Dec 8, 1987 - agents and conditions that activated c-fos also induced fra-1. Induction offra-1 ... stimulation by platelet-derived growth factor (PDGF) into.
MOLECULAR AND CELLULAR BIOLOGY, May 1988, p. 2063-2069 0270-7306/88/052063-07$02.00/0 Copyright © 1988, American Society for Microbiology
Vol. 8, No. 5
fra-1: a Serum-Inducible, Cellular Immediate-Early Gene That Encodes a Fos-Related Antigen DONNA R. COHEN AND TOM CURRAN* Department of Molecular Oncology, Roche Institute of Molecular Biology, Roche Research Center,
Nutley, New Jersey 07110 Received 8 December 1987/Accepted 1 February 1988
A set of proteins antigenically related to the c-fos protein (Fos) are induced by serum in fibroblasts. To isolate cDNA clones of genes encoding such proteins, a Agtll expression cDNA library constructed from serumstimulated rat fibroblasts was screened with antibodies raised against a hydrophilic region (amino acids 127 to 152) of Fos. One of the positive clones identified, termed fra-l (Fos-related antigen) was characterized. It encoded a protein that shared several regions of extensive amino acid homology with Fos (including the region that showed similarity to both the yeast GCN4 regulatory protein and the protein encoded by thejun oncogene), although its nucleotide sequence was considerably diverged from that of the c-fos gene. Only a subset of the agents and conditions that activated c-fos also induced fra-1. Induction of fra-1 expression following serum stimulation was delayed compared with that of c-fos. However, like c-fos, fra-1 was induced rapidly by serum in the presence of protein synthesis inhibitors. Thus, a family of Fos-related, inducible genes are involved in the cellular immediate-early transcriptional response to extracellular stimuli.
The c-fos gene is the cellular homolog of the oncogene (v-fos) carried by the FBJ and FBR murine osteogenic sarcoma viruses (9, 10, 12). The c-fos protein product (Fos) is a nuclear phosphoprotein (6) that forms noncovalent complexes with several other cellular proteins (6, 14, 37). The Fos protein complex is associated with chromatin in vivo (35, 37) and demonstrates both nonspecific and sequence-specific DNA-binding properties in vitro (13, 35, 37). In most cell types, the basal levels of c-fos mRNA and protein are quite low. However, c-fos expression is induced rapidly (generally within 5 min [16]) and transiently after stimulation by a variety of agents in many different situations, including those resulting in mitosis, differentiation, and neuronal cell depolarization (for a review, see T. Curran, in E. P. Reddy, A. M. Skalka, and T. Curran, ed., The Oncogene Handbook, in press). Furthermore, c-fos expression can be induced in the presence of protein synthesis inhibitors (16, 31), which appear to extend the time course of transcription and increase the stability of the transcripts, resulting in an increased accumulation of mRNA (5, 15, 16). The expression of c-fos in the presence of protein synthesis inhibitors is a major criterion for its classification as a cellular immediate-early response gene (8a, 29). The concept of the cellular immediate-early transcriptional response to extracellular stimuli (8a, 29) arose by analogy to studies of viral genes that are expressed very early in the cascade of events that occur during viral infection (34, 36) and that often play a role in the regulation of expression of other viral genes. It has long been recognized that the modulation of eucaryotic gene expression probably involves a series of gene activator proteins involved in the primary response of a cell to stimulation (see reference 43 for a review). In 1977, Pledger et al. dissected the response of mouse BALB/c 3T3 cells to extracellular stimulation by platelet-derived growth factor (PDGF) into two phases, namely competence and progression, which finally result in an induction of DNA synthesis (32). There followed the identification of a set of genes whose expression *
was induced by PDGF in the presence of protein synthesis inhibitors, including JE and KC (4), c-myc (26), and c-fos (5, 16, 27, 31). The term competence gene was coined to describe these genes, as their expression was thought to link PDGF stimulation to the acquisition of competence (4). More recently, it has been recognized that genes such as c-fos and c-myc may serve as nuclear signals in a more general sense (for a review, see Curran, in press). With the identification of several more cDNAs encoding genes that are expressed as part of this primary response (27a, 28, 29, 29a, 41a), the term "cellular immediate-early gene" has been coined to describe genes which respond rapidly to growth factors in the presence of protein synthesis inhibitors. Previously, we reported that immunoprecipitation or Western blot analysis of lysates from serum-stimulated fibroblasts and nerve growth factor-treated PC12 cells with antisera directed against a synthetic peptide (amino acids 127 to 152) identifies a set of Fos-related antigens (14, 37). These proteins migrate at positions corresponding to 46, 35, and 30 kilodaltons (kDa) on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the kinetics of their induction are generally delayed with respect to Fos, primarily being synthesized between 45 and 120 min poststimulation (as compared to Fos, which appears within 15 min [14]). Like Fos, some of the related antigens are acidic nuclear proteins that display nonspecific DNA-binding activity (14,
