Oncogene (1997) 14, 837 ± 847 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Transformation by ras modi®es AP1 composition and activity Fatima Mechta1, Dominique Lallemand, Curt M Pfarr2 and Moshe Yaniv Unite des Virus OncogeÁnes; URA 1644 du CNRS, DeÂpartement des Biotechnologies, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France. The Ras proteins play a central role in regulating cell Ras proteins play a central role in signal transduc- growth and their mutation can lead to abnormal tion (Feig and Cooper, 1988; Mulcahy et al., 1985; proliferation. To analyse the potential link betwen AP1 Smith et al., 1989). These proto-oncogenes, frequently activity, encoded by members of the jun and fos gene mutated in human tumors, connect receptors as the cell families, and Ras-mediated cellular transformation, we membrane with a cytoplasmic cascade of protein have studied several NIH3T3 clones which overexpress kinases followed by downstream nuclear events the Ha-Ras or Ki-Ras oncogenes. These transformed including induction of transcription and DNA synth- ®broblasts accumulated higher levels of cJun, JunB, esis. The proteins (p21Ras) encoded by the ras genes Fra1 and Fra2 proteins relative to their normal counter- (Harvey-ras/Ha-ras, Kirsten-ras/Ki-ras and N-ras) bind parts. They also displayed increased AP1 DNA binding guanine nucleotides, possess an intrinsic GTPase activity which was predominantly composed of cJun and activity and cycle between an active (GTP bound) Fra1 containing dimers. Following serum stimulation of form and an inactive (GDP bound) state (Barbacid, Ras clones, the elevated levels of cJun and Fra1 1987). Activated p21Ras proteins stimulate a cascade remained steady, while the induction of JunB and Fra2 of cytoplasmic protein kinases including Raf, MEK, was partially attenuated. Moreover, deregulated Ras MAPK (Mitogen Activated Protein Kinase) which in signaling resulted in a complete loss of the serum turn culminates in the phosphorylation of nuclear inducibility of cFos and FosB. Ectopic co-expression of transcription factors resulting in transcriptional activa- cJun and Fra1 in NIH3T3 ®broblasts led to a tion of growth-mediating genes (Bollag and McCor- transformed phenotype, attenuation of cFos serum mick, 1991; Boulton et al., 1991; de Vries-Smit et al., inducibility, increased AP1 activity and Cyclin D1 1992; Dent et al., 1992; De rijard et al., 1994; Hibi et accumulation, all characteristics of oncogenic Ras al., 1993; Howe et al., 1992; Kyriakis et al., 1992; expressing cells. These results demonstrate that cJun Lowenstein et al., 1992; Wood et al., 1992). and Fra1 are crucial mediators of the Ras-transforma- The induction of c-jun and c-fos transcription is one tion process. of the earliest nuclear responses to a wide variety of extracellular stimuli and is thought to be crucial in Keywords: AP1; cJun; Fra1; Ras; serum inducibility; mediating cellular proliferating (Curran and Franza, transformation 1988; Herschman, 1991). The mitogen stimulation of c-fos transcription is a paradigm for a gene regulated via the Ras pathway. The SRE (Serum Response Element) in the c-fos promoter is continuously bound by two nuclear proteins SRF and Elk/TCF (Herrera Introduction et al., 1989; KoÈ nig, 1991; Norman et al., 1988; Prywes and Roeder, 1987; SchroÈ ter et al., 1990; Shaw et al., The precise mechanism by which speci®c oncopro- 1989; Treisman, 1987). Upon Ras activation Elk1 is teins cause malignant transformation of cells by rapidly phosphorylated by MAPK-1 and MAPK-2 transmitting signals for aberrant growth is not fully (also called ERK1 and ERK2) resulting in a strong understood. Proto-oncogenes commonly participate in increase in its transcriptional activity (Hipskind et al., the regulation of normal cellular growth through 1994; Prywes et al., 1988; Zinck et al., 1993). A signal transduction pathways which convert extra- dierent mechanism has been proposed for c-jun cellular stimuli into a program of gene expression. transcriptional activation which involves a cJun/ Many of these genes are also activated during the G0 ATF2 heterodimer stably bound to a TRE element to G1 transition when quiescent cells are stimulated in the c-jun promoter (van Dam et al., 1993). Ras by mitogens to re-enter the cycle. The rapid activation promotes phosphorylation of the cJun induction of such genes, called `immediate early protein augmenting its transcriptional activity (Bine - genes' is followed by the transcription of down- truy et al., 1991; Smeal et al., 1991). stream target genes that are critical for the The jun and fos families of genes are composed of proliferative response of the cell. A number of these three jun members (c-jun, junB, and junD) (Bohmann et immediate early genes encode transcription factors, al., 1987; Hirai et al., 1989; Maki et al., 1987; Ryder et including Jun and Fos. al., 1988, 1989) and four dierent fos