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Two novel c-abl mRNAs are expressed in rat parotid salivary glands during in vivo β-adrenergic receptor stimulation
272
Biochimica et Biophysica Acta, 1049(1990) 272-277
Elsevier BBAEXP 92079
Two novel c-abl mRNAs are expressed in rat parotid salivary glands during in vivo fl-adrenergic receptor stimulation Prema M. Mertz 1, Tuula Backman 1,, Andre Bernards 2 and Eleni Kousvelari 1 Clinical Investigations and Patient Care Branch, National Institute of Dental Research, NIH, Bethesda, MD and 2 The Cancer Center of the Massachusetts Hospital and Harvard Medical School, Charlestown, MA (U.S.A.)
(Received24 January 1990)
Key words: Proto-oncogene;Isoproterenol;Rat salivarygland The c-abl proto-oncogene is transcribed in most cell lines and tissues into two mRNAs of 6.5 and 5.3 kb, which have different 5' ends and encode two 150 kDa proteins that are largely colinear, but have different N-termini. We show here that two unusually short and abundant c-abl-related mRNAs of 1.5 and 1.3 kb appear in rat parotid salivary glands, within 1 day of in vivo administration of the fl-adrenergic receptor agonist isoproterenol. These transcripts are not found in the submandibular salivary gland or in the heart and they are too short to encode the known c-abl proteins. RNA blot, S1 nuclease protection and primer extension analysis suggest that the isoproterenoi inducible parotid gland mRNAs do not contain the kinase domain, but represent part of the C-terminal segment of the abi reading frame.
Introduction The c-abl gene is the cellular homolog of the transforming v-abl sequence of Abelson murine leukemia virus and a member of the tyrosine kinase family of proto-oncogenes [1-3]. In most murine tissues and cell lines, two major c-abl mRNAs of 5.3 and 6.5 kb in length are produced of two transcriptional promoters [4,5]. These transcripts differ at the 5' end and encode proteins of 1123 and 1142 amino acids that are largely colinear, but have unique N-termini of 26 and 45 amino acids, respectively [6]. The human c-abl gene has a similar organization and encodes two very similar proteins [7,8]. The absence of obvious signal peptides or membrane-spanning domains suggests that both abl proteins belong to the group of the non-receptor kinases. One of the proteins is N-terminally myristoylated [9] and both share sequence similarity with other non-receptor kinases in the common protein segment upstream of the kinase domain [6]. Protein segments similar to these upstream conserved motifs are also found in phospholipase C-II [10]. A unique feature of the abl gene
* Present address: Universityof Oulu Institute of Dentistry, Department of Cardiology, Aapistie 3, SF-90220, Oulu, Finland. Correspondence: E. Kousvelari, Clinical Investigations and Patient Care Branch, Building 10, Room 1A-19, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20832, U.S.A.
products is a large C-terminal domain of about 650 amino acids beyond the kinase region. The function of this unique abl domain is unknown, but its deletion does not alter the ability of v-abl to transform fibroblasts or lymphoid cells [11]. Recent evidence suggests that this part of abl may contain a partial nuclear localization signal [12]. Chronic in vivo administration of the fl-adrenoreceptor agonist isproterenol in rats causes marked enlargement of the parotid salivary glands accompanied by transcriptional alterations on parotid gland-specific genes [13,14]. We now report that such treatment results in the appearance of two unusually small homologous c-abl mRNAs which represent sequences C-terminal to the abl kinase domain and are specifically induced in the parotid but not in the submandibular salivary glands. Materials and Methods A n i m a l s and reagents
Male Wistar 3 month-old rats were obtained from Harlan Sprague-Dawley. Mini-osmotic pumps, Model 2002, were purchased from Alza, (Paulo Alto, CA). Isoproterenol was from Sigma (St. Louis, MO). All molecular biological reagents were obtained from either BRL, Sigma or Boehringer-Mannheim, (Indianapolis, IN), unless otherwise noted. The c D N A probes used were a v-abl cDNA purchased from Oncor, Inc. (Gaithersburg, MD) of size 1.7 kb, encoding sequences in the tyrosine kinase domain. The two mouse c-abl
0167-4781/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
273 cDNA probes were clone IV (2 kb) and clone 4.4 (4.4 kb). These clones encode sequences in the 5' untranslated region, in the amino-terminus of the tyrosine kinase and in the carboxy-terminus of the tyrosine kinase along with the entire 3' untranslated region of the c-abl message, respectively. A schematic representation of the different abl cDNAs is shown in Fig. 1. In vivo administration of isoproterenol Rats were anesthetized with 5 mg of sodium phenobarbitol per kg body weight and a mini-osmotic pump was inserted subcutaneously into the back of the animal as per the manufacturers instructions. The mini pump delivered the fl-adrenergic agonist isoproterenol (1 m g / day) from day 0 to day 9. Parotid glands, submandibular glands and heart were removed at the end of each experimental period, trimmed of fat and connective tissue, weighed, frozen in liquid nitrogen and maintained at - 70 ° C. Isolation and Northern blot analysis of total R N A Total RNA was isolated from rat parotid, submandibular glands and heart by the method of Chirgwin et al. [15]. Equal amounts (20 /~g) of RNA from each sample were denatured for 20 min at 70 ° C and electrophoresed in 6% formaldehyde 1.2% agaroge gels [16]. After ethidium bromide staining, the RNA was transferred to nitrocellulose, v-abl and c-abl (clone IV and clone 4.4) inserts were isolated and nick-translated essentially as described in Ref. 17 using a nick-translation kit purchased from Boehringer-Mannheim, and [a-3zp]dCTP (3000 C i / m m o l ) obtained from ICN. Hybridizations were performed at 4 2 ° C overnight. Filters were washed and exposed to Kodak XAR film for the times stated in the figure legends. $1 nuclease analysis RNA was isolated from rat parotid or submandibular glands after 9 days of isoproterenol treatment as described previously. To determine the 5' ends of the transcribed mRNAs, S1 nuclear analysis was performed as described [18,19]. The cDNA fragments used as probes, isolated from the 4.4 c-abl done, were: EcoRIXhoI, EcoRI-HindlII and EcoRI-BamHI fragments, 5' end-labeled at the XhoI, HindlII and B a m H I sites, respectively. About 20 000 cpm of probe was added to 100/~g of total RNA and ethanol precipitated together. The resulting pellet was dissolved in 30 /~1 annealing buffer, denatured for 15 min at 8 5 ° C and hybridized for 16 h at 52°C. The samples were then diluted with 300 /~1 S1 nuclease buffer containing 100 units S1 nuclease. After 90 min at 14°C, the S1 nuclease-protected fragments were concentrated by ethanol precipitation and subjected to gel electrophoresis on 5% polyacrylamide, 7 M urea sequencing gel. The gel was dried and subjected to autoradiography.