37). We report here the cloning and characterization of a gene encoding one of these proteins (fra-1, Fos-related antigen). We show that fra-1 was a serum-inducible gene which was significantly diverged from the c-fos gene, but which encoded a protein having regions of extensive amino acid homology to Fos. Like c-fos, fra-1 was induced in the presence of protein synthesis inhibitors, indicating that it was also a cellular immediate-early response gene. MATERIALS AND METHODS Cell culture. Rat fibroblast 208F cells (33) were maintained in Dulbecco-Vogt modified Eagle minimum essential medium containing 10% fetal calf serum. Prior to serum induc-
Corresponding author. 2063
2064
COHEN AND CURRAN
tion, cells were serum deprived (Dulbecco-Vogt modified Eagle medium + 0.5% fetal calf serum) for 44 h and then stimulated by the addition of 20% fetal calf serum for various periods (see Fig. 1). Where indicated, cycloheximide was added to 10 ,ug/ml, and stimulation was carried out for 4 h. RNA isolation. Total RNA was prepared from cultured cells by a modification of the LiCl-urea procedure of Auffray and Rougeon (1). The cells were grown in 150-mm-diameter dishes just to the point of confluence and were then serum deprived or serum stimulated as described above. Following stimulation for the appropriate time, the medium was removed and the cells were washed briefly with phosphatebuffered saline and then lysed in the dishes with 3 M LiCI-6 M urea solution (10 ml/150-mm-diameter dish). The lysates were collected and homogenized (twice for 1 min) and then held overnight at 0°C. The RNA was pelleted by centrifugation with an SW4OTi-type rotor (25,000 rpm; 30 min, 4°C), and the pellet was suspended in 0.5 ml of 10 mM sodium acetate (pH 4.8)-0.5% SDS-1 mM EDTA containing 0.1% diethylpyrocarbonate. The RNA solution was extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1). The RNA was recovered finally by ethanol precipitation, and the pellet was dissolved in TE buffer (10 mM Tris hydrochloride [pH 7.5] and 1 mM EDTA) containing 0.1% diethylpyrocarbonate. Samples were stored at -70°C. Typical yields of total RNA were 100 to 150 ,ug/150-mm-diameter dish. cDNA library preparation and screening. Total RNA was prepared as described above from rat fibroblast 208F cells stimulated with serum for 75 min. Poly(A)+ RNA was selected by a single passage over an oligo(dT)-cellulose column (2), and double-stranded cDNA was prepared by the RNase H procedure of Gubler and Hoffman (17). cDNA molecules were repaired by using T4 DNA polymerase, and internal EcoRI sites were modified by treatment with EcoRI methylase. EcoRI linkers were then added to the methylated DNA, and excess linkers were cleaved and removed by chromatography on Sepharose 4B, which also served to size fractionate the cDNA molecules. cDNAs longer than approximately 0.5 kilobase pairs (kb) were ligated into the EcoRI site of Xgtll (44); the recombinant phage were packaged in vitro, and the packaged library was titrated and plated for screening purposes by using Escherichia coli Y1090r- (45; Promega Biotec). To screen the library, approximately 5 x 104 PFU were plated on each of 25 150-mm-diameter petri dishes, and the plates were incubated and overlaid with isopropyl-p-Dthiogalactopyranoside-impregnated nitrocellulose filters exactly as described by Hunyh et al. (24). After removal from the plates and a brief rinse in TBS (50 mM Tris hydrochloride [pH 8.0], 150 mM NaCl), the filters were incubated in blocking solution and treated with antibody as described by Sambucetti et al. (38). The antibody used for this work was the M-peptide antibody directed against a synthetic 27amino-acid peptide corresponding to Fos protein residues 127 to 152 (11). Agar plugs were removed from the plates for all putative positive signals; the phage were allowed to diffuse from the agar into 1 ml of SM buffer (100 mM NaCl, 8 mM MgSO4 7H20, 50 mM Tris hydrochloride [pH 7.5], and 0.01% gelatin) and were then plated at a lower density (approximately 200 PFU/100-mm-diameter dish) and rescreened with the M-peptide antibody. Analysis of positive clones. DNA was prepared from each of the confirmed positive clones by the small-scale procedure of Helms et al. (18). The clones were then analyzed by digestion with EcoRI restriction endonuclease to determine
MOL. CELL. BIOL.
