Proc. Nail. Acad. Sci. USA Vol. 88, pp. 8890-8894, October 1991 Cell Biology Overexpression of c-jun, junB, or junD affects cell growth differently (APi factor/growth suppression/in vitro transformation//retroviral vector) MARC CASTELLAZZI*, GIANNIS SPYROUt, NATHALIE LA VISTA*, JEAN-PIERRE DANGY*, FABRICE PIU*, MOSHE YANIVt, AND GILBERT BRUN* *Laboratoire de Biologie Mol6culaire et Cellulaire, Centre National de la Recherche Scientifique-Unite Mixte de Recherche 49, Ecole Normale Sup6rieure, 46, Allke d'Italie 69364, Lyon, France; and tUnite des Virus Oncogenes, Centre National de la Recherche Scientifique-Unitd de Recherche Associde 1149, Ddpartement des Biotechnologies, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris, France Communicated by Andre Lwoff, June 21, 1991 (receivedfor review March 21, 1991)

ABSTRACT The coding sequences of murine c-jun, junB, When quiescent fibroblasts are stimulated to enter the cell or junD, which code for with practically identical cycle, transcription of c-jun and junB is rapidly and tran- dimerization and DNA binding properties, were introduced siently activated in early G1 phase. They are therefore into a nondefective retroviral vector, and the phenotype of considered as "immediate early " (9, 10), like the primary avian fibroblasts chronically infected with each of members ofthefos family.junD transcription is only weakly these viruses was studied. Cells expressing c-jun grew in activated under these conditions (11). Furthermore, the three low-serum medium and developed into colonies in agar, two protooncogenes respond differently to activation of properties characteristic of in vitro transformation. Cells ex- kinase A- or protein kinase C-dependent signal transduction pressing junB grew in agar, with a reduced efficiency as pathways (11). Differences between the threejun genes were compared to c-jun, but did not grow in low-serum medium. also observed in their interactions with hormone receptors. Finally, no effect of junD expression on cell growth was Although excess c-Jun strongly suppressed estrogen- observed. These different phenotypes suggest that these three dependent transcriptional activation in MCF7 human breast closely related transcription factors play distinct roles during cancer cells, excess JunB only partially repressed estrogen normal cell growth. Analysis of c-jun deletion mutants and of activity, and JunD had no effect at all (12). These properties, c-jun/junB and c-jun/junD chimeric genes showed that the in addition to differential tissue distribution in adult orga- N-terminal portion (amino acids 2-168) of the c-Jun protein nisms (7) and during embryonic development (13), suggest that is involved in transcriptional activation is required for distinct functional roles for the three Jun proteins. efficient transformation. On the contrary, cells expressing a Human c-jun, and to a lesser extent humanjunB, have been truncated mouse c-Jun lacking this N-terminal domain grew shown to transform primary rat embryo cells, but only in slower than normal embryo fibroblasts. The reduced growth cooperation with an activated ras (14, 15). In contrast, rate may be related to the finding that expression of the intact overexpression of v-jun or of c-jun from avian, murine, or or the truncated mouse c-jun repressed the endogenous avian human origin was shown to be sufficient for the transforma- c-Jun homologue, suggesting that functional c-Jun product is tion of primary chicken embryo fibroblasts (CEFs) (16-18). required for normal cell growth. In the present study, we took advantage of this avian system to compare directly the oncogenic potential of each of the The c-jun protooncogene was first characterized as the murinejun genes. cellular counterpart of the v-jun oncogene carried by the avian sarcoma virus ASV17 (1, 2). The c-Jun protein together MATERIALS AND METHODS with c-Fos were shown to be components of the activator Vector Construction. The various jun coding sequences protein 1 (AP1) transcriptional complex. c-Jun can form were introduced into