CI.IV CI.4.4
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Fig. 1. A schematic comparison of the v-abl and c-abl sequences used in the Northern analyses. The restriction enzyme map of the c-abl type IV cDNA and the position of the c-abl clone IV cDNA, c-abl clone 4.4 cDNA and the v-abl sequence in A-MuL V P160 strain are shown. The hatched box in the v-abl sequence indicates the kinase region. LTR, long terminal repeats.
Primer extension analysis A 20-nucleotide primer, 5 ' - G C T G G C C T T T G G G G A C T T G G - Y , 221 nucleotides upstream of the XhoI site (Fig. 1), complimentary to the c-abl m R N A sequence, was used for primer extension experiments. The primer (100 ng) was radiolabeled with 100 /~Ci [732p]ATP in the presence of polynucleotide kinase. For one sequencing reaction, 5 ng of the labeled primer and 20/~g of total RNA (from parotid glands of rats treated with isoproterenol for 9 days) were annealed by slow cooling from 80 ° C to 42 ° C. Simultaneously doublestranded C1 4.4 cDNA, was annealed with the primer (which was not end-labeled) and for the sequencing reactions the K / R T sequencing systems (Promega, Madison, WI) was used. The primer extension reactions with RNA as template were performed using the chain termination procedure with each of the four dideoxynucleotide triphosphates (dd-NTPs) and reverse transcriptase as described [20]. A control reaction was also performed with all four dNTPs but without the ddNTPs. The reaction products were then size fractionated on 6% polyacrylamide, 7 M urea sequencing gels.
Results Expression of c-abl m R N A s in rat parotid glands during in vivo fl-adrenoceptor stimulation In a previous paper, we reported the appearance of two unusually small v-abl homologous mRNAs of 1.5 and 1.3 kb in the parotid glands of rats after 8 days treatment with isoproterenol [21]. The probe utilized in these earlier studies represents a 1.7 kb segment of the v-abl between HinclI and XholI sites which includes the entire kinase domain (Fig. 1). To characterize the parotid mRNAs further and to determine the kinetics of their appearance, RNA from the parotid glands of rats treated with isoproterenol for 0 - 9 days was analyzed by Northern blots. In panel A of Fig. 2 shows total parotid RNA from rats treated with isoproterenol for the indicated number of days, hybridized with the previously used v-abl probe. Of the two ubiquitous c-abl mRNAs (5.3 and 6.5 kb in mouse [4]) only one appears to be
274 A Treatment ISO T i m e (days)
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Fig. 2. Effect of isoproterenol on c-abl gene expression in the parotid glands. Total RNA was isolated from the parotid glands of rats chronically treated with isoproterenol (ISO) (1 mg/day) for the times indicated in the figure. 20 #g of total R N A was applied to each lane and analyzed by Northern blot hybridization with either v-abl (A), c-abl 4.4 (B) and c-abl C1 IV (C) cDNA probes.
present in parotid glands and is barely visible at this exposure. In contrast, two short mRNAs of 1.3, and 1.5 kb are very prominent and appear only after 1 day of
/3-adrenergic receptor stimulation. These mRNAs persist at virtually the same level during the entire 9-day course of isoproterenol treatment. To test what part of B
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Fig. 3. Northern hybridization analysis of rat, submandibular gland and heart RNA. Total RNA was isolated from the submandibular gland (A, B and C) and heart (D and E) of rats treated in vivo with isoproterenol (1 mg/day) for the times indicated in the figure. 20/~g of total RNA was applied to each lane and analyzed by Northern blot hybridization with either 32P-labeled v-abl (A) c-abl 4.4 (B, D) and IV c-abl (C and E) cDNA probe.