insert size and the presence of internal EcoRI sites. Agarose gels from these restriction digests were blotted to nitrocellulose (40) and used in cross-hybridization experiments to identify families of related inserts. Northern (RNA) and Southern blot analyses. Total RNA was analyzed by electrophoresis in 0.8% mini-agarose-formaldehyde gels (5 ,ug of RNA per lane), followed by transfer to nitrocellulose and hybridization with nick-translated probes as described by Morgan et al. (30). After hybridization, the filters were washed finally in 0.2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS solution. Genomic DNA or DNA from Xgtll cDNA clones was digested with restriction enzymes (see Fig. 2) and electrophoretically separated on 0.8% agarose gels, followed by transfer to nitrocellulose (40). Hybridization conditions were the same as those for Northern filters, and final posthybridization washes were carried out in 0.2x SSC-0.1% SDS solution. Probe preparation. DNA fragments for use in probe syntheses were prepared as follows. Phage or plasmid constructs containing the fragment of interest were digested with appropriate restriction enzymes, and the fragments were resolved on 0.8% low-melting-point agarose minigels. DNA was purified from melted agarose slices by Elutip-d columns (Schleicher & Schuell, Inc.) and then recovered by ethanol precipitation and suspended in TE buffer. Radioactively labeled probes with specific activity from 1 x 109 to 2 x 109 cpm/,ug were generated by nick translation (kit no. 5000, Amersham Corp.) of approximately 100 ng of purified DNA fragment. Generally, probe concentrations of 106 cpm/ml were used in hybridization experiments. Sequencing. The 1.5-kb EcoRI fra-1 cDNA insert was subcloned into the M13mpll vector that had been digested with EcoRI. One recombinant M13 clone for each orientation of the insert was isolated, and DNA was prepared. This DNA was used for the generation of overlapping deleted subclones on each strand by the Cyclone I Biosystem (International Biotechnologies, Inc.). Template DNA was prepared for each subclone, and sequence data were generated by the dideoxy chain termination method using the Sequenase DNA sequencing kit (United States Biochemical Corp.). In vitro transcription and translation. The 1.5-kb EcoRI fra-1 cDNA insert was subcloned into the EcoRI site of the pSP65 vector (Promega), and one recombinant plasmid was isolated for each orientation of the insert. Purified plasmid DNA was prepared for both isolates (designated pSP65/fra1.7 and pSP65/fra-1.8), and 20 ,ug of each DNA was linearized with AvaI restriction endonuclease. RNA transcripts of the two clones were synthesized in vitro by using SP6 RNA polymerase (Promega) under the conditions specified by the supplier and including 7mGpppG at the same concentration as the GTP to ensure that the synthetic RNAs were capped. This RNA was used to program mRNA-dependent rabbit reticulocyte lysates (Amersham) in the presence of [35S]methionine (ca. 300 ,uCi/mmol; Amersham). For immunoprecipitation analyses, the reticulocyte lysate translates were diluted in 0.3 ml of RIPA buffer (Sa) and treated with antibodies as described previously (6). RESULTS Screening the cDNA library with Fos antibody. A cDNA library was constructed in the Xgtll vector from poly(A)+ RNA isolated from 208F rat fibroblasts that had been stimulated with serum for 75 min. Approximately 106 indepen-
fra-1: A SERUM-INDUCIBLE, FOS-RELATED ANTIGEN GENE
VOL. 8, 1988
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B FIG. 1. Expression of fra-1 in serum-stimulated fibroblasts. Total RNA was isolated from 208F rat fibroblasts that were stimulated with 20%o fetal calf serum for 0, 15, 30, 45, 60, 90, 120, and 240 minutes or with cycloheximide (CHX) or cycloheximide plus 20%o serum (CHX +) for 240 minutes. Samples were separated electrophoretically on mini-0.8% agarose-formaldehyde gels (5 ,ug of RNA per lane), and the gels were blotted to nitrocellulose filters which were then hybridized to nick-translated probes. (A) Filter was hybridized to a probe synthesized from the 1.5-kb EcoRI fra-1 cDNA fragment isolated from the original Xgtll recombinant phage. (B) Filter was hybridized to a probe generated from the EcoRI-SalI 1.35-kb fragment of the chimericfos gene (38). The positions of the 28S and 18S RNAs are indicated. Film exposure was 2 h at -70°C with intensifying screens.
