THE JOURNAL OF BIOLOGICAL CHEMISTRY . © 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 31, Issue of November 5, pp. 19185-19191, 1990 Printed in U.S.A. A Growth Factor-inducible Nuclear Protein with a Novel Cysteine/ Histidine Repetitive Sequence* (Received for publication, April 16, 1990) Raymond N. DuBois, Michael W. McLanet, Kevin Ryder§, Lester F. Lauf, and Daniel Nathans From the Howard Hughes Medical Institute and the Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Growth factors rapidly induce transcription of a set of genes that encode regulatory proteins, many of which have been identified by cDNA cloning. Here we report the analysis of a cDNA corresponding to a gene induced in mouse 3T3 cells by growth factors and a variety of other extracellular signaling agents. The cDNA encodes a proline-, serine-, and glycine-rich nuclear protein designated Nup475 of 319 amino acids that contains two tandemly repeated cysteine- and his- tidine-containing sequences (CXsCX,CX3H) suggestive of a novel heavy metal-binding domain. Nup475 pro- duced in Escherichia coli binds zinc. Its mRNA is pres- ent in a number of mouse tissues and cell lines, being especially abundant in intestine, thymus, and regen- erating liver and in a macrophage cell line stimulated by 7-interferon. We hypothesize that Nup475 is a reg- ulatory protein with a novel zinc finger structure. Polypeptide growth factors and other extracellular ligands induce in target cells sequential changes in gene expression that appear to mediate long term cellular responses to the ligands. In mouse 3T3 fibroblastic cells, serum, platelet-de- rived growth factor, or fibroblast growth factor rapidly induce transcriptional activation of a set of “immediate early” genes that encode nuclear proteins (including known or probable transcription factors), putative cytokines, transmembrane proteins, and cytoskeletal proteins (reviewed in Refs. 1-3). Among the nuclear proteins are members of the Fos and Jun families, Myc, and three zinc finger proteins. Some of these proteins are thought to regulate the genomic response to growth factors by repressing immediate early genes and by activating genes expressed later. Here we report the identification and initial characteriza- tion of another immediate early nuclear protein designated Nup475. Like many of the other immediate early proteins, Nup475 was identified by analyzing cDNA clones from a library prepared from BALB/c 8T3 cells stimulated with serum in the presence of an inhibitor of protein synthesis (4). * This research was supported in part by National Cancer Institute Grant 5 POQ1 CA16519. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) J05669. + Present address: ICI/Americas, Wilmington, DE 19803. § Postdoctoral Fellow of the American Cancer Society. Present address: Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, PA 19111. { Present address: Dept. of Genetics, University of Illinois College of Medicine, Chicago, IL 60680. Nup475 has two tandemly repeated cysteine and histidine sequences of novel type suggestive of a heavy metal-binding domain and is shown to bind Zn**. On the basis of these properties and its nuclear location, we suggest that Nup475 may be a nucleic acid-binding protein involved in regulating the response to growth factors. EXPERIMENTAL PROCEDURES Cell Culture and Transfection—BALB/c 3T3 cells were maintained and stimulated with serum as described previously (4). Cos 1 cells (5) were maintained in minimal essential medium plus 10% fetal calf serum; for transfection, cells were treated with calcium phosphate- precipitated recombinant plasmid containing Nup475 cDNA (EcoRI- Bglll fragment) cloned into the EcoRI site of pMT2 (6). Partial Hepatectomy—Partial hepatectomy in which 70% of the liver was removed was carried out as described (7). At various times after hepatectomy, mouse livers were surgically removed and total RNA was isolated (8). DNA Sequencing—Nup475 cDNA and genomic DNA isolates were cloned into the pGEM-2 plasmid (Promega Biotec), and sets of 5’ and 3’ deletions were prepared. The resulting subclones were se- quenced as double-stranded plasmids by the dideoxynucleotide chain termination method (9) using deoxyadenosine 5’ -[a-*°S]thiotriphos- phate and