Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9369-9373, October 1993 Cell Biology JunD mutants with spontaneously acquired transforming potential have enhanced transactivating activity in combination with Fra-2 (AP-1/cellular transformation/spontaneous mutation/direct repeats/transactlvation) TAKASHI KAMEDA, ATSUKO AKAHORI, MARTHA H. SONOBE, TAKEHISA SUZUKI, ToSHINORI ENDO, AND HIDEO IBA* Department of Tumor Virus Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Communicated by Hidesaburo Hanafusa, July 8, 1993 ABSTRACT Although a replication-competent retrovirus transforming activity at all (20, 21). When fos family genes that carries junD has no transforming activity in chicken such as c-fos (mouse) (22),fra-2 (chicken) (7),fra-1 (rat), and embryo fibroblasts, we have isolated mutant viruses that have fosB (mouse) (unpublished results) were introduced into spontaneously acquired transforming activity. The molecularly chicken embryo fibroblasts (CEFs) by replication-competent cloned junD genes of three such mutant viruses (Ti, T2, and retroviruses, all of them induced cellular transformation of T3) were shown to be responsible for the cellular transforma- CEFs. With regard to endogenous AP-1 components in tion. DNA sequence analysis indicated that a specific polynu- logarithmically growing CEFs and quiescent CEFs, the basal cleotide in the junD sequence was tandemly multiplied three level expression offra-2 (23, 24), c-jun (18, 20, 25), andjunD times or five times in Ti and T2, respectively. The repeated (21) has been reported, whereas c-fos expression is hardly polynucleotide encodes 16 amino acid residues that are located detectable in the absence of growth stimulation. in a highly conserved region among Jun family proteins. The Although much has been learned about the transforming junD mutation in T3 involved an inversion, a translocation, and mechanisms of several oncogenes carried by retroviruses by nucleotide substitutions that caused drastic amino acid ex- comparing the structural and biochemical properties ofv-onc changes in another well-conserved region among Jun family and c-onc, this approach cannot readily be applied to most of proteins. The transcriptional activity of these mutants was thefos orjun family genes: aberrant expression ofmost ofthe analyzed by means of transient expression experiments in F9 members of AP-1 causes clear cellular transformation as cells using a reporter gene containing a single AP-1 binding site. described above. In the case of c-jun, for example, initial Compared with the wild-type JunD, none of them showed work was concentrated on the 8 region, which is specifically enhanced transactivating activity in the forms of homodimers deleted in v-Jun (16) and has been suggested to have several or of heterodimers with c-Fos or Fra-1. However, they did transcriptional functions (26, 27). But its biochemical func- tion in cellular transformation is not so clear because c-jun exhibit much higher transactivating activity than the wild type retains significant transforming activity (17, 18). Several when they formed heterodimers with Fra-2, indicating that the mutational analyses on Jun mutants and the Jun/JunD chi- mutated regions function as transactivation domains in a meras have revealed transactivation domains in the N-ter- partner-specific manner. Since we have previously reported minal portion (20) as well as the C-terminal portion (28) of that there is a basal level ofFra-2 expression in chicken embryo c-Jun, but the most critical domain has not yet been identi- fibroblasts, the results may indicate that protein complexes fied. Furthermore, there seems to be no simple correlation between JunD mutants and Fra-2 play a crucial role in the between the transforming potential and the AP-1 activity cellular transforming activity. measured in the same cells by means of transient expression experiments (28, 29). The c-jun and c-fos protooncogenes were first characterized In this report, we first describe the isolation and structural as the cellular counterparts of the viral oncogenes carried by analysis of spontaneous mutants ofjunD that have acquired avian sarcoma virus 17 (1) and Finkel-Biskis-Jinkins murine transforming activity. Since these gain-of-function mutants sarcoma virus (2), respectively. c-jun belongs to a multigene altered only a limited region in the coding sequence and have family that includesjunB (3) andjunD (4, 5), and thejun gene potential advantages for studies of the transforming mecha- family codes for nuclear proteins that dimerize with the Fos nism by Jun proteins, we next compared the transcriptional family proteins, such as c-Fos, Fra-1 (6), Fra-2 (7), and FosB regulatory function of these mutants with that of wild-type (8), to form the transcription factor complex AP-1 (9). Di- junD in F9 cells, which are reported to have only a marginal merization