dent recombinant phage were screened with antibody directed against the M-peptide region of the Fos protein (residues 127 to 152). From the positive signals obtained, clones encoding c-fos were identified by high-stringency hybridization with a c-fos (mouse) probe, and these were excluded from further studies. The remaining clones were organized into families of related inserts on the basis of cross-hybridization analyses. Characterization of the fra-1 cDNA insert. One family of five clones, encoding the cDNA now referred to as fra-1, contained inserts of approximately 1.5 kb which hybridized to a messenger RNA of 1.7 to 1.8 kb (Fig. 1A). Basal expression of this mRNA was found to be low; however, an increase in expression following serum stimulation was observed after 30 to 60 min, and expression peaked by 90 min. At 4 h after stimulation, mRNA levels had declined but were still elevated compared with basal values. Figure 1B shows, for comparison, the time course of c-fos expression following serum stimulation. Expression of this gene was significantly elevated within 15 min, peaked around 30 min, and had returned to basal values by 90 min poststimulation. Thus, the kinetics of expression of fra-1 were delayed and slightly protracted compared with those of c-fos. Furthermore, fra-1 was not expressed in all the circumstances in which c-fos expression could be induced. For example,fra-1 showed only modest increases in mRNA levels in PC12 cells treated with 1 mM BaCl2 plus cycloheximide and with nerve growth factor plus benzodiazepine, and there was no detect-
A
B
FIG. 2. Genomic Southern blot analysis of fra-1. High-molecular-weight genomic DNA isolated from 208F rat fibroblasts was digested with BamHI (B), EcoRI (E), or HindIII (H) restriction endonuclease (7.5 pLg per digest) at 37°C, and the samples were separated electrophoretically on 0.8% agarose gels and then blotted to nitrocellulose filters. The ifiters were hybridized to nick-translated probes synthesized from either the 1.5-kb fra-1 cDNA fragment (A) or the 1.35-kb chimeric fos gene fragment (B) under high-stringency conditions (42°C in buffer including 0.75 M NaCl and 509o formamide). DNA size markers of X DNA digested with HindIll and 4X174 DNA digested with HaeIII were used, and fragment positions are shown. Film exposure was 16 h at -70°C with intensifying screens.
able elevation in fra-1 mRNA levels in Metrazole-treated mouse brain RNA samples (data not shown). However, both c-fos and fra-1 mRNAs accumulated in fibroblasts in the presence of the protein synthesis inhibitor cycloheximide (Fig. 1). Southern blot analyses (Fig. 2) indicated that thefra-1 and c-fos genes were independent genetic loci and did not cross-hybridize under high-stringency (Fig. 2) or low-stringency conditions (data not shown). Restriction enzyme mapping of thefra-1 cDNA insert revealed the presence of a single HindlIl site within the insert, and therefore, the genomic Southern blot analysis which identified two fra-1hybridizing HindlIl fragments is consistent with the conclusion thatfra-1 is a single-copy gene. There were two BamHI fragments, and longer film exposure revealed a second, much fainter, EcoRI fragment at approximately 1.5 kb. As the cDNA insert had no internal EcoRI or BamHI sites, these sites must have been located within introns in thefra-1 gene. Synthesis of the fra-1 cDNA protein product in vitro. The fra-1 cDNA insert was subcloned into the EcoRI site of the pSP65 vector (Promega). An SP6-derived transcript of this insert was translated in vitro in a rabbit reticulocyte lysate system and gave rise to a protein migrating with an apparent molecular size of 35 kDa on SDS-polyacrylamide gel elec-
MOL. CELL. BIOL.