Sequenase (United States Biochemial Corp.). Sequence Homology Search—Homology search of the GenBank, EMBL, and NBRF databases was carried out with the use of the GenMenu program which incorporates the GCG sequence analysis software (10). Mapping the 5’ End of Nup475 mRNA—A ™P-radiolabeled oligo- nucleotide complementary to nucleotides 17 to 45 of the Nup475 mRNA was hybridized to 25 yg of total cellular RNA purified from BALB/c 373 cells 3 h after the addition of 20% serum and 10 yg of cycloheximide per ml. Primer extension was carried out as perviously described (11). The S1 nuclease assay was performed as described (12). The probe used was a “P-labeled DNA synthesized on a 3-kb! mouse genomic clone of nup475 (partial sequence shown in Fig. 5) using as primer an oligonucleotide complementary to nucleotides 17 to 45 of the mRNA. Southern Biot Analysis—Genomic DNA was digested with EcoRI or HindIII (New England Biolabs), fractionated on 1% agarose gels, and then transferred to nitrocellulose (Optibind, Schleicher and Schuell) in 20 x SSC (SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0). DNA probes were prepared from the Nup475 cDNA by isolating the Neol-ByglII fragment from a low melt agarose gel. The fragments were labeled with [a-??P]dCTP by nick translation (Boeh- ringer Mannheim). Prehybridization and hybridization incubations were carried out in 5 x SSC at 65 °C for 14-24 h. After hybridization, the blots were washed twice in a buffer containing 2 x SSC at room temperature for 30 min. Then, the filters were washed in 1 x SSC at room temperature for 120 min and exposed to x-ray film at —70 °C. Production of Nup475 in Escherichia coli and Preparation of Anti- sera—The coding region of the Nup475 cDNA (Ncol-Bglll) was cloned into the pET8C plasmid and expressed in the E. coli T7 polymerase expression system (13). EF. coli protein extracts were prepared by lysis in SDS sample buffer and electrophoresed in pre- ! The abbreviations used are: kb, kilobase(s); SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PDGF, platelet- derived growth factor. 19185 19186 parative SDS-polyacrylamide gels (14). The expressed protein was visualized by staining with Coomassie Blue, the Nup475 band was excised, and the gel segment was pulverized in liquid nitrogen and used to immunize New Zealand White rabbits {15}. To purify the antibody, purified bacterial Nup475 was covalently linked to Sepha- rose and used as an affinity column as described (45). Western blotting was carried out as described (16). Anti-peptide sera were raised against each of two synthetic peptides (corresponding to amino acids 84-99 and 804-319 of Nup475) coupled to bovine serum albumin. Northern Blot Analysis— Northern blot analysis was carried out as described (17). In Vitro Translation—JIn vitro translation of CDNA transcripts and immunoprecipitation were carried out as described (40). Comparison of Nup475 mRNA with the Cloned cCDNA~--Total RNA from regenerating mouse liver was annealed to a “P-labeled antisense transcript corresponding to a 338-nucleotide fragment of Nup475 cDNA derived from nucleotides 355 to 17 (Fig. 2). The RNA duplex was treated with RNase and fractionated by electrophoresis, as de- seribed (41). Amplification of cDNA prepared from total cellular RNA was carried out as described (42). The primers corresponded to nucleotides 2 to 22 (5’ primer) and nucleotides 1804 to 1783 (3° primer) of the Nup475 cDNA (Fig. 2). Immunofluorescence Staining of Cultured Cells—Serum-stimulated BALB/c 3T3 cells or Cos cells grown on 12-mm coverslips were processed for immunostaining, as described (18), using affinity-puri- fied anti-Nup475 antibody and rhodamine-conjugated donkey anti- rabbit IgG (Jackson Immunoresearch Laboratories). Zine Binding—Cell lysates and protein fractions were fractionated by discontinuous SDS-PAGE (14), and the proteins were electropho- retically transferred to nitrocellulose (Bio-Rad TransBlot). After transfer, the filters were washed in metal-binding buffer (100 mm Tris-HCl, pH 6.8, 50 mm NaCl) for 1 h, and the proteins were denatured in 6 M guanidine HC! for 30 min at 4°C, The proteins were then renatured by serial incubations in buffer containing de- creasing amounts of guanidine HCI. The filters were finally incubated in metal-binding buffer with ZnCl, and washed as described (19). RESULTS Analysis of Nupd75 cDNA-~Nup475 cDNA was initially identified by differential screening of a cDNA library prepared from poly(At) RNA of BALB/c mouse 3T3 cells stimulated with serum in the presence of cycloheximide (clone 475 of Ref. 4). As is typical of immediate early genes, nup475 is transcriptionally activated within minutes after stimulation of quiescent 3T3 cells with serum or PDGF, its mRNA in- creases transiently thereafter, and it is superinduced in the presence of an inhibitor of protein synthesis ((4) and Fig. 1). 