275
abl these mRNAs represent, we rehybridized the blot with two partially overlapping murine c-abl c D N A probes. In these experiments, the 4.4 probe, which extends 4.4 kb upstream of the poly(A) tail of the 5.3 and 6.5 kb c-abl mRNAs, to a position just upstream of the kinase domain (see Fig. 1), detected the two short mRNAs very prominently (Fig. 2; panel B). By contrast, clone IV, which begins just downstream of the kinase domain and extends to an EcoRI. site in the unique 5' part of the 6.5 kb mRNA [6], only detected a single transcript of about 5.3 kb in size (pane C of Fig. 2). We conclude that the inducible parotid mRNAs do not share significant sequence similarity with the kinase domain of c-abl. The two short rnRNAs are specific to parotid glands To examine whether the 1.3 and 1.5 kb abl-treated mRNAs are inducible in other tissues that possess /3adrenergic receptors, we analyzed R N A isolated from the submandibular salivary glands and the hearts of isoproterenol-treated rats. However, in neither of these tissues did any abl probe detect unusual mRNAs (Fig. 3). In submandibular RNA, all three v-abl and c-abl probes detect a single transcript of approx. 5.3 kb in length (top panel of Fig. 3). At least two c-abl mRNAs are detected in heart RNA (lower panel of Fig. 3). The additional bands in the over-exposed heart blot are presumably the 18 S and 28 S ribosomal RNAs. We conclude from the results in Figs. 2 and 3 that the 1.3 and 1.5 kb mRNAs are specific to parotid glands, and that parotid and submandibular salivary glands may only express one of the two conventional c-abl mRNAs. Further characterization of the inducible parotid m R N A s The RNA analysis suggest that the 1.3 and 1.5 kb mRNAs represent sequences downstream of the abl kinase domain. To characterize these transcripts further, we performed S1 nuclease protection and primer extension analysis. Because rat c-abl cDNAs were not available, we used 5' end-labeled fragments of the mouse c-abl 4.4 clone for the S1 analysis. Sites that were labeled include the XhoI site downstream of the kinase domain and the HindlII and B a m H I sites near the 3' end of the mRNAs (Fig. 1). The 1.6 kb EcoRI-XhoI, the 3.4 kb Eco,RI-HindlII and the 3.6 kb E c o R I - B a m H I were isolated and hybridized to parotid and submandibular RNA of 9 days isoproterenol-treated rats. Upon S1 digestion, the XhoI probe gave a clear protected fragment of about 360 bp only with parotid RNA (Fig. 4A). The HindlII and B a m H I probes did not give protected fragments (data not shown), either because the parotid mRNAs do not extend this far downstream, or because the rat and mouse c-abl sequences are not conserved in this region. The latter possibility seems likely, because both the HindlII and B a m H I sites map in the 3'-nontranslated part of the mRNAs. The protected fragment
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Fig. 4. S1 nuclease mapping (A) and primer extension analysis (B, C) of the isoproterenol inducible c-abl transcripts. The position of the probe, the sizes of the protected fragments and the reverse transcriptase extended sequences are indicated in the scheme below the autoradiogram. Lane 1, the undigested EcoRl probe; lane 2, fragment protected after hybridization of the probe to 100/.tg total submandibular RNA; lane 3, fragments protected after hybridization of the probe to 100 ~g total parotid RNA. For primer extension analysis, parotid total RNA (20/~g) was hybridized to 5 ng of a 5'-end-labeled 20 nucleotide primer. The reverse transcriptase reactions were carried out with all of the four deoxynucleotidetriphosphates and each of the four ddNTPs (B, lanes G, A, T, C) and in the absence of ddNTPs (lane N). For reference, dideoxy sequencing of CI 4.4 cDNA with the same 20-met was performed (C).
observed with the XhoI probe, however, confirms that an aberrant c-abl transcript is present in parotid glands. The S1 result suggests that there are no major sequence differences between rat and mouse c-abl in the 360 bp upstream of the XhoI site. We therefore synthesized a 20-mer oligonucleotide that represents a sequence 221 bp upstream of the XhoI site in mouse c-abl. The 5'-end-labeled primer was annealed to either parotid total R N A or to the double-stranded DNA containing the c-abl C1 4.4 insert (Fig. 4C lanes G, A, T,
276 C). The standard chain termination reactions in the presence of the d i d e o x y n u c l e o t i d e t r i p h o s p h a t e s (ddNTPs) were performed and the reaction products were analyzed on a sequencing gel. A representative autoradiogram is shown in Fig. 4B and C. The largest cDNA synthesized in the presence of dideoxynucletides (Figure 4B lanes G, A, T, C) or in the absence of the four dideoxynucleotides (Figure 4B lane N) was 115 nucleotides upstream of the end-labeled primer. Several lower faint bands are also seen in lane N which probably correspond to premature terminations. The results of the primer extension analysis confirm the S1 nuclease analysis and establish the position of the cap site to be 356 nucleotides upstream of the XhoI site.