the RCAS retroviral vector (noted R) either Jun/Jun homodimers or Jun/Fos heterodimers via the using the intermediate CLA12 adaptor plasmid (19). R-cJUN leucine repeat present in both proteins. Homo- and het- carries a 1148-base-pair (bp) Fsp I-Sca I fragment of the erodimers bind to the same consensus sequence, TGACTCA, murine c-jun gene, R-JUNB carries a 1036-bp Stu I-Ava I present in numerous promoters, initially defined as the phor- fragment of the mouse junB gene, and R-JUND carries a bol 12-myristate 13-acetate response element (3, 4). Two 1065-bp EcoRI-HindIII fragment ofjunD. R-cJUN^169 and other genes closely related to c-jun were characterized in R-cJUNCDL carry, respectively, the CJ169 and the CDL mouse and man, junB (5) and junD (6, 7). The three Jun defective c-jun genes described previously (20). CJ169 is proteins are almost identical in their C-terminal regions, deleted from amino acid 2 to 168. The leucine repeat region which are involved in dimerization and DNA binding, involved in dimerization (amino acids 284-311) has been whereas their N-terminal parts, which are involved in tran- deleted in CDL. c-jun/junB and c-jun/junD chimeric genes scriptional activation, diverge. All three form heterodimers were constructed using the internal Acc I site present in the among themselves or with c-Fos or other members of the three genes (Fig. 6). The corresponding viruses were named c-Fos family. The existence ofgene families with identical or R-JUNCB (with the N-terminal part of c-Jun and the C-ter- very similar DNA binding specificities is not restricted to the minal part ofJunB) and R-JUNBC (opposite configuration) as jun or fos families. It is rather frequent among nuclear well as R-JUNCD and R-JUNDc. proteins (8). One of the interesting issues will be to under- CEF Culture Conditions and Generation of Fully Infected stand the reasons for this diversity. Studies on the three jun Cultures. Cell culture conditions, preparation of primary genes bring some hints in this direction. CEFs, growth curve experiments, DNA transfections, and viral infections were as described (17). To measure plating The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CEF, chicken embryo fibroblast; RSV, Rous sar- in accordance with 18 U.S.C. §1734 solely to indicate this fact. coma virus. 8890 Downloaded by guest on September 25, 2021 Cell Biology: Castellazzi et al. Proc. Natl. Acad. Sci. USA 88 (1991) 8891 efficiency, 1 x 103 cells from each culture were seeded per 108 100-mm dish in regular medium and incubated for 2 weeks. The plates were then fixed with MeOH and stained with Giemsa, and the foci were counted. For the colony-forming - R-JUNB assay, cells were plated in 0.36% Difco agar in regular A R-JUND medium per 60-mm dish, on top of a 0.72% hard agar layer. uninfected Low-serum medium contained 0.5% fetal bovine serum as the only serum supply. To generate primary viral stocks, freshly prepared CEFs were transfected with the various plasmid DNAs carrying RCAS or the R-JUN constructs, expanded 106 for 1 week in regular medium, and the cell culture superna- tants were harvested. These primary stocks (5-10 x 104 infectious particles per ml on average) were used to infect freshly prepared CEFs and to generate within a week fully 0. .0o 105 infected cultures. In all experiments presented in this paper, 8 only such fully infected cultures were analyzed. The per- 8) centage of infected CEFs as well as the number of infectious co particles in the supernatant were routinely estimated using an E 107 -0C immunochemical assay with anti-p27915 antibodies (17). 7 When individual cell clones were isolated from infected o0 cultures and probed for integrated R-JUN genomes, we found no evidence for rearrangements or deletions ofthejun coding sequences (unpublished observations; ref. 17). Antisera. Affinity-purified polyclonal rabbit antisera react- ing against the mouse c-Jun (anti-Jun), JunB, or JunD were R-JUNB prepared as described (12). Anti-Jun antibodies were gener- R-JUND ated against the 143 C-terminal amino acids of the mouse uninfected c-Jun protein beginning at the AAGLAFP sequence. Anti- bodies against JunB (anti-PepB) or against JunD (anti-PepD) were generated, respectively, against the peptides ISYLPHAPPFAGG (amino acids 210-222) and GC- QLLPQHQVPAY (amino acids 329-341). 