6 0.8 1.9 30 6.0 he Serum TPA KCt + Cali, Forskoiin Fic, 1. Northern blot analysis showing response of Nup475 mRNA in BALB/c 3T3 cells to signaling agents. Total RNA prepared from cells at various times after stimulation with 20% fetal calf serum (15 ug of RNA), 10 eM forskolin (10 wg), 60 mM 12-0- tetradecanoylphorbol-13-acetate (TPA) (10 xg), or 60 mM KCI plus 10 mM CaCl, (10 yg) was fractionated by electrophoresis and blotted ont nitrocellulose, and the filter was probed with Nup475 cDNA. The numbers above each lane indicate the time in hours after stimu- lation. Only the 1.8-kb region of each autoradiogram is shown. Novel Zinc-binding Protein Nup475 mRNA also increases following treatment of 3T3 cells with a phorbol ester, forskolin, and depolarizing concen- trations (20) of K* and Ca®* (Fig. 1). Nup475 mRNA is about 1.8 kb in length, as estimated by gel electrophoresis. The nucleotide sequence of a nearly full length Nup475 cDNA is shown in Fig. 2. It contains a long open reading frame beginning at nucleotide 60 (the first ATG codon) that is 959 nucleotides in length and encodes a protein of 319 amino acids with a predicted molecular mass of 33,592 daltons. Following the termination triplet TGA at position 1,017, there are 785 nucleotides and a poly(A) tail. A typical poly(A) addition signal is present at nucleotides 1,781 to 1,786. Several ATTTA or related sequences are present in the 3’ untranslated region, which may contribute to the observed instability of Nup475 mRNA (4, 21). The 5’ end of the mRNA was identified by reverse tran- scription of RNA prepared from superinduced 3T9 cells using an oligonucleotide primer (nucleotides 45-17) and by $1 nu- clease analysis using a genomic DNA probe made with the same primer (Fig. 3). Both procedures revealed that the 5’ end of the mRNA maps predominantly to a site 16 nucleotides upstream of the cDNA clone; this site is 29 nucleotides downstream from the start of a TATA-like sequence (ATAAA) present in a nup475 genomic clone {Fig. 2). Predicted Protein Sequence—The protein encoded by the long open reading frame of the Nup475 cDNA Is rich in glycine, serine, and proline residues, including 3 PPPPG sequences. Another notable feature of the amino acid se- quence is the presence of a partial repeat of 26 amino acids from residue 95 to 120 and 133 to 158 (underlined in Fig. 2). Of the 26 amino acid residues in each repeat, 17 are identical or closely related (Fig. 4), including identically spaced cysteine and histidine residues of the form CXsCX,C.X3H, suggestive of a heavy metal-binding finger structure of novel type. Our initial search of the EMBL/GenBank/NBRF protein and nucleic acid databases (versions 21, 62, 23, 35, respec- tively) did not reveal significant homology between Nup475 and known proteins. However, just prior to submission of this manuscript, we discovered a similarity of parts of the Nup478 cDNA sequence to that for a recently described cDNA (des- ignated TIS11) derived from an mRNA induced by phorbol ester (43). The nucleotide sequence of Nup475 cDNA between positions 300 and 1731 (Fig. 2) is present in TIS11 cDNA except that Nup475 has an additional C at position 607 in its coding sequence (beyond which the reading frames of Nup475 and TIS11 differ), Upstream of the region of identity, the two cDNAs show no sequence similarity, and downstream of the identity region Nup475 cDNA has 62 additional nucleotides prior to the poly(A) tail, including an AATAAA polyadenyl- ation site. As a result of the above differences, Nup475 cDNA encodes a protein of 319 amino acids whereas TIS11 encodes a protein of 183 amino acids, with