COHEN AND CURRAN
2066
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FIG. 3. Synthesis of Fra-1 in vitro. RNAs tran scribed from pc-fos(rat)-1 (5a) and from pSP65/fra-1.7 in vitro were translated in rabbit reticulocyte lysates in the presence of [35S]mel the products were analyzed on an 8% SDS-polyacrylai iide gel (anes shown under c-fos and fra-1, respectively). Lanes: T, total labeled products of the reticulocyte lysate translation; NS5, translation products immunoprecipitated with nonimmune serum I; IS, translation products immunoprecipitated with M-peptide antisera; no RNA, translation products obtained when no in vivi o transcribed RNA was added to the reticulocyte lysate markers.
system; M,
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trophoresis (Fig. 3). This protein was immunopre cipitated by M peptide (Fos amino acids 127 to 152) antis era with an efficiency similar to that observed for the Fos pirotein (Fig. 3). Sequence analysis of fra-1. The 1.5-kb fra-1 insert was sequenced (Fig. 4), and an open reading frame e ncoding 275 amino acids was identified. Nucleotide sequence analysis of the 5' region of a longer cDNA clone identified an in-frame terminator codon immediately upstream of th e sequence shown in Fig. 4. This codon confirms the hypoth esis that the open reading frame most likely initiates at the mtethionine at nucleotide position 46. Primer extension analy,sis revealed the presence of two reverse transcripts, one of vvhich corresponded to the 5' end of the fra-1 cDNA and aniother which was 100 to 200 nucleotides longer. We are presen itly attempting to determine whether this result indicates th at there are two independent but related mRNA species or' whether the shorter reverse transcript simply represents a st rong stop in the extension reaction. The 3' untranslated reg,ion of fra-1 contained two AU-rich sequences between nuclleotide positions 1415 and 1445 that were similar to the putCitive regulatory sequences in the 3' untranslated region of c -fos thought to be involved in mRNA turnover (39). Aniother open reading frame encoding 81 amino acids was alsso identified within the fra-1 cDNA sequence, although thiis predicted protein did not show homology with any knc wn protein sequences.
fra-1 and c-fos proteins show significant homolcigy although the genes have diverged considerably. Comparison of the nucleotide sequence of fra-1 with that of c-f'os revealed several regions of identifiable homology, but th(ese homologous regions were disrupted by frequent mism atches, particularly in the third-base position of the codo ns (Fig. 5). This level of sequence similarity is not compatilble with the formation of stable hybrids in Southern, Norther-n, or plaque
hybridization experiments using large nick-translated probes, and probably accounts for the lack of cross-hybridization between fra-l and c-fos. In contrast, comparison of the amino acid homology through the same region (Fig. 5) shows a very high level (ca. 75%) of conservation between the Fos and Fra-1 proteins. There were two major regions of amino acid homology and two small peptide homologies detected by computer analysis (Fig. 6). As expected, there was significant similarity between the Fos and Fra-1 proteins in the region containing the M-peptide amino acids (Fos amino acids 127 to 152), and interestingly, the high level of conservation was maintained for a considerable number of residues beyond the M peptide (78 residues in total; Fra-1 amino acids 100 to 177). This region is the same as that for which homologies between Fos, the yeast GCN4 regulatory protein, and the jun oncogene protein have previously been reported (42). The Fra-1 and Fos proteins are 77% conserved in this 78-residue region, while Fra-1 shows 28% similarity to both the corresponding jun (amino acids 208 to 286) and GCN4 (amino acids 218 to 281) domains and Fos is about 22% conserved in these regions. Recently, a predicted partial amino acid sequence of the human c-jun protein was reported (3). The c-jun sequence contains an additional cysteine residue conserved between the Fos, Fra-1, and Jun proteins in the DNA-binding domain that is not conserved in the v-jun protein. This analysis does not take into account gaps or insertions required to maximize alignment. Vogt et al. (42) reported a higher percentage of conservation between Fos and GCN4; however, they used the v-fos rather than the c-fos predicted amino acid sequence. We infer from this similarity that there may be a functional relationship between these proteins, although not necessarily an evolutionary kinship. The domains of high-level amino acid homology between Fos and Fra-1 are separated by large regions of little or no amino acid conservation (Fig. 7). Nevertheless, comparison of the hydrophobicity profiles of the two proteins (Fig. 