1 0 20 Western Blotting and Immunofluorescence. Recovery of days cells, Western blotting, and immunofluorescence were done as described (12, 17). FIG. 1. Growth curves of CEF cultures, uninfected or infected with RCAS, R-cJUN, R-JUNB, or R-JUND, in regular medium (A) or in low-serum medium (B). Cell number corresponds to the average RESULTS value of two 100-mm Petri dishes. Phenotypes of CEFs Infected with Retroviruses Carrying c-junjunR, orjunD. CEF cultures fully infected with RCAS, low serum was usually more pronounced with RCAS- R-cJUN, R-JUNB, or R-JUND were generated (see Mate- infected cultures and in elder CEFs, as already reported (17). rials andMethods). They were healthy cultures, growing well Taken together, these data indicate that the R-JUN viruses in regular medium (Fig. lA). CEFs infected with R-cJUN alter to different degrees the growth capacities ofthe infected exhibited slightly enhanced growth potential. This was ob- CEFs. R-cJUN transforms CEFs, by promoting strong cell vious from (i) a shorter latency period after plating (between growth in agar and in low-serum medium. R-JUNB also day 0 and day 2), (ii) a higher saturation density (reaching 20 transforms CEFs, at a lower efficiency, promoting growth in x 106 cells per 100-mm dish compared with 10-15 x 106 with agar but not in low serum. Finally, R-JUND does not appear all other cultures), and (iii) a higher plating efficiency at low to affect the growth of cells. density (150-200 large and tight foci per 1 x 103 cells per Accumulation of Mouse Jun Proteins in the Infected CEFs. plate, instead ofabout 50 smallerfoci with the other cultures). To check if the absence of any visible effect of the junD- All of the cultures were contact inhibited and of limited containing virus or the differences between c-jun and junB life-span. Features characteristic of senescent CEFs were are not caused by drastic differences in their expression, the usually observed after 1.5-2 months (corresponding to pas- level of the mouse Jun products in the infected cells was sages 12-15), showing that they were not immortalized. analyzed. Whole cell extracts from uninfected CEFs or from The cultures were tested for their ability to grow in agar and CEFs infected with RCAS, Rous sarcoma virus (RSV), in low-serum medium, characteristic of in vitro transforma- R-cJUN, R-JUNB, and R-JUND were run on SDS/ tion. Single cells were plated in semisolid medium (Table 1, polyacrylamide gels, transferred to nitrocellulose mem- experiment 1). No colonies were observed even after 4 weeks branes, and tested for the presence of the different Jun with uninfected, RCAS-, and R-JUND-infected CEFs. In products. Proteins of the expected molecular weights were contrast, colonies were found with R-cJUN and R-JUNB, indeed seen in infected CEFs (Fig. 2). In Fig. 2A, anti-Jun although at different frequencies: in plates seeded with 1 x antibodies recognized the mouse c-Jun, JunB, and JunD 103 cells, 15-30% of the R-cJUN-infected cells formed col- virally encoded products as well as the avian endogenous onies, whereas only 0.5-3% of the R-JUNB-infected cells c-Jun. Since the sequence and the level of expression of the grew in agar. Colonies growing in agar were heterogeneous in chicken junB and junD (if they exist) are unknown, it is size, with those derived from R-JUNB-infected CEFs unclear if our general antibodies or JunB- and JunD-specific smaller, on average. antibodies will detect these proteins. If so, they should As shown in Fig. 1B, only R-cJUN-infected CEFs were comigrate with and be submitted to the same regulation as the capable of sustained growth in low-serum medium, doubling chicken c-jun since only one band was detected in control approximately every 4-5 days. The other cultures did not cells (see below). The