identical sequences corre- sponding to amino acids 81 to 183 of Nup475. That the predicted sequence of the C-terminal segment of Nup475 is correct is supported by the ability of antiserum raised against a peptide corresponding to the C-terminal 15 amino acids of Nup475 to precipitate the Nup475 in vitro translation product (see Fig. 7). Several lines of evidence indicate that the Nup475 cDNA clone actually corresponds to a cellular RNA. First, as shown in Fig. 3, the 5’ end of Nup475 mRNA has been mapped on the cloned gene, and overlapping genomic and cDNA se- quences have been identified (Fig. 2). Second, when the “P- antisense transcript of a 5’ Nup475 cDNA fragment of 338 nucleotides (nucleotides 355 to 17 of Fig. 2) was annealed with RNA from regenerating liver (in which Nup475 mRNA Novel Zinc-binding Protein 19187 +1 ATAAAAGGAGAAAGCTCCTGCTGCGGGCCACAGCCTGACTICTGCGAACCGACAGTCGGTCTCTTCACCAAGGCCATTCGCGCCACC ATG GAT CTC TCT GCC ATC TAC GAG CGAACCGACAGTCGGTCTCTTCACCAAGGCCATTCGCGCCACC ATG GAT CTC TCT GCC ATC TAC GAG MET Asp Leu Ser Ala Ile Tyr Glu 8 84 AGC CTC CAG TCG ATG AGC CAT GAC CTG TCA TCC GAC CAC GGA GGA ACC GAA TCC CTC GGA GGA CTT TGG AAC ATA AAC TCG GAC TCC ATC Ser Leu Gln Ser MET Ser His Asp Leu Ser Ser Asp His Gly Gly Thr Glu Ser Leu Gly Gly Leu Trp Asn Ile Asn Ser Asp Ser Ile 38 174 CCG TCT GGG GTC ACC TCT CGC CTG ACT GGC CGC TCC ACT AGC CTG GTG GAG GGC CGA AGC TGT GGC TGG GTA CCC CCA CCC CCT GGT TTT Pro Ser Gly Val Thr Ser Arg Leu Thr Gly Arg Ser Thr Ser Leu Val Glu Gly Arg Ser Cys Gly Trp Val Pro Pro Pro Pro Gly Phe 68 264 GCA CCT TTG GCT CCC CGC CCA GGC CCT GAG CTG TCA CCC TCA CCT ACT TCG CCT ACT GCA ACT CCc ACC ACC TCC TCT CGA TAC AAG ACC Ala Pro Leu Ala Pro Arg Pro Gly Pro Glu Leu Ser Pro Ser Pro Thr Ser Pro Thr Ala Thr Pro Thr Thr Ser Ser Arg Tyr Jys Thr 98 354 GAG CTC TGT CGG ACC TAC TCA GAA AGC GGG CGT TGT CGC TAC GGG GCC AAG TGC CAG TIT GCT CAC GGC CTG GGT GAA CTT CGC CAA GCC Glu Leu Cys Arg Thr Tyr Ser Glu Ser Gly Arg Cys Arg Tyr Gly Ala Lys Cys Gln Phe Ala His Gly Leu Gly Glu Leu Arg Gln Ala 128 444 AAT CGC CAC CCC AAG TAC AAA ACG GAA CTC TGC CAC AAG TTC TAC CTC CAG GGC CGC TGC CCC TAT GGC TCT CGA TGC CAC TTC ATC Cac Asn Arg His Pro i i i 158 334 AAC CCC ACC GAG GAC CTA GCT CTC CCT GGC CAG CCC CAT GTG CTG CGA CAA AGC ATC AGC TTC TCC GGC TTG CCC TCA GGC CGC AGA AGC Asn Pro Thr Glu Asp Leu Ala Leu Pro Gly Gln Pro His Val Leu Arg Gin Ser Ile Ser Phe Ser Gly Leu Pro Ser Gly Arg Arg Ser 188 624 TCG CCG CCA CCT CCA GGC TTT TCT GGC CCT TCC CTG TCC TCT TGT TCC TTT TCG CCT TCC AGC TCC CCA CCG CCC CCT GGG GAT CTT CCA Ser Pro Pro Pro Pro Gly Phe Ser Gly Pro Ser Leu Ser Ser Cys Ser Phe Ser Pro Ser Ser Ser Pro Pro Pro Pro Gly Asp Leu Pro 218 714 ¢eTT TCC CCT TCT GCC TTC TCT GCT GCC CCT GGG ACC CCT GTG ACT CGA AGA GAC CCT AAC CAG GCC TGT TGC CCC TCC TGC CGA AGG TCT Leu Ser Pro Ser Ala Phe Ser Ala Ala Pro Gly Thr Pro Val Thr Arg Arg Asp Pro Asn Gln Ala Cys Cys Pro Ser Cys Arg Arg Ser 248 804 ACT ACC CCC AGC ACC ATC TGG GGG CCC TTG GGT GGC CTG GCT CGG AGC CCA TCT GCC CAC TCT CTG GGA TCC GAT CCT GAT GAC TAC GCC Thr Thr Pro Ser Thr Ile Trp Gly Pro Lev Gly Gly Leu Ala Arg Ser Pro Ser Ala His Ser Leu Gly Ser Asp Pro Asp Asp Tyr Ala 278 894 AGC AGC GGC AGC AGC CTG GGG GGG TCA GAC TCA CCT GTC TIT GAG GCA GGG GTG TTT GGG CCT CCC CAG ACC CCT GCA CCC CCA AGG CGT Ser Ser Gly Ser Ser Leu Gly Gly Ser Asp Ser Pro Val Phe Glu Ala Gly Val Phe Gly Pro Pro Gln Thr Pro Ala Pro Pro Arg Arg 308 984 cTc CCC ATC TTC AAT CGT ATC TCT GTC TCT GAG TGA CAAGTGCCTACCTACCCAGTATGGATCAGCTAGATCTCAAAGAGAGGGCAGGGACTGCTCATTGCTGTGGG Leu Pro Ile Phe Asn Arg Ile Ser Val Ser Glu * 319 GACCTGGGGCACTCCTCTAAGTTAATAAGTCCCATCTTCTGGACATTCCAAGATGCAATAACCCAT IT TCCCTGGTGC TGGGCTGGGGCAGGTCCCTAGTTTGCAAATICAGTGTTTGGG TGGATCCGT TCCTAGGGTACCTAAGATGT TTGAGGGAGACAGT TGACAGTTGGTCTTCCAGGCCCCAAGTCTTCTGTIGITTT TGAGATAGGAGCT TATTATGGTACCCCAGGCTGGCT TTGAACTCAATATAATCCTGCCTTAGCCTITTCCAAGTTCTGGGGT TACAGGTATGCACCAGCCCCTCTGCAACTCTGGTICTCCTGGAATCT TAAGTGCTGTGAAGAGCCGGCTCCCAC AATACTATCCTAATTTITACTAGACCCTGAAGT TCAGTGTCCGGTGGTCGAAGCCTCTCCTGAGAATCCTGGTGCTCAAATTTCCCTCCTAAAGCAAATAGCCAAAGCCATTGCCAAAT CCCTTCTCCCCCAACCAGTGGGCCCTTTAITIATGACGACTTTATITAT TGTATTAAGATITIATAGTAITTATATATATTGGGICGTCTACTCCGTITTTICTTTTTGTAATGTTAAAA CTGATACTGTATTAAGTATATGCTATAATATATTAATATATTGCTACCGTACAAGTCTATITTTTGGGGGGGGT