A Jun-Binding Protein Related to a Putative Tumor Suppressor FELIPE S
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6726-6730, July 1993 Cell Biology A Jun-binding protein related to a putative tumor suppressor FELIPE S. MONTECLARO* AND PETER K. VOGTt Department of Microbiology, University of Southern California School of Medicine and Norris Cancer Center, 2011 Zonal Avenue, Los Angeles, CA 90033-1054 Contributed by Peter K. Vogt, April 12, 1993 ABSTRACT A Agtll cDNA library of chicken embryo were visible, nitrocellulose filters (Amersham; Hybond-C fibroblasts was screened with biotinylated Jun protein to extra) impregnated with isopropyl 3-D-thiogalactopyranoside identify Jun-binding clones. Eight such clones were isolated; (10 mM) were placed over the plaques and incubated over- one contains a gene referred to asjif-l that is homologous to the night at 30°C. The filters were then rinsed of bacterial debris putative tumor suppressor gene QM.jif-l codes for a protein of with wash buffer (10 mM Tris, pH 7.5/15 mM NaCl/0.1% 25 kDa that binds to the leucine zipper ofviral and cellular Jun. Tween 20) and blocked with 5% nonfat dry milk in incubation The Jif-1 protein also binds to itself. Jif-1 does not contain a buffer (20 mM Hepes, pH 7.2/50 mM NaCl/0.1% Nonidet leucine zipper, and it does not bind to the 12-O-tetradeca- P-40/5 mM dithiothreitol) for 1 hr at 4°C. Biotinylated Jun noylphorbol 13-acetate response element DNA sequence. Com- protein was premixed as a complex with avidin-alkaline plex formation of Jif-1 with Jun inhibits DNA binding and phosphatase (Vector Laboratories; 0.9 milliunits) and added reduces transactivation by Jun. Addition of Fos protein to to 5% nonfat dry milk in incubation buffer at a final concen- Jun-Jif-1 complexes restores DNA-binding activity. These tration of 100 ng/ml (':3 nM) before incubation with the filter observations suggest that Jif-1 is a negative regulator of Jun. at 4°C with gentle shaking for 5 hr. The filters were washed quickly three times with wash buffer containing 0.2% Triton Jun is a member of the transcription factor complex AP-1. It X-100 at 4°C, followed by four more washes at room tem- binds to DNA either as a homodimer or heterodimer with Jun perature for no more than 20 min. The filters were then B, Jun D, or any member of the Fos family of proteins (1). incubated in substrate solution [100 mM Tris, pH 9.5/100 mM These dimerizations are mediated by a heptad repeat of NaCl/5 mM MgCl2/nitroblue tetrazolium (0.33 mg/ml)/ leucine residues that interacts with a similar leucine repeat in bromochloroindolyl phosphate (0.165 mg/ml)] until color the partner molecule, forming a coiled-coil structure referred development (1-2 hr). to as the leucine zipper (2). Jun appears to occupy a nodal Cloning and Sequencing. Inserts ofclones selected from the point in transcriptional regulation in the cell because it also Agtll library of CEF cDNA were amplified by PCR using interacts with unrelated transcription factors. It can form forward and reverse primers located upstream and down- dimers with cAMP response element binding proteins and stream, respectively, of the unique EcoRI site of the lacZ then bind to cAMP response element sequences (3-5). It can gene in A (Promega). Sequences of PCR products were interact with helix-loop-helix proteins such as MyoD (6, 7), determined by automated sequencing (Applied Biosystems) and it can affect transcriptional regulation by steroid and using the upstream A primer. The cloned insert was se- retinoic acid receptors (8-10). quenced by using a combination of automated and manual Jun is also the target of cellular regulatory proteins. A protocols described by the manufacturer (Sequenase; United negative regulator has been postulated to interact with the 8 States Biochemical). PCR products were cloned into pCRII domain ofJun (11-13), whereas a different inhibitory protein, (Invitrogen) using the manufacturer's recommended proce- IP-1, has been found to interfere with DNA binding of dure. Fos/Jun, acting through the leucine zipper (14, 15). Another Plasmid Construction. Plasmids VJ-0, VJ-9, VJ-8, VJ-4, cellular protein targets a highly conserved cysteine in the VJ-1, CJ-1, and CJ-4, used for in vitro transcription and basic domain in a redox control mechanism that regulates translation, were described (31). The transcription and trans- Jun-DNA binding (16, 17). Jun is also the substrate ofseveral lation vector for jif-1, pCRII-ATGJIF, was synthesized by protein kinases (18-27). PCR amplification of the cloned phage insert using oligonu- Despite these numerous protein-protein interactions in- cleotides that fused a terminal HindIII site and the first five volving Jun, the interplay of Jun with the cellular transcrip- codons of the human QM gene to the 5' coding sequence of tional machinery and the control of Jun activation potential jif-) and a phage T7 oligonucleotide (reverse primer) com- are only incompletely understood. Other still unknown pro- plementary to sequences in pCRII and located