mouse c-Jun protein (334 amino acids) grow at all. In several independent experiments, cell death in could be clearly distinguished from its endogenous chicken Downloaded by guest on September 25, 2021 8892 Cell Biology: Castellazzi et al. Proc. Natl. Acad. Sci. USA 88 (1991) Table 1. Colony formation of CEF cultures fully infected with PepB antibodies revealed bands only in R-JUNB-infected various RCAS derivatives cells. However, in addition to the expected murine JunB Number of colonies per 60-mm product, another band of variable intensity was consistently plate in semisolid medium observed. The origin of this latter product remains unclear. It may be a degradation product of the mouse protein or an Time Plating Plating Plating avian protein induced by excess mouse JunB, such as an CEF after plating, with 1 x 105 with 1 x 104 with 1 x 103 avian JunB homologue. In any case, this band was never infection weeks cells cells cells detected in uninfected CEFs or in CEFs infected with the Experiment I other viruses and is therefore specifically related to infection None 3-4 - - - with R-JUNB. These results obtained by Western blots RCAS 3-4 - - - indicate that the three mouse Jun products were expressed R-cJUN 1-2 + + 231/312* and accumulated in cells infected with the corresponding R-JUNB 2-3 + 520/562t 20/14 R-JUN viruses. R-JUNB 2-3 + 115/77t 5/7 To look for possible heterogeneity ofmouse Jun expression R-JUNB 2-3 + + 39/30 at the single cell level, immunofluorescence experiments R-JUND 3-4 - - - were performed. As shown in Fig. 3, nuclear staining was Experiment 2 indeed observed in every R-JUN-infected CEF population, None 3-4 - - - when the corresponding antibody was used. At least 80o of RCAS 3-4 - - - the infected cells showed an immunofluorescent signal in the R-cJUN 1-2 + + 162/176* nuclei. Low levels ofJun protein may also have been present R-cJUN"l69 3-4 - - - in the cytoplasm, but it was not detectable above the back- R-cJUNCDL 3_4 - - ground fluorescence. As seen with the Western blots, the R-JUNB 2-3 + 376/330t 35/54 anti-PepB and anti-PepD antibodies were highly specific: no R-JUNCB 1-2 + + 72/78t nuclear signal was detected in uninfected or in RCAS- R-JUNBC 3-4 + 98/57§ - infected CEFs or in R-JUN-infected CEFs probed with the R-JUND 3-4 - - - noncorresponding antibody (Fig. 3 C and E). The anti-Jun R-JUNCD 1-2 + + 126/143* antibodies reacted with JunB and JunD, but only very faintly R-JUNDc 3-4 - - - with uninfected orRCAS-infected cells (Fig. 3G). In all ofour -, Absence of colonies; +, too many colonies to be counted. immunofluorescence studies, the intensity ofthe Jun-specific *Fast-growing colonies, with 20% of them visible by eye. signal was significantly higher in cells infected with the tSlow-growing colonies, with about 1% of them visible by eye. differentjun-containing viruses than in control chicken cells, tFast-growing colonies, with about 1-5% visible by eye. demonstrating that the murine Jun proteins were expressed §Microcolonies, about 90%o being necrosed. and accumulated in the nucleus. Transforming Ability of c-jun Deletion Mutants and of homolog (314 amino acids). The avian product alone was c-jun/junB and c-jun/junD Chimeras. In an attempt to lo- found in the uninfected CEF control, in RCAS-infected cells, calize the portion(s) of the c-jun gene responsible for trans- and in CEFs transformed by RSV. In independent experi- formation, viruses carrying deleted c-jun or c-jun/junB and ments, this avian c-Jun band was consistently less intensely c-jun/junD chimeras were constructed (see Fig. 6 for their stained than the mouse c-Jun band (by a factor of 2-5 as structures) and their effect on transformation was analyzed. estimated visually; Fig. 2A; see Fig. 4; ref. 17), demonstrating Western blotting experiments with extracts from CEFs in- clear overexpression of the virally encoded mouse product. fected with R-cJUNCDL or R-cJUN^l69 showed the accumu- In Fig. 2B, anti-PepD antibodies detected a single band, lation of c-Jun-specific bands, with migrations in accordance present only in R-JUND-infected CEFs. In Fig. 2C, anti- to their calculated molecular weights (Fig. 4). Both products