TGGAATT TT TAaat aaaATCTTGAGTGIGAACTGAAAAAAAARA AA Fic. 2. The cDNA sequence and predicted amino acid sequence of Nup475. Numbers appearing in the left margin refer to the first nucleotide listed on that line. Numbers appearing in the right margin refer to the last amino acid on that line. The principal start of the mRNA is indicated by +1. Nucleotides in the top line were determined by sequencing a genomic clone. The underlined amino acids represent a conserved repeat of 26 amino acids. The underlined nucleotides in the 3’ untranslated region represent sequences associated with unstable mRNAs (20). Lowercase letters indicate a poly(A) addition signal. is induced, see below), treated with RNase, and fractionated by gel electrophoresis, the cellular RNA protected the anti- sense transcript from RNase digestion (data not shown), indicating that Nup475 mRNA contains a continuous se- quence corresponding to nucleotides 17 to 355 of the cDNA. Finally, when oligonucleotide primers corresponding to the 5’ and 3’ ends of Nup475 cDNA (nucleotides 2-22 and 1804- 1783, respectively) were used to amplify cDNA prepared from total RNA of serum-stimulated 3T3 cells and the resulting amplification product was analyzed, the product was of the predicted size for a nearly full length Nup475 cDNA and gave identical BamHI and Ball restriction patterns as the cloned cDNA (data not shown). We conclude that the Nup475 cDNA whose sequence is shown in Fig. 2 is derived from a single cellular mRNA species. Nucleotide Sequence of the Upstream Genomic Region— Murine genomic clones of Nup475 were isolated using the cDNA as a probe, and the 5’-most clone was identified. The nucleotide sequence just upstream of the transcription start site is shown in Fig. 5. The sequence ATAAAA at —29 relative to the start site presumably functions as a TATA element. Also present are two core Spl sequences at —34 and —362 (22) and two binding sites for another immediate early pro- tein, Zif268, at —45 and —77 (23). There is also a core glucocorticoid-like response element (24) present at nucleo- tide position —705. The sequence GATTTC which has been shown to bind transcription factor H4Tf-1 (25) is present at nucleotide —831, and there is a potential serum response element (CCATAAAAGG) (26) at position —31, overlapping the TATA-like sequence. There are two potential AP-2 sites (27), one at —43 which overlaps a Zif268 site and another at —363 which overlaps an Sp1 site. The functional significance of these various elements is not presently known. Southern Blot Analysis of Genomic DNA—Southern blot analysis of mouse genomic digests under stringent conditions (see “Experimental Procedures”) revealed single intensely hybridizing EcoRI and HindIII fragments (8.8 kb and 7.1 kh, respectively), data not shown. Human and rat DNA also showed simple EcoRI and HindIII digest patterns. These results are consistent with the presence of a single copy of nup475 in the mouse, rat, and human genomes. However, other faintly hybridizing fragments were observed in all di- gests, which could represent predominantly intronic or gene- flanking regions, or related genes. Presence of Nup475 mRNA in Mouse Cell Lines and Tis- sues—To determine how widely expressed the nup475 gene is in mouse cell lines and tissues, total RNA samples from various proliferating lines, adult mouse tissues, and mouse liver after partial hepatectomy were analyzed by Northern blot analysis for the presence of Nup475 RNA (Fig. 6). The RNA was detected in many murine cell lines including a macrophage line (RAW, Ref. 28) and B cell lines (29-31). In the macrophage cell line stimulated with spleen extract (RAW S of Fig. 7) or y-interferon (data not shown), the amount of Nup475 mRNA was severalfold greater than that in unstim- ulated cells. Of the tissues examined, thymus, regenerating liver, and small intestine had a high level of mRNA, while quiescent liver, kidney, and spleen had lower levels (Fig. 6). The RNA was also present in 8- and 12-day placenta but was barely detectable in 16-day placenta. None was detectable in whole brain RNA. We conclude that nup475 is expressed in many tissues and cell lines and its mRNA level increases in response to diverse signals. 