Discussion Chronic administration of the/3-adrenergic receptor agonist isoproterenol leads to a marked enlargement of the parotid salivary glands in rats [22,13]. The gland size starts increasing after 1 day and doubles after 3 days of drug administration. By 10 days, the gland size increases about 500% [23]. In a recent study, we observed the appearanee of two unusually short v-abl related mRNAs in the parotid glands of rats that had been treated for 8 days with isoproterenol [21]. The results presented here demonstrate, that these short mRNAs can be detected in parotid glands after 1 day of treatment with the drug and that their levels remain constant during 9 days of adminstration. Beside their inducibility, these short mRNAs are unusual for two reasons. First, these transcripts are much too short to encode the two conventional c-abl proteins of 1123 and 1142 amino acids [6]. Secondly, these mRNAs do not hybridize with a probe that represents the kinase domain of c-abL Instead, the S1 and primer extension data suggest that at least one, and possibly both, of the short m R N A s represent the C-terminal half of the abl reading frame. This is intriguing, because abl is unique among tyrosine kinases in having a C-terminal domain of more than 600 amino acids beyond the kinase region. Perhaps this unique abl domain is expressed as a separate polypeptide in rat parotid glands after stimulation of the fl-adrenergic receptor, but this remains to be confirmed. Isoproterenol stimulation of rat parotid acinar cells in vitro leads to a significant increase in the steady state level of c-los m R N A within 60 rain. This increase is probably mediated by increased cAMP levels, since the effect is blocked by the fl-adrenergic antagonist propanolol [21]. Similar results have been reported by Barka et al. [24] on the c-los gene expression in mouse submandibular glands, after a single injection of isoproterenol. However, the unusual c-abl mRNAs have so far only been seen after in vivo administration of isoproterenol in rats, and were not detected in acinar cells that were treated in vitro with this drug for up to 4 h [21].
Hence, the induction of the short c-abl mRNAs is a relatively late effect of fl-adrenergic receptor stimulation. Two major c-abl mRNAs are expressed in most murine cell lines and tissues [4]. It has been suggested that the level of the 6.5 kb m R N A is rather constant, whereas the 5.3 kb m R N A level varies [25]. In this respect, it is interesting to note that we have only found a c-abl m R N A of approx. 5.3 kb in either parotid or submandibular salivary glands of rats. When rat c-abl c D N A probes become available, it will be interesting to test the identity of this m R N A species. Several aberrant c-abl mRNAs have been described recently. In a mouse pre-B lymphocyte library, two major and two minor types of c-abl cDNA were found to be colinear for approx. 5 kb upstream of the poly(A) tail, but to diverge at a common position upstream of the kinase domain, and to predict abl proteins with different N-termini [6]. However, a m R N A corresponding to one of the minor cDNA types has never been seen, and the sequence represented by the other minor type is not conserved between mouse and man [5]. The biological significance of these minor cDNA types is doubtful, therefore. An abundant 4.0 kb c-ab! mRNA in the haploid cells of the mouse testes uses a cryptic polyadenylation site, but has an intact abl reading frame [26-28]. To this collection of unusual mRNAs we now add the short mRNAs that appear in the parotid glands of isoproterenol-treated rats. At this point it is difficult to comment on the biological significance of the parotid c-abl transcripts. The C-terminus of the kinase domain of c-abl maps approx. 650 bp upstream of the internal XhoI site in the mouse. The primer extension and S1 analysis results suggest that one or both of the parotid mRNAs start approx. 360 bp upstream of the XhoI site. At least potentially, the parotid mRNAs may encode part of the C-terminal domain of abl. Experiments aimed at isolating and analyzing rat parotid c-abl cDNAs are in progress.
Acknowledgements We would like to thank Dr. Bruce J. Baum for his constant support during these studies. We would also like to thank Dr. David Baltimore for.his interest in our work. We are grateful to Mrs. Rosemary Perkins for the skillful typing of this manuscript.
References 1 Abelson, H.T. and Rabstein, L.S. (1970) Cancer Res. 30, 22132222. 2 Rosenberg, N., Baltimore, D. and Schrer, C. (1975) Proc. Natl. Acad. Sci. USA 72, 1932-1936. 3 Witte, O.N., Dasgupta, A. and Baltimore, D. (1980) Nature 283, 826-831. 4 Wang, J.Y.J. and Baltimore,D. (1983) Mol. Cell. Biol. 3, 773-779.
277 5 Bernards, A., Paskind, M. and Baltimore, D. (1988) Oncogene 2, 297-304. 6 Ben-Neriah, Y., Bernards, A., Paskind, M., Daley, G.Q. and Baltimore, D. (1986) Cell 44, 577-586. 7 Shtivelman, E., Lifshitz, B., Gale, R.P., Roe, B.A. and Canaani, E. (1986) Cell 47, 277-284. 8 Bernards, A., Rubin, C.M., Westbrook, C.A., Paskind, M. and Baltimore, D. (1987) Mol. Cell. Biol. 7, 3231-3236. 9 Jackson, P. and Baltimore, D. (1989) EMBO J. 8, 449-456. 10 Stahl, M.L., Ferenz, C.R., Kelleher, K.L., Kriz, R.W. and Knopf, J.L. (1988) Nature 332, 269-272. 11 Prywes, R., Foulkes, J.G., Rosenberg, N. and Baltimore, D. (1983) Cell 34, 569-579. 12 Van Etten, R.A., Jackson, P. and Baltimore, D. (1989) Cell 58, 669-678. 13 Schneyer, C.A. (1972) In Regulation of Organ and Tissue Growth (Goss, R.J., ed.), pp. 211-232, Academic Press, New York. 14 Ann, D.K., Clements, D., Johnstone, E.M. and Carlson, D.M. (1987) J. Biol. Chem. 262, 899-904. 15 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. 16 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 202-203, Cold Spring Harbour Laboratory Press, Cold Spring Harbor, New York.