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-4-a-----anti-JuLn to 4 - aPepD _ FIG. 2. Analysis of the Jun products in the various cell lines by Western blotting. Total cell extracts were prepared from uninfected CEFs and from CEFs infected with RCAS, RSV, R-JUND, R-cJUN, and R-JUNB. Equal amounts ofprotein (10 ,ug) were separated on polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-Jun antibodies recognizing all three Jun products (A), with anti-PepD (aPepD) antibodies (B), and with anti-PepB (aPepB) antibodies (C). The positions ofthe endogenous avian and ofthe murine c-Jun products, respectively, are given by the small arrow and the large arrowhead. Molecular weights are given as Mr X 10-3. Downloaded by guest on September 25, 2021 Cell Biology: Castellazzi et aL Proc. Natd. Acad. Sci. USA 88 (1991) 8893 In a second series ofexperiments, viruses carrying the four juncB, junBc, junCD, and jun1' chimeric genes were con- structed (Fig. 6). Immunofluorescent staining of CEFs, ex- I pressing these chimeric proteins produced the expected specificity with the different anti-Jun antibodies -(see, for example, Fig. 3 F and C). CEFs infected with R-JUNCB or R-JUcDIC, which carry the N-terminal part ofc-Jun, behaved similarly to CEFs infected with R-cJUN: they showed en- hanced growth potential in regular and low-serum medium (Fig. 5 A and B for R-JUNCB) and formed large and fast- growing colonies in agar. Only a slightly (2- to 3-fold) lower plating efficiency in agar was observed with R-JUNCB as compared with R-cJUN and R-JUNcD (Table 1, experiment 2). On the contrary, transformation capacity was lost in CEFs infected with R-JUNDc and strongly reduced in R-JUNBC infected cells (Table 1, experiment 2). Reprision of the EndCgenousAvan c-Jun ProdWct. As previously reported (17), the endogenous c-Jun product was undetectable in cells overexpressing the mouse c-Jun protein (Figs. 2A and 4). Interestingly, repression was also observed FIG. 3. Iminunofluorescent staining in CEFs. The following with cells expressing the N-terminal deleted c-JunA460 mutant cultures are shown: R-cJUN-infected CEFs probed with anti-Jun but not with cells expressing c-JuncDL. Partial repression was antibodies recognizing all three Jun proteins (A) or with anti-PepB (C); R-JUNB-infected CEFs probed with anti-PepB (B); R-JUND- observed with the cells expressing JunD (Fig. 2A). In cells infected CEFs probed with anti-PepD (D) or with anti-PepB (E); expressing JunB, the presence of an additional band of R-JUNCB-infected CEFs probed with anti-PepB (F); RCAS-infected unknown nature that migrates at the position of the endog- CEFs probed with anti-Jun antibodies recognizing all three Jun enous avian c-Jun did not allow us to conclude in favor of a proteins (G). (A-C, F, and G, x55; D and E, x220.) similar repression (Figs. 2C and 4).

also accumulated in the nucleus, as judged from immunoflu- DISCUSSION orescence studies (data not shown). The accumulation of c-JunCDL did not cause any visible phenotypic change (Fig. 5 The transforiming potential of each member of the mousejun A and B; Table 1). This result was expected, since dimer family of transcription factors was compared. Thejun genes were independently expressed ftom a self-replicating retro- formation is considered a prerequisite for c-jun-mediated RSV, in which the such as DNA binding and tran- viral vector, RCAS (19), derived from activities so far discovered, natural oncogene v-src was replaced with jun coding se- scriptional activation. On the contrary, the growth rate of cells expressing the truncated c-JunAl69 product was slowed 108 down (Fig. 5A) and plating efficiency was lowered to around one-fifth of the plating efficiency observed with uninfected CB CEFs, with very small clones observed after 2 weeks. No R-JUN growth was detected in agar (Table 1, experiment 2) or under 107 L low-serum conditions (Fig. SB) where dead cells accumu- R CJUNCJ lated.