downstream of teins probably bind to and regulate Jun. Here, we report on the jif-) termination codon. The resulting PCR product was the search for such additional Jun-interacting factors (Jifs) digested with HindIII/EcoRI and then cloned into HindIIII and describe Jif-1,j which exhibits properties of a Jun regu- EcoRI sites in pCRII. pRC/RSV-JIF was constructed by latory protein. cloning the HindIII/Xba I insert of pCRII-ATGJIF into the HindIII/Xba I site of pRC/RSV (Invitrogen). The bacterial MATERIALS AND METHODS Probe Preparation and Library Screening. The chicken Jun Abbreviations: Jif, Jun-interacting factor; RSV, Rous sarcoma virus; GST, glutathione S-transferase; CEF, chicken embryo fibroblasts; protein was expressed in Escherichia coli and purified by TRE, 12-O-tetradecanoylphorbol 13-acetate response element [the nickel chromatography (28). The purified protein was bio- target DNA sequence of the AP-1 transcription factor (ATGACT- tinylated as described by Bayer and Wilchek (29). The Agtll CAT)]; CAT, chloramphenicol acetyltransferase. library of chicken embryo fibroblast (CEF) cDNA (30) was *Present address: Gladstone Institute for Cardiovascular Disease, plated in the Y1090 bacterial strain. Soon after the plaques San Francisco General Hospital, 2550 23rd Street, San Francisco, CA 94110. tPresent address: The Scripps Research Institute, 10666 North The publication costs ofthis article were defrayed in part by page charge Torrey Pines Road, La Jolla, CA 92037. payment. This article must therefore be hereby marked "advertisement" *The Jif-1 sequence has been deposited in the GenBank data base in accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession no. L13234). 6726 Downloaded by guest on September 28, 2021 Cell Biology: Monteclaro and Vogt Proc. Natl. Acad. Sci. USA 90 (1993) 6727 expression vector of chicken c-Jun, pDSH6JUN, was con- Jif-l ----P-RCYR YCKNKPYPKS RFCRGVPDPK IRIFDLGRKK AKVDEFPLCG 45 structed by PCR amplification of CJ1, using oligonucleotides QM MGRRPARCYR YCKNKPYPKS RFCRGVPDAK IRIFDLGRKK AKVDEFPLCG 50 that placed an Sph I site and six histidine codons immediately upstream of the jun coding sequence at residue 5 and a Jif-l HMVSDEYEQL SSEALEAARI CANKYMVKSC GKDGFHIRVR LHPFHVIRIN 95 BamHI site after the termination codon. The resultant PCR QM HMVSDEYEQL SSEALEAARI CANKYMVKSC GKDGFHIRVR LHPFHVIRIN 100 product was digested with Sph I/BamHI, and the fragment was cloned into the Sph I/BamHI sites of pDS56 (32). The Jif-l KMLSCAGADR LQTGMRGAFG KPQGTVARVH MGQVIMSIRT KAQNKEHVVE 145 GST-Jif-1 and GST-cJun fusion proteins were made from QM KMLSCAGADR LQTGMRGAFG KPQGTVARVH IGQVIMSIRT KLQNKEHVIE 150 pGEXlJif-1 and pGEXlcJun-1, respectively, which contain the glutathione S-transferase (GST) coding sequence fused, Jif-1 ALRRAKFKFP GRQKIHISKK WGFTKFNADA FEEMVAQKRL IPDGCGVKYV 195 * * * * respectively, to the 5' end of the jif-) and jun coding se- QM ALRRAKFKFP GRQKIHISKK WGFTKFNADE FEDMVAEKRL IPDGCGVKYI 200 quences in-frame with GST in the E. coli expression vector pGEX1. Jif-l PGRGPLDRWR ALHAA 210 Binding Assays. In vitro-synthesized mRNA transcripts QM PSRGPLDKWR ALHS- 214 were translated in the presence of 25 ,uCi (1 Ci = 37 GBq) of [35S]methionine (1200 Ci/mmol) by using rabbit reticulocyte FIG. 1. Alignment of the predicted amino acid sequences ofJif-1 lysate following the manufacturer's recommendations and QM. Mismatched residues are marked with asterisks. (Promega). The lysates were examined by SDS/PAGE, fol- lowed by fluorography. The GST-Jif and GST-Jun fusion peptide including the missing residues is 215 amino acids. The proteins were expressed in E. coli strain TG-1. Bacterial amino acid homology to QM is 92%. Thirteen amino acid lysates were prepared by sonication, and GST fusion proteins residues are substituted but are conserved with the exception were analyzed by SDS/PAGE and Coomassie staining. of residues 26 (alanine to proline), 128 (methionine to iso- Equal amounts of fusion proteins or GST control were leucine), 177 (glutamic acid to glutamine), and 199 (glycine to incubated with glutathione-agarose at 4°C. The agarose pel- serine) in Jif-1. The stop codon results in a different carboxyl- lets were rinsed and aliquoted. Translated 35S-labeled pro- terminal residue. Computer analysis of the amino acid se- teins were incubated with glutathione-bound fusion protein quence suggests that the protein is primarily basic, with (typically containing 2 ug of fusion protein as judged by predominantly a-helical structure, containing potential SDS/PAGE and Coomassie staining) for 30 min at 4°C. myristoylation and protein kinase C phosphorylation sites Unbound proteins were removed by washing four times in (data not shown). The Jif-1 protein does not contain heptad phosphate-buffered saline with 1% Triton X-100. The agarose repeats of leucine residues or of similar hydrophobic amino pellet was resuspended in sample buffer, and the boiled acids, and it does not show any structural resemblance to extract was analyzed by SDS/PAGE and fluorography.