17 Rigby, P.W., Dieckman, M., Rhodes, C. and Berg, P. (1977) J. Mol. Biol. 113, 237-251. 18 Berk, A.J. and Sharp, P.A. (1977) Cell 12, 721-732. 19 Favaloro, J., Treisman, R. and Kamen, R. (1980) Methods Enzymol. 65, 718-749. 20 Geliebter, J. (1987) Focus 9, 5-8. 21 Kousvelari, E., Louis, J., Huang, L-H. and Curran, T. (1988) Exp. Cell Res. 179, 194-203. 22 Selye, H., Cantin, M. and Veilleux, R. (1961) Growth 25, 243-248. 23 Schneyer, C.A. (1969) Proc. Soc. Exp. Biol. Med. 131, 71-75. 24 Barka, T., Gubits, R.M. and Van Der Noen, H.M. (1986) Mol. Cell. Bil. 6, 2984-2989. 25 Renshaw, M.W., Capozza, M.A. and Wang, J.Y.J. (1988) Mol. Cell. Biol. 8, 4547-4551. 26 Ponzetto, C. and Wolgemuth, D.J. (1985) Mol. Cell. Biol. 5, 1791-1794. 27 Meijer, D., Hermans, A., Von Lindern, M., Van Agthoven, T., De Klein, A., Mackenbach, P., Grootegoed, A., Talarico, D., Della Valle, G. and Grosveld, G. (1987) EMBO J 6, 4041-4048. 28 Oppi, C., Shore, S.K. and Reddy, P.E. (1987) Proc. Natl. Acad. Sci. USA 84, 8200-8204.
Biochimica et Biophysica Acta, 1049(1990) 272-277
Elsevier BBAEXP 92079
Two novel c-abl mRNAs are expressed in rat parotid salivary glands during in vivo fl-adrenergic receptor stimulation Prema M. Mertz 1, Tuula Backman 1,, Andre Bernards 2 and Eleni Kousvelari 1 Clinical Investigations and Patient Care Branch, National Institute of Dental Research, NIH, Bethesda, MD and 2 The Cancer Center of the Massachusetts Hospital and Harvard Medical School, Charlestown, MA (U.S.A.)
(Received24 January 1990)
Key words: Proto-oncogene;Isoproterenol;Rat salivarygland The c-abl proto-oncogene is transcribed in most cell lines and tissues into two mRNAs of 6.5 and 5.3 kb, which have different 5' ends and encode two 150 kDa proteins that are largely colinear, but have different N-termini. We show here that two unusually short and abundant c-abl-related mRNAs of 1.5 and 1.3 kb appear in rat parotid salivary glands, within 1 day of in vivo administration of the fl-adrenergic receptor agonist isoproterenol. These transcripts are not found in the submandibular salivary gland or in the heart and they are too short to encode the known c-abl proteins. RNA blot, S1 nuclease protection and primer extension analysis suggest that the isoproterenoi inducible parotid gland mRNAs do not contain the kinase domain, but represent part of the C-terminal segment of the abi reading frame.
Introduction The c-abl gene is the cellular homolog of the transforming v-abl sequence of Abelson murine leukemia virus and a member of the tyrosine kinase family of proto-oncogenes [1-3]. In most murine tissues and cell lines, two major c-abl mRNAs of 5.3 and 6.5 kb in length are produced of two transcriptional promoters [4,5]. These transcripts differ at the 5' end and encode proteins of 1123 and 1142 amino acids that are largely colinear, but have unique N-termini of 26 and 45 amino acids, respectively [6]. The human c-abl gene has a similar organization and encodes two very similar proteins [7,8]. The absence of obvious signal peptides or membrane-spanning domains suggests that both abl proteins belong to the group of the non-receptor kinases. One of the proteins is N-terminally myristoylated [9] and both share sequence similarity with other non-receptor kinases in the common protein segment upstream of the kinase domain [6]. Protein segments similar to these upstream conserved motifs are also found in phospholipase C-II [10]. A unique feature of the abl gene
* Present address: Universityof Oulu Institute of Dentistry, Department of Cardiology, Aapistie 3, SF-90220, Oulu, Finland. Correspondence: E. Kousvelari, Clinical Investigations and Patient Care Branch, Building 10, Room 1A-19, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20832, U.S.A.