19188 1 2 ‘ea: Fic. 3. Mapping the 5’ end of Nup475 mRNA. The autoradiogram on the left shaws the results of reverse transcription in which an antisense ’P- labeled oligonucleotide primer (nucleo- tides 45 to 17 of the cDNA, Fig. 2) was annealed to total 3°73 cell RNA and ex- tended with reverse transcriptase, and the products were fractionated by elec- trophoresis in a sequencing gel. Lane J, primer alone; fane 2, RNA from quies- cent. cells: lane 3, RNA from cells stim- ulated for 3 h with serum in the presence of 10 yg/mi cycloheximide; lanes 4-7, genomic DNA sequence: reaction prod- ucts with the oligonucleotide primer in- dicated above. Arrows indicate the primer extension products on the RNA template. The autoradiogram on the right shows the results of $1 mapping of 3T3 RNA on a “P-labeled DNA probe prepared from cloned Nup475 genomic DNA using the oligonucleotide primer indicated above (nucleotides 45 to 17). After annealing of prebe with total 3T3 cell RNA, the Si-resistant DNA was fractionated by electrophoresis in a se- quencing gel. Lane J, DNA probe; lane 2, RNA from quiescent cells; lane 3, RNA from cells stimulated for 3 h with serurn in the presence of 10 ug/ml cyclohexi- mide; lanes 4-7, genomic DNA sequence: reaction products with the oligonucleo- tide primer indicated above. Arrows in- dicate the S1-resistant probe. @ ® A @° Ss R ® P ® § C8, © oO Q O84 © 4 — N 4 L Nf ml ey /" © 7 @ ra @ & ‘ © K 4 oe ® © G N -B8O @0® L 6@x,8 © BO® Pr@- Fic. 4. Repetitive amine acid sequence of Nup475 depicted as hypothetical zinc fingers. The circled amino acids are either identical or similar in the repetitive sequence. Production of Nup475 in E. coli and Preparation of Anti- sera—~To produce Nup475 proteins, E. coli strain BL21 (DE3) was transformed with a recombinant pET8C-Nup475 plas- mid, as detailed under “Experimental Procedures.” Induced plasmid-containing strains produced a protein of approxi- mately 37 kDa not present in vector-transformed bacteria (Fig. 7). The 37-kDa protein band was excised and used for immunization of rabbits. In addition, antisera against two synthetic peptides (residues 304 to 319 and 84 to 99, respec- tively) were also prepared). The sera reacted with the £. cali- produced 37-kDa protein (data not shown) and with a 37-kDa protein synthesized in reticulocyte lysates programmed with RNA transcripts of Nup475 cDNA (Fig. 7). These results establish the identity of the &. coli protein and the activity of the antisera, Subceilular Localization of Nupd75—To determine the cel- 3 GAT C Novel Zinc-binding Protein 123 G A TF C waned A cS G c J: c LS a 6 - c S c T c & G T 6 cy T 3 G 8 tT c G @ _ lular localization of Nup475, BALB/C 3T3 cells were stimu- lated with serum, and Nup475 was detected by immunofluo- rescence with affinity-purified antibody raised against the &. coli 37-kDa protein (Fig. 8). Quiescent cells showed a faint signal in their nuclei, which increased markedly by 2 h after serum stimulation and returned to baseline by 4 h. Partially purified Nup475 blocked the immunofluorescence seen in stimulated cells. Similar experiments were carried out in cos cells transfected with a Nup475/pMT plasmid, which repli- cates in cos cells. Nup475 antigen was localized to nuclei of transfected cells (Fig. 8); similar results were obtained by analyzing cytoplasmic and nuclear fractions for Nup475 an- tigen (data not shown). We conclude that Nup475 is predom- inantly a nuclear protein. Zinc Binding by Nup475—As indicated above, the predicted amino acid sequence of Nup475 suggests that the protein may have two heavy metal-binding fingers of novel type. To deter- mine whether the protein binds metal ions, an extract of E. coli containing Nup475 was fractionated by SDS-PAGE, the separated proteins were transferred to nitrocellulose, rena- tured, and then incubated with ®Zn"*, as described under “Experimental Procedures.” As shown in Fig. 9, although many £. coli proteins bind zinc, in the pET8C-Nup475 strain there is an additional zinc-binding protein that corresponds in mobility to the predominant protein that reacts with anti- Nup475 serum. The intensity of zinc labeling of Nup475 is similar to that seen with an E. coli extract containing Zif268 (Fig. 9), a DNA-binding zine