occurs quite specifically through a leucine zipper level of endogenous AP-1 activity. structure (10): Jun family members can form low-affinity homodimers and high-affinity heterodimers with the Fos family, whereas Fos-related proteins do not form stable MATERIALS AND METHODS homodimers (11-13). Although these hetero- and ho- Cell Growth Conditions and Transfection Assay. CEFs were modimers bind to similar DNA binding sites (TGACTCA, prepared from 10-day embryos and were maintained in min- AP-1 binding sites), each dimer was shown to have a distinct imal essential medium supplemented with 5% calf serum, transcriptional regulatory function, so that transcription can 10% tryptose phosphate broth, and 1.0% dimethyl sulfoxide be positively and negatively modulated (13-15). (DMSO) at 38.50C. The virus infection, virus titer assay, and High-level expression of c-jun alone has been reported to colony formation in soft agar were essentially the same as cause cellular transformation in chicken primary cultures described (22). CEFs (1.0 x 106 cells per 60-mm plate) were (16-18) and established cell lines (19). Although junB was stably transfected with expression plasmids by the Polybrene shown to have partial transforming activity (20), junD has no DMSO method (30), transferred after 2 days, and kept under The publication costs ofthis article were defrayed in part by page charge Abbreviations: CEF, chicken embryo fibroblast; RSV, Rous sar- payment. This article must therefore be hereby marked "advertisement" coma virus; CAT, chloramphenicol acetyltransferase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9369 9370 Cell Biology: Kameda et al. Proc. Natl. Acad. Sci. USA 90 (1993) hard agar (0.8%) for 2 weeks before counting the number of Table 1. Focus and colony formation by viruses carrying junD foci. F9 teratocarcinoma cells were maintained in Dulbecco's or its derivatives modified Eagle's medium supplemented with 10% fetal bo- Focus number,* Colony formation vine serum and were transfected with pcolTRECAT (3 ,g) Virus 10-6 x ffu/ml per 3 x 103 cells (13) and expression plasmids by the modified calcium phos- phate coprecipitation method as described (13). DNA used DS3 (vector alone) ND ND for transfection experiments was made up to 9 ,g by addition JH1 (c-jun) 1.1 86 of the control empty expression plasmid, pRSV2B. The JDM1 (junD) NDt NDt transfected F9 cells were disrupted after 48 hr, and chlor- Clone 5 0.62 37 amphenicol acetyltransferase (CAT) activity in cell lysates Ti 3.5 101 containing the same amount of protein was determined. T2 2.9 78 Plasmid Construction. For construction of a retrovirus Clone 12 0.51 28 vector carryingjunD, the 1.1-kb EcoRI-Hinfl fragment con- T3 1.4 64 taining the entire mouse junD coding region preceded by the T3A NDt NDt 5' noncoding sequence from 476 (for higher translation effi- T3B 1.02 58 ciency) was isolated from pYN3150 (kind gift from D. ffu, Focus-forming units; ND, not detected. Nathans). The fragment was blunt-ended by Klenow frag- *CEFs were kept under soft agar for 7 days after infection and the ment, ligated to Bgl II linkers, and cloned into the Bgl II site number of foci was counted. of pDS3 in the sense orientation to generate pJDM1. All tWhen CEFs were kept under hard agar for >10 days, foci became mutants were the detectable at a low frequency (about 103 ffu/ml). molecularly cloned junD introduced into tVery small background colonies were detectable (5-20) when 105 vector in a similar manner after Bgl II digestion. Sal I digests infected cells were plated. of these plasmids were ligated with the Sal I digest of pREP to generate the replication-competent viral genome structure. transforming activity spontaneously, 15 colonies were picked The ligated DNAs were transfected into CEFs and stocks of up from agar layers, trypsinized, and passaged as monolayer replication-competent viruses (subgroup A) were harvested culture under soft agar for 3 weeks. Among them, 5 mono- as described (22). A parental expression plasmid, pRSV-2B, layer cultures assumed clearly transformed morphology. The was generated by digesting pRSV-2 (31) with Sal I, filling in culture fluids were recovered and tested for the presence of with Klenow fragment, and inserting a Bgl II linker. All Bgl transforming virus by colony-forming activity assay in soft II fragments ofjunD and its derivatives were inserted into the agar. Two of the virus stocks (nos. 5 and 12) were shown to single Bgl II site of pRSV-2B and the constructs were contain high titers of colony-forming activity, comparable to designated according to the insert-e.g., pRSV-junD. that of wild-type Rous sarcoma virus (RSV) (SRA) or JH1, Molecular Cloning ofjunD Mutants. Using the PCR tech- which carries the human c-jun gene, whereas the others nique, the.junD sequences were molecularly cloned from showed a moderate or low colony-forming activity.