones were derived from the same mRNA. To ensure the correctness of the cDNA sequence, PCR followed by DNA sequencing were repeated three times. The composite sequence, based on clones 1 and 2, contained an open reading frame of 2289 nucleotides including a start and a stop codon, 14 nucleotides of the 59-untranslated region, and 1025 nucleotides of the 39-untranslated region (Fig. 1B). The cDNA encoded a protein with a calculated molecular mass of 87.4 kDa, and the deduced amino acid sequence was 96% identical to mouse and rat type II cGK (9, 10), indicating that the isolated cDNA clones represented a human isoform of type II cGK. A Northern filter containing polyA + RNA from different human tissues was hybridized with a 1.1 kb EcoRI fragment of clone 1, corresponding to nucleotides 799 to 1910 of the open reading frame. The probe detected a major 6 kb mRNA in prostate, small intestine and colon, in addition to a 4.4 kb mRNA in thymus and prostate (Fig. 2). Low levels of the 6 kb mRNA was also detected in brain and placenta (data not shown). The type II cGK has previously been shown to be present in large amounts in the rat intestinal brush border (10) and is thought to be involved in the regulation of chloride secretion (11). The high levels of type II cGK found in human prostate could very well be related to the secretory function of this organ. However, this has to be verified. Polymerase chain reaction using oligo 3 and oligo 4, corresponding to nucleotides 2384 to 2403 and 3080 to 3099, respectively, of the human type II cGK cDNA, resulted in a 716 base pair product from human genomic DNA (data not shown). In contrast, no detectable product was amplified using hamster or mouse genomic DNA. The human product was subcloned, sequenced and found to be identical to the human type II cGK cDNA sequence. A cell hybrid panel, consisting of 44 different human/mouse and human/hamster hybrids, was tested using these primers in an amplification reaction (Table 1). The PCR product mainly segregated with chromosome 4. Low amounts of product were amplified from a cell line that had not been shown to contain chromosome 4. However, karyotyping had only been performed on 20 cells from each cell culture. A small fraction of the cells containing chromosome 4 could be responsible for the weaker signal observed in this cell line. Furthermore, one cell line had karyotypic evidence of chromosome 4 while testing negative for the PCR product. This could be due to a deletion of chromosome 4 in the region containing type II cGK, undetected by karyotyping. To be able to unambiguously determine the chromosomal localization of type II cGK, a Southern blot consisting of BgIII-digested DNA from another 15 somatic cell hybrids was hybridized with a 1.4-kb EcoRI fragment from clone 1. A 6.0-kb human specific fragment was detected and found to be present in ten of the cell hybrids, segregating with chromosome 4 (Table 1). To determine the subchromosomal localization, a somatic cell hybrid panel with defined regions of chromosome 4 was tested using PCR and the type II cGK specific oligonucleotides as described earlier. The PCR product was amplified only from the cell lines containing the region 4q13.1-21.1 (Fig. 3), showing this to be the area where the type 761
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FIG. 1. (A) Cloning strategy of the human type II cGK cDNA. Filled bars represent the open reading frame, while open bars represent the 59- and 39-untranslated regions. Numbers correspond to the nucleotide sequence, where positions +1 to +3 indicate the start codon. Clone 1 was isolated using the mouse type II cGK as a probe, and clone 2 was generated using 59-RACE (see text for details). (B) Nucleotide sequence and deduced amino acid sequence of the human type II cGK. 762
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FIG. 2. Northern blot analysis of type II cGK in various human tissues. Hybridization was performed using a 1.1 kb EcoRI fragment from clone 1 as a probe. Migration of RNA markers is indicated on the right. TABLE 1 Chromosomal Localization of Human Type II cGK; Segregation of the Type II cGK PCR Product (P) or a Specific 6.0-kb Southern Fragment (S) with Human Chromosomes in DNA from Somatic Cell Hybrids (See Text for Details) Concordancy P Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Discordancy S
P
Discordancy %
+/+
−/−
+/+
−/−
+/−
−/+
+/−
S −/+
P %
S %
1 0 0 3 3 0 2 1 0 0 0 0 2 1 1 0 0 1 2 0 1 1 1 1
36 38 37 39 28 37 38 36 38 38 38 37 36 34 37 38 37 37 35 37 35 37 38 37
6 2 9 10 4 5 6 6 2 5 6 6 4 7 4 5 5 5 5 3 8 7 10 3
4 3 2 5 3 2 3 4 4 5 2 4 2 2 3 5 4 3 4 5 3 4 3 5
3 4 4 1 1 4 2 3 4 4 4 4 2 3 3 4 4 3 2 4 3 3 3 3
4 2 3 1 12 3 2 4 2 2 2 3 4 6 3 2 3 3 5 3 5 3 2 3
4 8 1 0 6 5 4 4 8 5 4 4 6 3 6 5 5 5 5 7 2 3 0 7
1 2 3 0 2 3 2 1 1 0 3 1 3 3 2 0 1 2 1 0 2 1 2 0
16 14 16 5 30 16 9 16 14 14 14 16 14 20 14 14 16 14 16 16 18 14 11 14
33 67 27 0 53 53 40 33 60 33 47 33 60 40 53 33 40 47 40 47 27 27 13 47
Note. Concordance indicates the presence of both the PCR product/6.0-kb Souther band and the chromosome (+/+) or the absence of both (−/−). Discordance indicates the presence of the PCR-product/6.0-kb Southern band in the absence of the chromosome (+/−) or vice versa (−/+). 763
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FIG. 3. Chromosomal sublocalization of the gene encoding type II cGK. The sections of chromosome 4 present in the various cell lines are indicated schematically. The type II cGK specific PCR product was amplified from cell lines indicated by “+,” and no product was amplified from cell lines indicated by “−.” The arrow points to the region where the gene encoding type II cGK is located. The cell lines used (HHW 416-1659) have been described in (16).
II cGK locus is located. This region also contains the locus for the polycystic kidney disease type II, an autosomal dominant disease for which the gene has not yet been identified (22). ACKNOWLEDGMENTS This work was supported by The Norwegian Cancer Society, the Research Council of Norway (NFR), Anders Jahre’s Foundation for the Promotion of Science, Abbott Norway, and Nordisk Insulin Foundation Committee.
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