finger protein of the TF IH A type (23). Novel Zine-binding Protein 19189 ~997 -« FAGTCTGACA TTGAACGCCT GATCECCOTT CCTCCACCTC CAARTGCCAA GATTACAACC ATATGCCACT GTCTTGTCTT -$17 «-PYGPITTGTT TOTCAAGACA SGGTTTCTCA GCCGAGCATG GTGACACACG COTTTGATCO CAGCACTCCE GAGGCAGAGG ~837 CAGGCGGATT _SCTGAGTICA ASGCCASCCT GGTCTACAAA GTGAGTTICCA GGACAGCCAS AGCTATACAG AGAAACCOTG PHATE S$ “757 TCTCGAABAA CAAAAACAAC AAACAAACAA AAAAGACAGG GTTTCTGTGT GTAGICCIGG CTGTTCTGGA ACTCACTCTG 2GRE ma oN . Fig. 5. Nucleotide sequence Of the — 657 -zaGACCAGGC TASCCTCGAA CTCATAGABA TCCOTCTGCT TCTGCCTTCC AAGTGCTGAA ATTAAAAGTT TGGGCCATCA nup475 8’ flanking region. Numbers € @ left xt ‘ : ‘irs cleo- on the left side refer to the first nucleo “S97 - GIGATOTGGC ATCTTTTTAC TTCCTAAGAA ARGGGTCTCA CCAAGTTACT CAAGETGOCO TAGAACECAC TGTATAGTGS tide listed on the line with the mRNA start site designated as +1. The TATA- : 1B “S17 - AGCCAGGCCT TGAACTACCA GTCTCCTGCC TCAGCCTCCC PGAGGEGCCG GAATCACAGT CCTAAACCAG CAGATCCTAC like element is labeled, and other poten- tial protein binding sites are underlined 439 6 - or 2 : wenn es ak GARTGT CTCYCACTGT CTCTTIGTTT TTC GOT c 2 CE and labeled appropriately (Zif268, Spl, TIGTUAATOT CT KCTGT CICTTIGTTT TTCTCTOGGC TECTGGCCAT CICTGTCTCT TCCAACTTGT CTC are GRE (glucocorticoid-like response ele- ment), H4TF1, AP-2). The last 24 nucle- 7327-—«—« SEECateTeT crcAcAceca CACGECCTCA STCTCTGCHE TIGTCAATIG TCCCTAGAGC CCTGCCCOEA CITCTECCCE ), , 2). ‘ otides correspond to the first 8 codons in the cDNA. “277 - AGTTCTOTTC COSACTGODCT TCCACCTCTG AAAAACACTA GGACGCURUG CTACCATCAC CTCSAGTTGS TCGTACACAA “19? CUCTCCUTAC TACCCCCCGA GAGGGGECGG AGCTGCSSGA GGGCCAAGTT CAGGCACATT TTGCATGCTA ACCAGCAGAT _2aR2 “117 AGGCGGAAGG GGCCTAGTAC CAGGCCGGCC GAGCTACGCE GEGEGGGCEC GTCCCGGAAG CTCTAGTGGC CACGCECCCA 2iti2es 2if/268 enenuesmena TATA ~37 GSEEGCCOOA TARAAGGAGA AAGCTCCTGC TGCGGGCCAC AGCCTGACTT CTGCGAACCG ACAGTCGGTC TCTTCACCAA spi FaRE $k +44 GGCCATTCGC GCCACC ATG GAT Cre TCT GCC ATC TAC GAG Placenta Liver 1 2 12345 67 r 3 r ’ Wo >» z2>%%G ¢ a ee x oO 4 E & z = & Oa «@ & o & £9 = 4 e ee we oe o FN FT wo ad Fic. 6. Northern blot analysis of RNA from mouse tissues or cell lines. Total RNA (15 ug) was fractionated by electrophoresis and blotted onto nitrocellulose, and the filter was probed with Nup475 cDNA. The tissue or cell source of the RNA is listed above each lane. 8-, 12-, and 16-day samples were prepared from placentas at 8, 12, and 16 days of gestation. The time in hours after partial hepatectomy is Hsted above each lane of the liver panel. RAW T, and RAW S represent, respectively, quiescent and stimulated macrophages in culture, as noted in the text. DISCUSSION Of the genes so far defined that. are rapidly activated in mouse 3T3 cells by serum growth factors, several encode regulatory proteins that modulate gene expression, including cellular counterparts of viral oncoproteins; others encode potential cytokines, transmembrane proteins, or cytoskeletal proteins (1-3). A number of activated genes, identified by cDNA cloning, remain to be analyzed. In this report, we describe the analysis of one of these previously isolated cDNAs (4) and its encoded protein, designated Nup475. The nup475 gene is a typical growth factor-induced, im- mediate early gene, i.e. it is transeriptionally activated in JT3 cells within minutes of addition of serum or PDGF and is superinduced by growth factor in the presence of an inhibitor of protein synthesis (4). The level of Nup475 mRNA rises rapidly after growth stimulation and rapidly declines. The decline is due to two processes: shut-off of transcription and lability of the RNA (4). Such lability may be related to AUUUA sequences present in the 3’ untranslated region of the mRNA, as suggested for cytokine mRNAs (38) and other immediate early RNAs (3). 