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FIG. 4. Analysis of the Jun products by Western blotting in cells 10 20 infected with R-cJUNCDL and R-cJUNA169. Positions of the endog- days enous avian and ofthe murine c-Jun products, respectively, are given by the small arrow and the large arrowhead. Dots indicate the FIG. 5. Growth curves of CEF cultures, uninfected or infected positions of the truncated c-JunCDL and c-JunAl69 mouse products. with R-cJUN, R-JUNcn, R-cJUN L, or R-cJUN~ l9, in regular Molecular weights are given as Mr X 10-3. medium (A) or in low-serum medium (B). Downloaded by guest on September 25, 2021 8894 Cell Biology: Castellazzi et al. Proc. Natl. Acad Sci. USA 88 (1991)

DNA binding & growth In their distinct capacities to interact with other growth- dimerization agar serum""low regulatingAn interestingfactors.growth behavior was observed with cells NH2 accl expressing the N-terminal truncated c-JunAl69product. These - c - j u n cells grew significantly slower, accumulated dead cells under c - j u n A 1 6 9 -- low-serum condition, and exhibited poor plating efficiency. C-ju nCDL - - Since this c-JunAl9 truncated protein maintains its dimeriza- tion and DNA binding domains, it may compete with the endogenous c-Jun for dimerization with members of the Fos + _ family and create a partially defective AP1 factor (20). The * | >= junB effect ofthis cryptic AP1 on growth may be amplified because ~ junCB of the repression of the endogenous avian c-Jun product. Its BC -( ) - absence could be caused either by repression of the endoge- nous c-jun transcription by excess murine c-Jun protein or by its increased degradation, perhaps as a result ofthe formation of mixed heterodimers between avian and mouse c-Jun. Ex- _ _...., a jun + + periments are necessary to distinguish between these alterna- junDC - - tives. The decreased growth rate and plating efficiency ofcells containing predominantly a truncated form of c-Jun strongly FIG. 6. Structures of c-jun deletion mutants that is summary of the growth properties exhibited by 4 Ens hinfetendwith argue intact c-Jun essential for normal cell growth. the different viruses. +, Growth; -, absence of detectable growth; Finally, these experiments show that a truncated derivative of (*), CEFs infected with R-JUNnC still retain the ability to develop a nuclear factor can be used to slow down cell growth, microcolonies.~~~~ behaving in a certain sense as an antioncogene. quence. The phenotypes of primary CEF cultures, chroni- We are grateful to L. Loiseau for skillful technical assistance and to C. Pfarr and J. Samarut for valuable comments on the manuscript. cally infected with each of these viruses, were studied. This work was supported by grants from Institut National de la Sante Antibodies raised against either all murine Jun products or et de la Recherche Mddicale, Association pour la Recherche sur le specifically against JunB or JunD allowed us to show (i) Cancer, Ligue National Franqaise contre le Cancer, Federation accumulation ofthe expected mouse products and (ii) expres- National des Centres de Lutte contre le Cancer, and Fondation pour sion of each of these products at the single cell level as well la Recherche Medicale. G.S. was supported by afellowship from the as clear nuclear localization. Primary cells expressing c-jun, European Molecular Biology Organization. junB, or junD exhibited clearly different degrees of growth alterations related to in vitro transformation, depending upon 