products is a large C-terminal domain of about 650 amino acids beyond the kinase region. The function of this unique abl domain is unknown, but its deletion does not alter the ability of v-abl to transform fibroblasts or lymphoid cells [11]. Recent evidence suggests that this part of abl may contain a partial nuclear localization signal [12]. Chronic in vivo administration of the fl-adrenoreceptor agonist isproterenol in rats causes marked enlargement of the parotid salivary glands accompanied by transcriptional alterations on parotid gland-specific genes [13,14]. We now report that such treatment results in the appearance of two unusually small homologous c-abl mRNAs which represent sequences C-terminal to the abl kinase domain and are specifically induced in the parotid but not in the submandibular salivary glands. Materials and Methods A n i m a l s and reagents
Male Wistar 3 month-old rats were obtained from Harlan Sprague-Dawley. Mini-osmotic pumps, Model 2002, were purchased from Alza, (Paulo Alto, CA). Isoproterenol was from Sigma (St. Louis, MO). All molecular biological reagents were obtained from either BRL, Sigma or Boehringer-Mannheim, (Indianapolis, IN), unless otherwise noted. The c D N A probes used were a v-abl cDNA purchased from Oncor, Inc. (Gaithersburg, MD) of size 1.7 kb, encoding sequences in the tyrosine kinase domain. The two mouse c-abl
0167-4781/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
273 cDNA probes were clone IV (2 kb) and clone 4.4 (4.4 kb). These clones encode sequences in the 5' untranslated region, in the amino-terminus of the tyrosine kinase and in the carboxy-terminus of the tyrosine kinase along with the entire 3' untranslated region of the c-abl message, respectively. A schematic representation of the different abl cDNAs is shown in Fig. 1. In vivo administration of isoproterenol Rats were anesthetized with 5 mg of sodium phenobarbitol per kg body weight and a mini-osmotic pump was inserted subcutaneously into the back of the animal as per the manufacturers instructions. The mini pump delivered the fl-adrenergic agonist isoproterenol (1 m g / day) from day 0 to day 9. Parotid glands, submandibular glands and heart were removed at the end of each experimental period, trimmed of fat and connective tissue, weighed, frozen in liquid nitrogen and maintained at - 70 ° C. Isolation and Northern blot analysis of total R N A Total RNA was isolated from rat parotid, submandibular glands and heart by the method of Chirgwin et al. [15]. Equal amounts (20 /~g) of RNA from each sample were denatured for 20 min at 70 ° C and electrophoresed in 6% formaldehyde 1.2% agaroge gels [16]. After ethidium bromide staining, the RNA was transferred to nitrocellulose, v-abl and c-abl (clone IV and clone 4.4) inserts were isolated and nick-translated essentially as described in Ref. 17 using a nick-translation kit purchased from Boehringer-Mannheim, and [a-3zp]dCTP (3000 C i / m m o l ) obtained from ICN. Hybridizations were performed at 4 2 ° C overnight. Filters were washed and exposed to Kodak XAR film for the times stated in the figure legends. $1 nuclease analysis RNA was isolated from rat parotid or submandibular glands after 9 days of isoproterenol treatment as described previously. To determine the 5' ends of the transcribed mRNAs, S1 nuclear analysis was performed as described [18,19]. The cDNA fragments used as probes, isolated from the 4.4 c-abl done, were: EcoRIXhoI, EcoRI-HindlII and EcoRI-BamHI fragments, 5' end-labeled at the XhoI, HindlII and B a m H I sites, respectively. About 20 000 cpm of probe was added to 100/~g of total RNA and ethanol precipitated together. The resulting pellet was dissolved in 30 /~1 annealing buffer, denatured for 15 min at 8 5 ° C and hybridized for 16 h at 52°C. The samples were then diluted with 300 /~1 S1 nuclease buffer containing 100 units S1 nuclease. After 90 min at 14°C, the S1 nuclease-protected fragments were concentrated by ethanol precipitation and subjected to gel electrophoresis on 5% polyacrylamide, 7 M urea sequencing gel. The gel was dried and subjected to autoradiography.
CI.IV CI.4.4
ATG
I
I EcoRI ATG
I Hincll
I Xhot
I Sail
I I HindlllBamHI
I--~ gag
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Fig. 1. A schematic comparison of the v-abl and c-abl sequences used in the Northern analyses. The restriction enzyme map of the c-abl type IV cDNA and the position of the c-abl clone IV cDNA, c-abl clone 4.4 cDNA and the v-abl sequence in A-MuL V P160 strain are shown. The hatched box in the v-abl sequence indicates the kinase region. LTR, long terminal repeats.
Primer extension analysis A 20-nucleotide primer, 5 ' - G C T G G C C T T T G G G G A C T T G G - Y , 221 nucleotides upstream of the XhoI site (Fig. 1), complimentary to the c-abl m R N A sequence, was used for primer extension experiments. The primer (100 ng) was radiolabeled with 100 /~Ci [732p]ATP in the presence of polynucleotide kinase. For one sequencing reaction, 5 ng of the labeled primer and 20/~g of total RNA (from parotid glands of rats treated with isoproterenol for 9 days) were annealed by slow cooling from 80 ° C to 42 ° C. Simultaneously doublestranded C1 4.4 cDNA, was annealed with the primer (which was not end-labeled) and for the sequencing reactions the K / R T sequencing systems (Promega, Madison, WI) was used. The primer extension reactions with RNA as template were performed using the chain termination procedure with each of the four dideoxynucleotide triphosphates (dd-NTPs) and reverse transcriptase as described [20]. A control reaction was also performed with all four dNTPs but without the ddNTPs. The reaction products were then size fractionated on 6% polyacrylamide, 7 M urea sequencing gels.
Results Expression of c-abl m R N A s in rat parotid glands during in vivo fl-adrenoceptor stimulation In a previous paper, we reported the appearance of two unusually small v-abl homologous mRNAs of 1.5 and 1.3 kb in the parotid glands of rats after 8 days treatment with isoproterenol [21]. The probe utilized in these earlier studies represents a 1.7 kb segment of the v-abl between HinclI and XholI sites which includes the entire kinase domain (Fig. 1). To characterize the parotid mRNAs further and to determine the kinetics of their appearance, RNA from the parotid glands of rats treated with isoproterenol for 0 - 9 days was analyzed by Northern blots. In panel A of Fig. 2 shows total parotid RNA from rats treated with isoproterenol for the indicated number of days, hybridized with the previously used v-abl probe. Of the two ubiquitous c-abl mRNAs (5.3 and 6.5 kb in mouse [4]) only one appears to be
274 A Treatment ISO T i m e (days)
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2
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c-abl CI 4 . 4 ~,' , "'~'~ ~
c-abl CI IV
5 . 3 kb --
1 . 5 kb -1.3 kb --
Fig. 2. Effect of isoproterenol on c-abl gene expression in the parotid glands. Total RNA was isolated from the parotid glands of rats chronically treated with isoproterenol (ISO) (1 mg/day) for the times indicated in the figure. 20 #g of total R N A was applied to each lane and analyzed by Northern blot hybridization with either v-abl (A), c-abl 4.4 (B) and c-abl C1 IV (C) cDNA probes.