7), as calculated from the values of Hopp and Woods (23), indicates that except for a large region within the carboxy half of Fos, the two proteins showed a very high level of similarity. DISCUSSION We have previously described a set of Fos-related antigens that are induced in serum-stimulated fibroblasts and nerve growth factor-plus-benzodiazepine-treated PC12 cells and that are immunoprecipitated by an antibody directed against amino acids 127 to 152 (the M peptide) of Fos (14). This set includes a cluster of at least two proteins at 46 kDa, as well as several protein species at 35 and 30 kDa, as resolved on high-resolution two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins are not detected in uninduced cells, nor are they immunoprecipitated with other antibodies directed against Fos (14, 27; unpublished observations). In addition, it appears that there are differences between the sets of Fos-related proteins induced by different treatments in different cell types (7, 8, 14, 27, 31). The Fos-related antigens are not thought to represent cleavage products of Fos, on the basis of two observations: (i) they do not accumulate following turnover of Fos in pulse-chase analyses (8, 31) and (ii) tryptic peptide mapping studies (27; our own unpublished data) do not identify any peptides shared between Fos and the related antigens. The isolation of thefra-1 cDNA clone reported here, therefore, represents confirmation that at least one of the Fos-related antigens is encoded by a gene other than c-fos. Preliminary tryptic
fra-1: A SERUM-INDUCIBLE, FOS-RELATED ANTIGEN GENE
VOL. 8, 1988
2067
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FIG. 4. Nucleotide sequence of fra-1. The nucleotide sequence of the fra-1 cDNA insert was determined on both strands by the dideoxy chain termination method using the Sequenase DNA sequencing kit. The sequence shown proceeds in the 5' to 3' direction, and the deduced amino acid sequence starting from the putative initiator codon is shown below the nucleotide sequence. The AT-rich sequences similar to those in the 3' untranslated region of c-fos and thought to be involved in mRNA turnover (39) are indicated by dashes. The potential
polyadenylation signal is underlined. The boxed region represents the likely site of recognition for the Fos M-peptide antibody.
peptide mapping analysis suggests thatfra-1 may share some peptides with the cluster of 46,000-molecular-weight antigens (data not shown). However, the effects of posttranslational modification on the size and mobility of the tryptic peptides have not been determined, and therefore, confirmation of this finding awaits the production of antibodies specific for Fra-1. There has previously been a report of another cDNA sequence related to the third exon of c-fos, namely, r-fos (5). Unfortunately, the r-fos-containing clone has subsequently been lost (C. D. Stiles, personal communication), and so, further characterization of this sequence is not possible. fra-I shows no significant homology (nucleotide or amino acid) to the partially determined r-fos sequence, even through the regions in each predicted protein that are homologous to the Fos M-peptide domain. In contrast, Fra-1 showed extensive similarity to Fos, and the nucleotide conservation in the regions of amino acid similarity was significant enough to allow us to suggest that c-fos andfra-1 may have evolved from a common ancestral gene. The longest conserved amino acid domain, the 78-residue portion of the protein which contains the M-peptide sequence of the
Fos protein, is the same region of Fos for which homology has been detected with both the jun oncogene protein and the yeast GCN4 regulatory protein (42). It has been shown that this domain of the GCN4 protein has sequence-specific DNA-binding activity (19-22), and it has been speculated that the jun protein might have a similar DNA-binding function, on the basis of its homology with the GCN4 protein DNA-binding region (42). This hypothesis has recently been substantiated by the finding that this region of the jun protein could replace the GCN4 protein DNA-binding domain in functional assays (41). Structure-function analysis of the Fos protein indicates that although the N and C termini of the protein are required for stability, the functionally indispensable regions of the protein lie between amino acids 111 and 219 (25). This region encompasses the domain of Fos that is homologous to the GCN4,jun, and Fra-1 proteins. Although the function of Fos is not known, it is a nuclear protein (6) which demonstrates nonspecific and sequence-specific DNA-binding activity (13, 35, 37), and it is thought to be involved in linking extracellular stimuli to long-term adaptive responses by regulating the expression of target genes. It is reasonable to speculate
2068
MOL. CELL. BIOL.