partners (c-fos, fosB, fra1 and fra2) (Cohen and Curran, 1988; Nishina et al., 1990; Zerial et al., 1989). These genes encode Correspondence: M Yaniv components of the AP1 transcription factor formed by 1Present addresses: 1Unite INSERM 368, Biologie mole culaire du homo- or hetero-dimerization of the Jun and Fos De veloppement, ENS, 46 rue d'Ulm. 75230 Paris Cedex 05 France proteins. Dimerization occurs through a leucine-repeat 2Department of Cell Biology and Biochemistry, Texas Tech University Health Science Center, 3601 4th Street, Lubbock, Texas located in the highly conserved C-terminal part of the 79430 USA Jun proteins or in an internal position in the Fos API composition and activity in Ras-transformed fibroblasts FMechtaet al 838 proteins (Landschulz et al., 1988; Sassone-Corsi et al., dimers exhibit similar DNA binding speci®cities but 1988a). As the Fos proteins do not form dimers among dier in their transactivation eciencies (Chiu et al., themselves, AP1 is a set of low anity Jun/Jun and 1989; Hirai et al., 1990; Suzuki et al., 1991). The high anity Jun/Fos dimers (Nakabeppu et al., 1988). binding anities for dierent dimer combinations is This complex set of dimers recognizes and binds to the dependent on the speci®c TRE sequence and on the TRE element (TPA Responsive Element or TGACT- promoter context (Halazonetis et al., 1988; Ryseck and CA) through the basic domain of both proteins, Bravo, 1991). Moreover, the jun and fos immediate adjacent to the Leucine-zipper. AP1 recognition early genes are dierentially expressed during develop- sequences are found in many promoters and enhancers ment and in adult tissues (Dony and Gruss, 1987; Hirai of cellular and viral genes, including Collagenase, et al., 1989; Wilkinson et al., 1989). They also respond Stromelysin, Metallothionein IIA, Interleukin 2, dierently to mitogenic stimuli, genotoxic or differ- Transforming Growth Factor b, Simian Virus 40, entiating agents (Almendral et al., 1988; de Groot et Polyomavirus and Papillomavirus. The dierent AP1 al., 1990; Devary et al., 1991; Lau and Nathans, 1987; 3 Clone Ras Protein — NIH3T —R5 —R4 —R3 —R2 —R1 NIH3T3 L Ras L R5 R4 R3 R2 R1 Figure 1 Characterization of Ras expressing ®broblasts. Dierent neomycin-resistant clones were isolated after stable transfection of an activated Ha-ras gene in NIH3T3 ®broblasts. The level of Ras expression was analysed by immuno¯uorescence and immunoblot staining with a speci®c anti-Ras monoclonal antibody (see Material and methods). The plating eciency of soft agar clones was de®ned as the ratio of the number of macroscopic colonies after 15 days of growth to the number cells initially seeded. The plotted numbers represent data of one typical experiment. There was a good correlation between the amount of activated Ras and transformation as measured by a soft agar assay (ie: normal NIH3T3 ®broblasts or clone R5 that did not show accumulation of Ras protein do not grow in agar, whereas clones expressing the higher levels of Ras produced the higher number of colonies) API composition and activity in Ras-transformed fibroblasts FMechtaet al 839 3 3 — NIH3T —R5 —R4 —R3 —R2 —R1 —R5 —R4 —R3 —R2 —R1 — NIH3T L L cJun L cFos L L FosB L L L L L L L D L JunB L FosB Fra1 L JunD L Fra2 Figure 2 Expression pattern of the Jun and Fos proteins after transformation by Ras. The Jun and Fos protein levels were determined in the various Ras overexpressing clones by immunoblots probed with speci®c anti-Jun and anti-Fos antibodies as described in Material and methods. Identical amounts of whole-cell extracts were loaded in each lane, transferred to nitrocellulose, incubated with the indicated primary antibody and developed using enhanced chemiluminescence. The same extracts were used to performed each blot. The arrows or brackets indicate the position of the speci®c immuno-reactive band(s). Multiplicity of bands observed for JunB, Fra1 and Fra2 is caused by phosphorylation (DL, unpublished results). Note that cFos, FosB and DFosB are undetectable in these extracts. The weak bands reacting with the anti-cFos or anti-Fra1 antibodies in the NIH3T3 cell extracts are non speci®c. The dierences observed for the migration of JunB and Fra2 between the DT (R1) and the R2-R5 clones concerns probably their degree of phosphorylation and may depend on the nature of the Ras protein (Kirsten versus Harvey ras) Mechta et al., 1989; Ryseck et al., 1988; Lallemand et Results al., 1997). These observations suggest that, under various conditions, the members of the Jun and Fos Increased expression of cJun, JunB, Fra1 and Fra2 in families present dierent properties and have distinct Ras transformed cells functions. Several lines of evidence suggest that AP1 couples To study the consequences of oncogenic Ras expression signals from activated Ras proteins to the transcription on AP1 composition, we isolated a set of Ras- of target genes in transformed cells.