1. Maki, Y., Bos, T. J., Davis, C., Starbruck, M. & Vogt, P. K. (1987) Proc. Nat!. Acad. Sci. USA 84, 2848-2852. the jun gene considered. Cells expressing c-jun Were trans- 2. Nishimura, T. & Vogt, P. K. (1988) Oncogene 3, 659-663. formed, in that they grew efficiently in low-serum medium 3. Curran, T. & Franza, R. J. (1988) Cell 55, 395-397. and exhibited high plating efficiency in agar. With junB, the 4. Vogt, P. K. & Bos, T. J. (1990) Adv. Cancer Res. 55, 1-35. transformed phenotype was milder and restricted to growth 5. Ryder, K. & Nathans, D. (1988) Proc. Natd. Acad. Sci. USA 85, agar, a than with 8464-8467. in with lower plating efficiency c-jun. 6. Ryder, K., Lanahan, A., Perez-Albuerne, E. & Nathans, D. (1989) Differences in the transformation efficiencies were also noted Proc. Natl. Acad. Sci. USA 86, 1500-1503. between human c-jun and junB in a system requiring the 7. Hirai, S. I., Ryseck, R. P., Mechta, F., Bravo, R. & Yaniv, M. cooperation with an activated ras gene for the transformation (1989) EMBO J. 8, 1433-1439. of primary rat cells (14, 15). However, the growth properties 8. Karin, M. (1990) New Biol. 2, 126-131. 9. Ryseck, R. P., Hirai, S. I., Yaniv, M. & Bravo, R. (1988) Nature of the jun/ras-transformed cells were not studied in detail. (London) 334, 535-537. Finally, no effect on cell growth was observed with junD. 10. Bravo, R. (1990) Cell Growth Differ. 1, 305-309. These data demonstrate that overexpression of the three 11. Mechta, F., Piette, J., Hirai, S.-I. & Yaniv, M. (1989) New Biol. 1, proteins with identical dimerization and DNA binding prop- 297-304. erties can affect the growth properties of the cell differently. 12. Doucas, V., Spyrou, G. & Yaniv, M. (1991) EMBOJ. 10, 2237-2245. 13. Wilkinson, D. G., Bhatt, S., Ryseck, R.-P. & Bravo, R. (1989) Whereas c-jun and, to a lesser extent,junB contribute toward Development 106, 465-471. a fully transformed phenotype, such as the phenotype ex- 14. Schutte, J., Minna, J. D. & Birrer, M. J. (1989) Proc. Natl. Acad. hibited by RSV v-src'-infected CEFs (21),junD is incapable Sci. USA 86, 2257-2261. of such an effect. In this respect, junD may not be a 15. Schutte, J., Viallet, J., Nau, M., Segal, S., Fedorko, J. & Mina, J. protooncogene. (1989) Cell 59, 987-997. of deletion mutants and of and 16. Bos, J. T., Monteclaro, F. S., Mitsunobu, F., Ball, A. R., Chang, Analysis c-jun c-jun/junB C. H. W., Nishimura, T. & Vogt, P. (1990) Genes Dev. 4, 1677- c-jun/junD chimeric genes clearly showed that (i) the ability 1687. of c-Jun to efficiently transform cells resides in the N-termi- 17. Castellazzi, M., Dangy, J. P., Mechta, F., Hirai, S. I., Yaniv, M., nal part of the molecule (amino acids 2-168) and that (ii) the Samarut, J., Lassailly, A. & Brun, G. (1990) Oncogene 5, 1541- DNA binding and dimerization sequences of JunB and JunD 1547. can substitute for the corresponding c-Jun sequences in this 18. Suzuki, T., Hashimoto, Y., Okuno, H., Sato, H., Hishina, H. & Iba, H. (1991) Jpn. J. Cancer Res. 82, 58-64. process. It is reasonable to assume that, whereas c-Jun and 19. Hughes, S., Greenhouse, J. J., Christos, J., Petropoulos, J. & JunD can efficiently transactivate artificial promoters in Sutrave, P. S. (1987) J. Virol. 61, 3004-3012. transient chloramphenicol acetyltransferase assays (7), their 20. Hirai, S. I., Bourachot, B. & Yaniv, M. (1990) Oncogene 5, 39-46. different behavior in our transformation assays may reside in 21. Jove, R. & Hanafusa, H. (1987) Annu. Rev. Cell. Biol. 3, 1172-1174. Downloaded by guest on September 25, 2021