present in parotid glands and is barely visible at this exposure. In contrast, two short mRNAs of 1.3, and 1.5 kb are very prominent and appear only after 1 day of
/3-adrenergic receptor stimulation. These mRNAs persist at virtually the same level during the entire 9-day course of isoproterenol treatment. To test what part of B
A Treatment ISO
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Time (days)
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Tissue : Submandibular Gland --
D Tissue
5.3
kb
E
: Heart 6.5kb -- 5 . 3 k b
--
Fig. 3. Northern hybridization analysis of rat, submandibular gland and heart RNA. Total RNA was isolated from the submandibular gland (A, B and C) and heart (D and E) of rats treated in vivo with isoproterenol (1 mg/day) for the times indicated in the figure. 20/~g of total RNA was applied to each lane and analyzed by Northern blot hybridization with either 32P-labeled v-abl (A) c-abl 4.4 (B, D) and IV c-abl (C and E) cDNA probe.
275
abl these mRNAs represent, we rehybridized the blot with two partially overlapping murine c-abl c D N A probes. In these experiments, the 4.4 probe, which extends 4.4 kb upstream of the poly(A) tail of the 5.3 and 6.5 kb c-abl mRNAs, to a position just upstream of the kinase domain (see Fig. 1), detected the two short mRNAs very prominently (Fig. 2; panel B). By contrast, clone IV, which begins just downstream of the kinase domain and extends to an EcoRI. site in the unique 5' part of the 6.5 kb mRNA [6], only detected a single transcript of about 5.3 kb in size (pane C of Fig. 2). We conclude that the inducible parotid mRNAs do not share significant sequence similarity with the kinase domain of c-abl. The two short rnRNAs are specific to parotid glands To examine whether the 1.3 and 1.5 kb abl-treated mRNAs are inducible in other tissues that possess /3adrenergic receptors, we analyzed R N A isolated from the submandibular salivary glands and the hearts of isoproterenol-treated rats. However, in neither of these tissues did any abl probe detect unusual mRNAs (Fig. 3). In submandibular RNA, all three v-abl and c-abl probes detect a single transcript of approx. 5.3 kb in length (top panel of Fig. 3). At least two c-abl mRNAs are detected in heart RNA (lower panel of Fig. 3). The additional bands in the over-exposed heart blot are presumably the 18 S and 28 S ribosomal RNAs. We conclude from the results in Figs. 2 and 3 that the 1.3 and 1.5 kb mRNAs are specific to parotid glands, and that parotid and submandibular salivary glands may only express one of the two conventional c-abl mRNAs. Further characterization of the inducible parotid m R N A s The RNA analysis suggest that the 1.3 and 1.5 kb mRNAs represent sequences downstream of the abl kinase domain. To characterize these transcripts further, we performed S1 nuclease protection and primer extension analysis. Because rat c-abl cDNAs were not available, we used 5' end-labeled fragments of the mouse c-abl 4.4 clone for the S1 analysis. Sites that were labeled include the XhoI site downstream of the kinase domain and the HindlII and B a m H I sites near the 3' end of the mRNAs (Fig. 1). The 1.6 kb EcoRI-XhoI, the 3.4 kb Eco,RI-HindlII and the 3.6 kb E c o R I - B a m H I were isolated and hybridized to parotid and submandibular RNA of 9 days isoproterenol-treated rats. Upon S1 digestion, the XhoI probe gave a clear protected fragment of about 360 bp only with parotid RNA (Fig. 4A). The HindlII and B a m H I probes did not give protected fragments (data not shown), either because the parotid mRNAs do not extend this far downstream, or because the rat and mouse c-abl sequences are not conserved in this region. The latter possibility seems likely, because both the HindlII and B a m H I sites map in the 3'-nontranslated part of the mRNAs. The protected fragment
A
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GA
C
C
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GATC
b
1631-- ~
t
517-506 - 396 - 344--
t
--1631
~
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298--
11000 bP I : end-labeled fragment
1.6 kb • ATG !
0.36 kb P-e protected fragment
EO0 I~ H~nc I I . / ' "
X~or'.
B"m HI '~''"
. . . . .