109 K 72K 46 K mae ae 18 ae 14 18K Fic. 7. Left, expression of Nup475 in E. coli strain BL21(DE9). Extracts of E. coli harboring vector (pET8c) or Nup475 recombinant plasmid (pE'T8c-475) were fractionated by electrophoresis in 12.4% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane I, pET&c-transformed E. cali; lane 2, pETRe Nup47T5-trans- formed E. coli. Arrowhead indicates the Nup475 protein band. Right, {*S}methionine-labeled in vitro translation products of transcripts of Nup475 after immunoprecipitation. Lane J, protein markers (kDa); lane 2, antiserum prepared against Nup475 from E. coli; lane 3, antiserum prepared against peptide corresponding to amino acids 84- 99 of Nup475; lane 4, antiserum against peptide corresponding to amino acids 304-319 of Nup475; lanes 5-7, the respective preimmune sera. The protein encoded by Nup475 cDNA is rich in glycine, serine, and proline, including three copies of the sequence PPPPG. A notable feature is the presence of two tandem copies of a 26-amino acid sequence in which 17 of the 26 amino acids are identical or closely related. Each sequence repeat contains 3 spatially conserved cysteine residues and 1 histidine residue of the form CX,CX,CX.H, suggesting a novel 19190 Fic. 8. lmmunofluorescence of Nup475 in BALB/c 3T3 cells (left) and transfected cos cells (right). BALB/c 3T3 cells were grown to confluence and maintained for 2 days in medium containing 0.5% serum (panel A), stimulated for 2 h with medium containing 20% fetal calf serum (B and C), and then reacted sequen- tially with affinity-purified Nup475 rabbit antibody and rhedarnine- conjugated anti-rabbit IgG. In C, Nup475 was added prior to reaction with anti-Nup476 antibody. Cos cells were transfected with pMT2 vector (A) or with the pMT2/Nup475 construct (B and C) and then assayed for Nup475 antigen. In C, Nup475 was added prior to reaction with anti-Nup475 antibody. type of heavy metal-binding finger structure (32-34). Al- though Nup475 binds zinc ions, we do not yet know if zinc is bound to the putative fingers or to other parts of the molecule. Compared to the classes of known zinc finger proteins, ex- emplified by TFIIIA and Xfin (35, 36), steroid hormone receptors (37), GAL 4 and related fungal proteins (38), and one of the retroviral gag proteins (33, 39), the suspected fingers of Nup475 have a different order and spacing of cysteine and histidine residues. Further work will be required to determine whether Nup475 represents a new class of zinc finger proteins and whether its nuclear localization is an indication that it is a nucleic acid-binding protein involved in regulating transcription. If Nup475 is a transcription factor, its proline-rich sequence could be part of an effector domain (44). Little is known about the regulation of nup475 expression. It is widely, but not universally, expressed in mouse tissues and cell lines, and its expression is markedly stimulated in appropriate cells by PDGF, forskolin, depolarizing ionic con- ditions, y-interferon, or 12-0-tetradecanoylphorbol-13-ace- tate, and in regenerating liver following partial hepatectomy. Thus Nup475 appears to be part of a rather general response to extracellular signaling agents. The promoter region of nup47& contains a number of potential binding sites for known transcription regulators, including Sp] sites (22), a core glucocorticoid-like response element (24), two Zif268 binding sites (23), a possible serum response element (26), and two potential AP-2 binding sites (27). A number of immediate early genes have Zif268 sites (23), and it has been suggested that binding to these sites may play a role in down- regulating gene transcription after initial growth factor-in- duced activation. Further experiments should clarify the role Novel Zinc-binding Protein pET 8e-zif 266 pET 80-475 west itt 5 3 - uw a Fic. 9. Binding of Zn" by Nup475. Extracts of E. coli har- boring vector (pETS8c) or Nup475 recombinant plasmid (pETS8c-475} or Zif268 recombinant plasmid (pPET8C-Zif268) (23) were fraction- ated by electrophoresis in 12.5% SDS-polyacrylamide gel, and the proteins were transferred to nitrocellulose. The nitrocellulose sheet was then incubated with ZnCh, and zine-binding proteins were visualized by autoradiography. The same sheet was then reacted sequentially with affinity-purified anti-Nup475 antibody, alkaline phosphatase-conjugated goat anti-rabbit IgG, and phosphatase sub- strate to visualize Nup474 antigen (pET8c-475 west). On the /eft are 4C-protein markers (numbers in kDa). Arrowheads indicate the po- sitions of ZifZ68 (upper) and Nup475 (lower). of the various protein-binding sites in regulating the nupd75 gene. 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