COHEN AND CURRAN
NUCLEOTIDE SEQUENCE HOMOLOGY
1 FOS/FRA-l
342 GAGCAGATCAGCCCGGAGGAGGAAGAGCGCCGCAGGGTGAGACGCGAG GAGCAGCTATCTCCTGAAGAGGAAGAGAAACGGAGAATCCGAAGGGAA 522
CGGAACAAGCTAGCAGCTGCTAAGTGCAGAAACCGAAGAAAGGAATTG ****
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that the DNA-binding domain of Fos may correspond to the functionally indispensable region. Given that the Fra-1 protein is homologous to Fos in this region, then we might conclude that fra-1 is likely to also encode a DNA-binding protein. Evidence supporting this idea includes the primarily nuclear location of the Fos-related antigens (37) and the observation of nonspecific DNA-binding activity by these proteins with affinities similar to that displayed by Fos (37). The hydrophilic domain of Fra-1 (amino acids 178 to 200) that shows little or no structural similarity to the corresponding portion of Fos (amino acids 208 to 305) might confer specificity. In conclusion, fra-1 was a serum-inducible gene that was induced as part of the cellular immediate-early response. It is
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FIG. 7. Structural similarities between Fra-1 and Fos. The top line shows the complete comparison of the Fra-1 and Fos proteins. Each vertical line indicates an amino acid identity, and blocks, therefore, represent stretches of 100% conservation. Dashed lines indicate those regions of Fos which have been deleted in Fra-1. Below this is shown an alignment of the hydrophobicity profiles for Fos and Fra-1 predicted by the values of Hopp and Woods (23). Values were averaged over five-residue blocks, and the dashed lines indicate where gaps have been introduced in the Fra-1 profile to allow alignment with similar structures in the Fos protein.
EQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKS 130 177
FIG. 5. Nucleotide sequence and amino acid homologies betweenfra-1 and c-fos. The nucleotide homology between c-fos and fra-1 and the corresponding amino acid homology between Fos and Fra-1 are shown for a portion of the homologous region that encompasses the M-peptide sequence (Fos amino acids 127 to 152). Identical nucleotides or amino acid residues are indicated by asterisks between the sequences.
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FIG. 6. Major regions of homology between Fra-1 and Fos. The four major blocks of amino acid homology detected by computer analysis between the predictedfra-1 and c-fos proteins are shown. Identical residues are boxed. Also shown for region II are the DNA-binding regions of the yeast GCN4 and jun oncogene proteins.
interesting that fra-1 was not expressed in all circumstances in which c-fos expression was detected and that differences were observed in the sets of Fos-related antigens detected following stimulation of cells by various treatments (14). A possible interpretation of this finding is that Fos functions in signal transduction processes by interacting with various subsets of proteins, depending on the nature of the stimulus and the differentiated state of the stimulated cell. The isolation of the fra-1 cDNA supports the concept that c-fos may be regarded as a marker for a family of genes, some of which are closely related to c-fos (such as fra-1), while others are more distantly related (such asjun), that function in coupling membrane signals to the long-term adaptive responses. ACKNOWLEDGMENTS We thank Ueli Gubler for advice and help in cDNA cloning and K. Rubino and D. Luk for technical assistance.
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VOL. 8, 1988
fra-1: A SERUM-INDUCIBLE, FOS-RELATED ANTIGEN GENE
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