-
0
, c-abl type IV
~" Xho I
J
t lOObp t
Fig. 4. S1 nuclease mapping (A) and primer extension analysis (B, C) of the isoproterenol inducible c-abl transcripts. The position of the probe, the sizes of the protected fragments and the reverse transcriptase extended sequences are indicated in the scheme below the autoradiogram. Lane 1, the undigested EcoRl probe; lane 2, fragment protected after hybridization of the probe to 100/.tg total submandibular RNA; lane 3, fragments protected after hybridization of the probe to 100 ~g total parotid RNA. For primer extension analysis, parotid total RNA (20/~g) was hybridized to 5 ng of a 5'-end-labeled 20 nucleotide primer. The reverse transcriptase reactions were carried out with all of the four deoxynucleotidetriphosphates and each of the four ddNTPs (B, lanes G, A, T, C) and in the absence of ddNTPs (lane N). For reference, dideoxy sequencing of CI 4.4 cDNA with the same 20-met was performed (C).
observed with the XhoI probe, however, confirms that an aberrant c-abl transcript is present in parotid glands. The S1 result suggests that there are no major sequence differences between rat and mouse c-abl in the 360 bp upstream of the XhoI site. We therefore synthesized a 20-mer oligonucleotide that represents a sequence 221 bp upstream of the XhoI site in mouse c-abl. The 5'-end-labeled primer was annealed to either parotid total R N A or to the double-stranded DNA containing the c-abl C1 4.4 insert (Fig. 4C lanes G, A, T,
276 C). The standard chain termination reactions in the presence of the d i d e o x y n u c l e o t i d e t r i p h o s p h a t e s (ddNTPs) were performed and the reaction products were analyzed on a sequencing gel. A representative autoradiogram is shown in Fig. 4B and C. The largest cDNA synthesized in the presence of dideoxynucletides (Figure 4B lanes G, A, T, C) or in the absence of the four dideoxynucleotides (Figure 4B lane N) was 115 nucleotides upstream of the end-labeled primer. Several lower faint bands are also seen in lane N which probably correspond to premature terminations. The results of the primer extension analysis confirm the S1 nuclease analysis and establish the position of the cap site to be 356 nucleotides upstream of the XhoI site.
Discussion Chronic administration of the/3-adrenergic receptor agonist isoproterenol leads to a marked enlargement of the parotid salivary glands in rats [22,13]. The gland size starts increasing after 1 day and doubles after 3 days of drug administration. By 10 days, the gland size increases about 500% [23]. In a recent study, we observed the appearanee of two unusually short v-abl related mRNAs in the parotid glands of rats that had been treated for 8 days with isoproterenol [21]. The results presented here demonstrate, that these short mRNAs can be detected in parotid glands after 1 day of treatment with the drug and that their levels remain constant during 9 days of adminstration. Beside their inducibility, these short mRNAs are unusual for two reasons. First, these transcripts are much too short to encode the two conventional c-abl proteins of 1123 and 1142 amino acids [6]. Secondly, these mRNAs do not hybridize with a probe that represents the kinase domain of c-abL Instead, the S1 and primer extension data suggest that at least one, and possibly both, of the short m R N A s represent the C-terminal half of the abl reading frame. This is intriguing, because abl is unique among tyrosine kinases in having a C-terminal domain of more than 600 amino acids beyond the kinase region. Perhaps this unique abl domain is expressed as a separate polypeptide in rat parotid glands after stimulation of the fl-adrenergic receptor, but this remains to be confirmed. Isoproterenol stimulation of rat parotid acinar cells in vitro leads to a significant increase in the steady state level of c-los m R N A within 60 rain. This increase is probably mediated by increased cAMP levels, since the effect is blocked by the fl-adrenergic antagonist propanolol [21]. Similar results have been reported by Barka et al. [24] on the c-los gene expression in mouse submandibular glands, after a single injection of isoproterenol. However, the unusual c-abl mRNAs have so far only been seen after in vivo administration of isoproterenol in rats, and were not detected in acinar cells that were treated in vitro with this drug for up to 4 h [21].
Hence, the induction of the short c-abl mRNAs is a relatively late effect of fl-adrenergic receptor stimulation. Two major c-abl mRNAs are expressed in most murine cell lines and tissues [4]. It has been suggested that the level of the 6.5 kb m R N A is rather constant, whereas the 5.3 kb m R N A level varies [25]. In this respect, it is interesting to note that we have only found a c-abl m R N A of approx. 5.3 kb in either parotid or submandibular salivary glands of rats. When rat c-abl c D N A probes become available, it will be interesting to test the identity of this m R N A species. Several aberrant c-abl mRNAs have been described recently. In a mouse pre-B lymphocyte library, two major and two minor types of c-abl cDNA were found to be colinear for approx. 5 kb upstream of the poly(A) tail, but to diverge at a common position upstream of the kinase domain, and to predict abl proteins with different N-termini [6]. However, a m R N A corresponding to one of the minor cDNA types has never been seen, and the sequence represented by the other minor type is not conserved between mouse and man [5]. The biological significance of these minor cDNA types is doubtful, therefore. An abundant 4.0 kb c-ab! mRNA in the haploid cells of the mouse testes uses a cryptic polyadenylation site, but has an intact abl reading frame [26-28]. To this collection of unusual mRNAs we now add the short mRNAs that appear in the parotid glands of isoproterenol-treated rats. At this point it is difficult to comment on the biological significance of the parotid c-abl transcripts. The C-terminus of the kinase domain of c-abl maps approx. 650 bp upstream of the internal XhoI site in the mouse. The primer extension and S1 analysis results suggest that one or both of the parotid mRNAs start approx. 360 bp upstream of the XhoI site. At least potentially, the parotid mRNAs may encode part of the C-terminal domain of abl. Experiments aimed at isolating and analyzing rat parotid c-abl cDNAs are in progress.
Acknowledgements We would like to thank Dr. Bruce J. Baum for his constant support during these studies. We would also like to thank Dr. David Baltimore for.his interest in our work. We are grateful to Mrs. Rosemary Perkins for the skillful typing of this manuscript.
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